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The Effects of ADF/Cofilin and Profilin on the Conformation of the ATP-Binding Cleft of Monomeric Actin
Abstract
Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer's ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound epsilon-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound epsilon-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound epsilon-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.
... The ADF/cofilins can bind actin monomers as well . During complex formation with G-actin ADF/cofilin can shift the nucleotide binding cleft of actin into a "closed" conformation (Kardos et al., 2009), a change that can probably be responsible for the decreased nucleotide exchange on monomeric actin in the presence of ADF/cofilin (Nishida, 1985;Paavilainen et al., 2008). Although the ADF-H domain proteins are mainly localized in the cytosol, some of them can be found in the nucleus of the cells, as well (Castano et al., 2010;Chhabra and dos Remedios, 2005). ...
... Consistently, fluorescence quenching of the G-actin bound -ATP revealed that the accessibility of the nucleotide is antagonistically regulated by ADF/cofilins and profilin. The binding of S. cerevisiae cofilin decreased, while the binding of profilin increased the accessibility of the bound nucleotide to the quencher (Kardos et al., 2009). Temperature-dependent fluorescence resonance energy transfer measurements showed that both mouse cofilin-1 and profilin decreased the flexibility of the protein matrix in the small domain (SD1 and SD2) of actin, suggesting that this region of actin works autonomously as a rigid unit during the opening and closing of the nucleotide binding cleft (Kardos et al., 2013). ...
... Actin-binding proteins profilin and cofilin, when bound to G-actin between subdomains 1 and 3, have antagonistic effects on the conformation of the nucleotide-binding cleft. Profilin stabilizes the 'open' conformation of the cleft [7,39,40], whereas cofilin appears to lock the cleft in its 'closed' conformation [39][40][41][42]. Previous studies on the thermal unfolding of G-actin showed that profilin binding decreased the actin thermal stability [23], whereas significant stabilization of G-actin was observed in its complexes with cofilin [25,26]. ...
... Actin-binding proteins profilin and cofilin, when bound to G-actin between subdomains 1 and 3, have antagonistic effects on the conformation of the nucleotide-binding cleft. Profilin stabilizes the 'open' conformation of the cleft [7,39,40], whereas cofilin appears to lock the cleft in its 'closed' conformation [39][40][41][42]. Previous studies on the thermal unfolding of G-actin showed that profilin binding decreased the actin thermal stability [23], whereas significant stabilization of G-actin was observed in its complexes with cofilin [25,26]. ...
Differential scanning calorimetry was used to investigate the thermal unfolding of actin specifically cleaved within the DNaseI-binding loop between residues Met47-Gly48 or Gly42-Val43 by two bacterial proteases, subtilisin or ECP32/grimelysin (ECP), respectively. The results obtained show that both cleavages strongly decreased the thermal stability of monomeric actin with either ATP or ADP as a bound nucleotide. An even more pronounced difference in the thermal stability between the cleaved and intact actin was observed when both actins were polymerized into filaments. Similar to intact F-actin, both cleaved F-actins were significantly stabilized by phalloidin and aluminum fluoride; however, in all cases, the thermal stability of the cleaved F-actins was much lower than that of intact F-actin, and the stability of ECP-cleaved F-actin was lower than that of subtilisin-cleaved F-actin. These results confirm that the DNaseI-binding loop is involved in the stabilization of the actin structure, both in monomers and in the filament subunits, and suggest that the thermal stability of actin depends, at least partially, on the conformation of the nucleotide-binding cleft. Moreover, an additional destabilization of the unstable cleaved actin upon ATP/ADP replacement provides experimental evidence for the highly dynamic actin structure that cannot be simply open or closed, but rather should be considered as being able to adopt multiple conformations.
Structured digital abstract
... Actin polymerization, depolymerization branching, and nucleation depend on the activity of several actin-binding partners [27][28][29] and we therefore investigated for their importance for axonal swelling formation in the Cdk12 depleted AIS. We found that RNAi-mediated knockdown of either Chickadee (Profilin), Arp2 or Arp3 resulted in a complete rescue of axonal swellings in Cdk12 −/− neuronal clones (Fig. 4H, I), demonstrating that regulated F-actin formation and branching is a key determinant in regulating axon size proximal to the soma. ...
The role of cyclin-dependent kinases (CDKs) that are ubiquitously expressed in the adult nervous system remains unclear. Cdk12 is enriched in terminally differentiated neurons where its conical role in the cell cycle progression is redundant. We find that in adult neurons Cdk12 acts a negative regulator of actin formation, mitochondrial dynamics and neuronal physiology. Cdk12 maintains the size of the axon at sites proximal to the cell body through the transcription of homeostatic enzymes in the 1-carbon by folate pathway which utilize the amino acid homocysteine. Loss of Cdk12 leads to elevated homocysteine and in turn leads to uncontrolled F-actin formation and axonal swelling. Actin remodeling further induces Drp1-dependent fission of mitochondria and the breakdown of axon-soma filtration barrier allowing soma restricted cargos to enter the axon. We demonstrate that Cdk12 is also an essential gene for long-term neuronal survival and loss of this gene causes age-dependent neurodegeneration. Hyperhomocysteinemia, actin changes, and mitochondrial fragmentation are associated with several neurodegenerative conditions such as Alzheimer's disease and we provide a candidate molecular pathway to link together such pathological events. Cell Death Discovery (2023) 9:348 ; https://doi.
... The plasmid construction of His-tagged mouse profilin (Pro) (MW: 13.5 kDa) and Rattus norvegicus full-length cardiac leiomodin (Lmod2) (MW: 61.7 kDa) were expressed in Escherichia coli strains and purified as described previously [74,70]. The concentration of Pro and Lmod2 was estimated at 280 nm with photometry (Jasco V-550 spectrophotometer) by using a calculated absorption coefficient of ε Pro = 1.472 mL (mg cm) −1 and ε Lmod2 = 0.388 mL (mg cm) −1 , respectively. ...
The monomeric (G-actin) and polymer (F-actin) forms of actin play important role in muscle development and contraction, cellular motility, division, and transport processes. Leiomodins 1–3 (Lmod1–3) are crucial for the development of muscle sarcomeres. Unlike tropomodulins that localize only at the pointed ends, the striated muscle specific Lmod2 shows diffuse distribution along the entire length of the thin filaments. The G-actin-binding profilin (Pro) facilitates the nucleotide exchange on monomeric actin and inhibits the polymerization at the barbed end, therefore contributes to the maintenance of the intracellular pool of polymerization competent ATP-G-actin. Cyclophosphamide (CP) is a cytostatic drug that can have potential side effects on muscle thin filaments at the level of actin in myofilaments. Here, we aimed at investigating the influence of CP on actin and its complexes with actin-binding proteins by using differential scanning calorimetry (DSC). We found that upon CP treatment, the denaturation of the Pro-G-actin and Lmod2-F-actin complexes was characterized by an increased enthalpy change. However, after the CP treatment, the melting temperature of F-actin was the same as in the presence of Lmod2, seems like Lmod2 does not have any effect on the structure of the CP alkylated F-actin. In case of Pro bound G-actin the melting temperature did not respond to the CP addition. The intracellular function of Lmod2 in muscle cells can be modified within CP drug treatment.
... Like in muscle cells [7] , actin is also one of most abundant proteins in neurons. Numerous findings demonstrated that actin cytoskeleton plays a pivotal role in dendritic spine morphology [7][8][9] and the motility of the growth cone [10] , and their activities are regulated by a wide variety of ABPs like Arp2/3 [11][12] , ADF/cofilin [13][14] and fascin [15][16][17] . Corresponding to the expression of actin in muscle and nerve cells, those ABPs are expressed in nerve cells, most also in muscle cells, such as profilin [18] , cofilin, gelsolin [19][20] , tropomysin [21] and fascin. ...
Previous studies show that actin-binding Rho activating protein (Abra) is expressed in cardiomyocytes and vascular smooth muscle cells. In this study, we investigated the expression profile of Abra in the central nervous system of normal adult rats by confocal immunofluorescence. Results showed that Abra immunostaining was located in neuronal nuclei, cytoplasm and processes in the central nervous system, with the strongest staining in the nuclei; in the cerebral cortex, Abra positive neuronal bodies and processes were distributed in six cortical layers including molecular layer, external granular layer, external pyramidal layer, internal granular layer, internal pyramidal layer and polymorphic layer; in the hippocampus, the cell bodies of Abra positive neurons were distributed evenly in pyramidal layer and granular layer, with positive processes in molecular layer and orien layer; in the cerebellar cortex, Abra staining showed the positive neuronal cell bodies in Purkinje cell layer and granular layer and positive processes in molecular layer; in the spinal cord, Abra-immunopositive products covered the whole gray matter and white matter; co-localization studies showed that Abra was co-stained with F-actin in neuronal cytoplasm and processes, but weakly in the nuclei. In addition, in the hippocampus, Abra was co-stained with F-actin only in neuronal processes, but not in the cell body. This study for the first time presents a comprehensive overview of Abra expression in the central nervous system, providing insights for further investigating the role of Abra in the mature central nervous system.
... Crystallographic and fluorometric studies have demonstrated that proteins inhibiting the nucleotide exchange on the actin monomer keep the cleft between actin subdomains 2 and 4 in a "closed " conformation, although those promoting the nucleotide exchange maintain this site in an "open" state, thus allowing nucleotide exchange on the bound actin monomer (48,49). We thus propose that the -sheet domain of CAP induces a conformational change in the actin molecule upon binding and that this would enhance the exchange of the nucleotide, which is located in the cleft between subdomains 2 and 4 of actin. ...
Cyclase-associated proteins (CAPs) are among the most highly conserved regulators of actin dynamics, being present in organisms from mammals to apicomplexan parasites. Yeast, plant, and mammalian CAPs are large multidomain proteins, which catalyze nucleotide exchange on actin monomers from ADP to ATP and recycle actin monomers from actin-depolymerizing factor (ADF)/cofilin for new rounds of filament assembly. However, the mechanism by which CAPs promote nucleotide exchange is not known. Furthermore, how apicomplexan CAPs, which lack many domains present in yeast and mammalian CAPs, contribute to actin dynamics is not understood. We show that, like yeast Srv2/CAP, mouse CAP1 interacts with ADF/cofilin and ADP-G-actin through its N-terminal α-helical and C-terminal β-strand domains, respectively. However, in the variation to yeast Srv2/CAP, mouse CAP1 has two adjacent profilin-binding sites, and it interacts with ATP-actin monomers with high affinity through its WH2 domain. Importantly, we revealed that the C-terminal β-sheet domain of mouse CAP1 is essential and sufficient for catalyzing nucleotide exchange on actin monomers, although the adjacent WH2 domain is not required for this function. Supporting these data, we show that the malaria parasite Plasmodium falciparum CAP, which is entirely composed of the β-sheet domain, efficiently promotes nucleotide exchange on actin monomers. Collectively, this study provides evidence that catalyzing nucleotide exchange on actin monomers via the β-sheet domain is the most highly conserved function of CAPs from mammals to apicomplexan parasites. Other functions, including interactions with profilin and ADF/cofilin, evolved in more complex organisms to adjust the specific role of CAPs in actin dynamics.
Background: CAP recycles actin monomers from ADF/cofilin and catalyzes their nucleotide exchange through an unknown mechanism.
Results: The β-sheet domain of mouse CAP1 and a “mini-CAP” from the malaria parasite promote nucleotide exchange on actin.
Conclusion: The most conserved function of CAPs is re-charging actin monomers with ATP.
Significance: Actin dynamics from mammals to apicomplexan parasites rely on CAP-catalyzed nucleotide exchange.
... Exogenously expressed LIMK1 in neurons localizes to the Golgi membranes, and cofilin is reported to play a role in the export of p75-GFP to the apical surface of tissue culture (S2) cells (Bard et al., 2006). This procedure revealed several new components, including twinstar (the Drosophila homologue of cofilin), which regulates actin polymerization (Kueh et al., 2008; Chan et al., 2009; Kardos et al., 2009). Apart from the finding that twinstar knockdown inhibited secretion of the soluble secretory protein HRP, we could not deduce anything else about its role in protein secretion (Bard et al., 2006). ...
Knockdown of the actin-severing protein actin-depolymerizing factor (ADF)/cofilin inhibited export of an exogenously expressed soluble secretory protein from Golgi membranes in Drosophila
melanogaster and mammalian tissue culture cells. A stable isotope labeling by amino acids in cell culture mass spectrometry–based protein profiling revealed that a large number of endogenous secretory proteins in mammalian cells were not secreted upon ADF/cofilin knockdown. Although many secretory proteins were retained, a Golgi-resident protein and a lysosomal hydrolase were aberrantly secreted upon ADF/cofilin knockdown. Overall, our findings indicate that inactivation of ADF/cofilin perturbed the sorting of a subset of both soluble and integral membrane proteins at the trans-Golgi network (TGN). We suggest that ADF/cofilin-dependent actin trimming generates a sorting domain at the TGN, which filters secretory cargo for export, and that uncontrolled growth of this domain causes missorting of proteins. This type of actin-dependent compartmentalization and filtering of secretory cargo at the TGN by ADF/cofilin could explain sorting of proteins that are destined to the cell surface.
Profilin (13–20 kDa) is an actin-sequestering protein specializing in maintenance of actin into proto-filamentous form. In this chapter, we discuss the role of profilin in actin elongation and its association with the formin protein. In order to understand the function of profilin in relation to its structure, we studied the sequence and structure of the protein. This chapter also discusses different binding partners of profilin to better understand its role in actin polymerization and in turn the gliding motility in apicomplexan. We also talk about the apicomplexan-specific mini-domain present in profilin in order to deduce the functional aspect of these domains. Apart from discussing the actin-profilin and actin-formin interactions from structure-function perspective, we also report various regulatory proteins of profilin.
This chapter talks about the various isoforms of ADF that exist in different higher eukaryotes and its variation from apicomplexan ADF. We have discussed the structure, the function and the stability of the protein and the way it is regulated in different actin-mediated cellular processes to control the machinery of actin polymerization. ADF, an actin depolymerizing factor which exist in two isoforms in Plasmodium, has been well-studied in the current chapter. The structural difference of ADF from apicomplexan to its higher eukaryotic counterparts has been elucidated in this chapter. We have deduced a unique motif present in the apicomplexan ADF and have discussed about its plausible role in the regulation of actin depolymerization process. Finally, we also have briefed about the various regulators of ADF known in the literature.
Actin is a ubiquitous cytoskeletal protein and is a central player in the actin-mediated gliding motility phenomenon. In this chapter, we have explored the structural aspect of actin in general and various aspects of actin polymerization. We briefly discuss the different forms of actin found in apicomplexans. Further we present the various structural details known about the molecule and have summarized the functional role of every section of the protein and its metal ions. We also have discussed about the actin polymerization process in general by providing the brief of conformational changes of actin molecule (G-actin). We also provide an overview of the actin polymerization process followed by kinetics and thermodynamics of actin polymers (F-actin). Finally, we conclude by summarizing the interactions of actin regulatory drugs and by discussing their plausible modes of action on actin.
In the present work, the thermodynamic characterisation of toxofilin–G-actin complex was completed with differential scanning calorimetry. The relative change in the under curve area of the un-complexed G-actin in the presence of varying toxofilin concentrations was used as an indirect indicator of the complex formation. The toxofilin could efficiently bind to G-actin with a KD value of 15.7 µM. Besides its binding activity, toxofilin stabilised the attached actin molecules as the Tm value of G-actin increased to 64.19 °C after the complex formation. Based on the findings, it is possible to conclude that even non-mammalian actin-binding proteins can efficiently modify the basic structural and dynamic properties of actin monomers.
Differential scanning calorimetry was applied to compare the effects of mammalian twinfilin-1 or toxofilin on the thermodynamic properties of actin monomer. Although twinfilin and toxofilin have different structure and actin-binding sites, they similarly increased the thermodynamic stability of monomeric actin. The mammalian twinfilin increased, while the toxofilin did not significantly change the T1/2 value (the width at half-height of the transition peak) during the complex formation between the actin and the monomer binding proteins. In case of toxofilin, the EA value (activation energy) significantly increased compared to twinfilin where the activation energy was nearly insensitive to the complex formation. It seems that toxofilin can achieve its main function as an actin monomer sequestering protein by more effectively and consistently modifying the basic thermodynamic properties of the monomeric actin.
The effect of mammalian twinfilin-1 on the structure and dynamics of actin filaments were studied with steady state fluorescence spectroscopy, total internal reflection fluorescence microscopy and differential scanning calorimetry techniques. It was proved before that the eukaryotic budding yeast twinfilin-1 can efficiently bind and severe actin filaments in vitro at low pH values. In the present work steady-state anisotropy measurements revealed that twinfilin can bind efficiently to F-actin. Dilution-induced depolymerization assay proved that mammalian twinfilin-1 has an actin filament severing activity. This severing activity was more pronounced at low pH values. Total internal reflection fluorescence microscopy measurements could support the severing activity of mouse twinfilin-1. The average rate of depolymerization was more apparent at low pH values. The differential scanning calorimetry measurements demonstrated that mammalian twinfilin-1 could reduce the stiffness within the actin filaments before the detachment of the actin protomers. The structural and dynamic reorganization of actin can support the twinfilin-1 induced separation of actin protomers. The measured data indicated that mammalian twinfilin-1 was able to accelerate the monomers dissociation and/or sever the filaments effectively at low pH values.
It was concluded that twinfilin-1 can affect the F-actin in biological processes or under stress situations when the pH is markedly under the physiological level.
The effect of twinfilin-1 on the structure and dynamics of monomeric actin was investigated with fluorescence spectroscopy and differential scanning calorimetry experiments. Fluorescence anisotropy measurements proved that G-actin and twinfilin-1 could form a complex. Due to the formation of the complexes the dissociation of the nucleotide slowed down from the nucleotide-binding pocket of actin. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of twinfilin-1. Temperature dependent fluorescence resonance energy transfer and differential scanning calorimetry experiments revealed that the protein matrix of actin become more rigid and more heat resistant in the presence of twinfilin-1. The results suggest that the nucleotide binding cleft shifted into a more closed and stable conformational state of actin in the presence of twinfilin-1.
A neuron receives large numbers of input stimuli from other neurons and generates an output action potential in response to those inputs. In order to preserve past learning the integration algorithm that determines outputs from inputs must be relatively stable, but to implement short term or longer term learning it must be changeable on various timescales. Hence the algorithm and changes to the algorithm must be tightly controlled. There are two general types of change that can be made. One is change to individual synaptic weights, the other is change to the algorithm that determines the way in which the individual weights of currently active synapses are integrated.
The cytoskeleton is a polymer network composed of three linear biopolymers: F-actin, microtubules, and intermediate filaments. The polymerization of these filaments from their subunits is controlled by a host of regulatory proteins. Cytoskeletal filaments are strong polyelectrolytes, and electrostatic interactions help create networks and bundled structures within the cell. The bending stiffnesses of cytoskeletal polymers are much larger than those of conventional polymers, and, therefore, motivate new theoretical models to explain the elastic response of the cytoskeleton. The incorporation of energy-consuming motor proteins within cytoskeletal networks leads to further novel material properties in these systems.
The effects of toxofilin (an actin binding protein of Toxoplasma gondii) on G-actin was studied with spectroscopy techniques. Fluorescence anisotropy measurements proved that G-actin and toxofilin interact with 2:1 stoichiometry. The affinity of toxofilin to actin was also determined with a fluorescence anisotropy assay. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of toxofilin. The results can be explained by the shift of the nucleotide binding cleft into a closed conformational state. Differential scanning calorimetry measurements revealed that actin monomers become thermodynamically more stable due to the binding of toxofilin.
Eukaryotic cells contain three distinct cytoskeletal filament systems that exhibit very different assembly properties, supramolecular architectures, dynamic behaviour, and mechanical properties. Actin, which is involved in a plethora of functions, is the most abundant protein in most cell types, and is impressively conserved across species. The highest concentrations of actin (about 20% of total protein) are found as stable microfilament systems assembled within myofibrillar contractile structures in striated muscles. In addition to its specialized role in muscle contraction, actin is present in all muscle and non-muscle cells, where it plays a variety of roles thanks to its ability to assemble and disassemble depending on cell requirements. Actin plays an important role in maintaining cell structure and function by conferring mechanical strength and enabling intracellular contraction and/or tension. In non-muscle cells, microfilaments are involved in cell motility and cytokinesis. The dynamics of the actin cytoskeleton is maintained by two factors: (1) the ability of actin to undergo reversible transformation from the monomeric state (G-actin) to the polymeric state (F-actin), and (2) the interaction of actin with actin binding proteins (ABPs), that can inhibit or stimulate actin polymerisation, sever the polymers, cross-link actin filaments into bundles or in filamentous three-dimensional networks, and bind them to cell membrane. Considering the multiple cellular functions of actin, alterations in the organization of microfilaments will result in disorganized cell arrangement and orientation, uncontrolled cell growth, and abnormal responses to the environment. Higher vertebrates express six different highly conserved actin isoforms. Over the last decades, numerous studies have tried to elucidate the specific expressions, localizations, regulations, properties, and functions of the different isoactins. The understanding of their specific underlying mechanisms would be of major relevance not only for fundamental research but also for clinical applications, since modulations of actin isoforms are directly or indirectly correlated with severe pathologies.
The main goal of the work was to uncover the dynamical changes in actin induced by the binding of cofilin and profilin. The change in the structure and flexibility of the small domain and its function in the thermodynamic stability of the actin monomer were examined with fluorescence spectroscopy and differential scanning calorimetry (DSC). The structure around the C-terminus of actin is slightly affected by the presence of cofilin and profilin. Temperature dependent fluorescence resonance energy transfer measurements indicated that both actin binding proteins decreased the flexibility of the protein matrix between the subdomains 1 and 2. Time resolved anisotropy decay measurements supported the idea that cofilin and profilin changed similarly the dynamics around the fluorescently labeled Cys-374 and Lys-61 residues in subdomain 1 and 2, respectively. DSC experiments indicated that the thermodynamic stability of actin increased by cofilin and decreased in the presence of profilin. Based on the information obtained it is possible to conclude that while the small domain of actin acts uniformly in the presence of cofilin and profilin the overall stability of actin changes differently in the presence of the studied actin binding proteins. The results support the idea that the small domain of actin behaves as a rigid unit during the opening and closing of the nucleotide binding pocket in the presence of profilin and cofilin as well. The structural arrangement of the nucleotide binding cleft mainly influences the global stability of actin while the dynamics of the different segments can change autonomously.
Differential scanning calorimetry (DSC) was applied to investigate the thermal unfolding of rabbit skeletal muscle G-actin in its complexes with actin-binding proteins - cofilin, twinfilin, and profilin. The results show that the effects of these proteins on the thermal stability of G-actin depends on nucleotide, ATP or ADP, bound in the nucleotide-binding cleft between actin subdomains 2 and 4. Interestingly, cofilin binding stabilizes both ATP-G-actin and ADP-G-actin, whereas twinfilin increases the thermal stability of ADP-G-actin, but not ATP-G-actin. By contrast, profilin strongly decreases the thermal stability of ATP-G-actin, but it has no appreciable effect on ADP-G-actin. Comparison of these DSC results with literature data reveals a relationship between the effects of actin-binding proteins on the thermal unfolding of G-actin, stabilization or destabilization, and their effects on the rate of nucleotide exchange in the nucleotide-binding cleft, decrease or increase. These results suggest that the thermal stability of G-actin depends, at least partially, on the conformation of the nucleotide-binding cleft: the actin molecule is more stable when the cleft is closed, while an opening of the cleft leads to significant destabilization of G-actin. Thus, DSC studies on the thermal unfolding of G-actin can provide new valuable information on the conformational changes induced by actin-binding proteins in the actin molecule.
We have raised antibodies against the profilin of Chironomus tentans to study the location of profilin relative to chromatin and to active genes in salivary gland polytene chromosomes. We show that a fraction of profilin is located in the nucleus, where profilin is highly concentrated in the nucleoplasm and at the nuclear periphery. Moreover, profilin is associated with multiple bands in the polytene chromosomes. By staining salivary glands with propidium iodide, we show that profilin does not co-localize with dense chromatin. Profilin associates instead with protein-coding genes that are transcriptionally active, as revealed by co-localization with hnRNP and snRNP proteins. We have performed experiments of transcription inhibition with actinomycin D and we show that the association of profilin with the chromosomes requires ongoing transcription. However, the interaction of profilin with the gene loci does not depend on RNA. Our results are compatible with profilin regulating actin polymerization in the cell nucleus. However, the association of actin with the polytene chromosomes of C. tentans is sensitive to RNase, whereas the association of profilin is not, and we propose therefore that the chromosomal location of profilin is independent of actin.
Apicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multifaceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here, we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.
Apicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multi-faceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.
Actin is a protein abundant in many cell types. Decades of investigations have provided evidence that it has many functions in living cells. The diverse morphology and dynamics of actin structures adapted to versatile cellular functions is established by a large repertoire of actin-binding proteins. The proper interactions with these proteins assume effective molecular adaptations from actin, in which its conformational transitions play essential role. This review attempts to summarise our current knowledge regarding the coupling between the conformational states of actin and its biological function.
The nucleotide state of actin (ATP, ADP-P(i), or ADP) is known to impact its interactions with other actin molecules upon polymerization as well as with multiple actin binding proteins both in the monomeric and filamentous states of actin. Recently, molecular dynamics simulations predicted that a sequence located at the interface of subdomains 1 and 3 (W-loop; residues 165-172) changes from an unstructured loop to a beta-turn conformation upon ATP hydrolysis (Zheng, X., Diraviyam, K., and Sept, D. (2007) Biophys. J. 93, 1277-1283). This region participates directly in the binding to other subunits in F-actin as well as to cofilin, profilin, and WH2 domain proteins and, therefore, could contribute to the nucleotide sensitivity of these interactions. The present study demonstrates a reciprocal communication between the W-loop region and the nucleotide binding cleft on actin. Point mutagenesis of residues 167, 169, and 170 and their site-specific labeling significantly affect the nucleotide release from the cleft region, whereas the ATP/ADP switch alters the fluorescence of probes located in the W-loop. In the ADP-P(i) state, the W-loop adopts a conformation similar to that in the ATP state but different from the ADP state. Binding of latrunculin A to the nucleotide cleft favors the ATP-like conformation of the W-loop, whereas ADP-ribosylation of Arg-177 forces the W-loop into a conformation distinct from those in the ADP and ATP-states. Overall, our experimental data suggest that the W-loop of actin is a nucleotide sensor, which may contribute to the nucleotide state-dependent changes in F-actin and nucleotide state-modulated interactions of both G- and F-actin with actin-binding proteins.
Actin-depolymerizing factor (ADF)/cofilin is a well-conserved actin-modulating protein, which induces reorganization of the actin cytoskeleton by severing and depolymerizing F-actin. ADF/cofilin also binds to G-actin and inhibits nucleotide exchange, and hence, is supposed to regulate the nucleotide-bound state of the cellular G-actin pool cooperating with profilin, another well-conserved G-actin-binding protein that promotes nucleotide exchange. In this report, we investigated the biochemical properties of the ADF/cofilin-like protein Adf73p from ciliate Tetrahymena thermophila. Adf73p also binds to both G- and F-actin and severs and depolymerizes F-actin. Unlike canonical ADF/cofilin, however, Adf73p accelerates nucleotide exchange on actin and allows repolymerization of disassembled actin. These results suggest that the actin cytoskeleton of T. thermophila is regulated by Adf73p in a different way from those of mammals, plants, and yeasts.
The fluorescence lifetime of a substance usually represents the average amount of time the molecule remains in the excited state prior to its return to the ground state. Lifetime measurements are frequently necessary in fluorescence spectroscopy. These data can reveal the frequency of collisional encounters with quenching agents (Chapter 9), the rate of energy transfer, and the rate of excited state reactions (Chapters 10 and 12). Moreover, calculation of rotational correlation times from fluorescence anisotropics requires knowledge of the fluorescence lifetime (Chapters 5 and 6). The precise nature of the fluorescence decay can reveal details about the interactions of the fluorophore with its environment. For example, multiple decay constants can be a result of a fluorophore being in several distinct environments, or a result of excited state processes. The measurement of fluorescence lifetimes is difficult because these values are typically near 10 nsec, necessitating the use of high-speed electronic devices and detectors. However, because of the importance of these data, a great deal of effort has been directed towards developing reliable means for measurement of fluorescence lifetimes. In this chapter we will discuss in detail the theory and practical aspects of lifetime measurements.
Profilin, an essential G-actin-binding protein, has two opposite regulatory functions in actin filament assembly. It facilitates
assembly at the barbed ends by lowering the critical concentration (Pantaloni, D., and Carlier, M.-F.(1993) Cell 75, 1007-1014); in contrast it contributes to the pool of unassembled actin when barbed ends are capped. We proposed that
the first of these functions required an input of energy. How profilin uses the ATP hydrolysis that accompanies actin polymerization
and whether the acceleration of nucleotide exchange on G-actin by profilin participates in its function in filament assembly
are the issues addressed here. We show that 1) profilin increases the treadmilling rate of actin filaments in the presence
of Mg ions; 2) when filaments are assembled from CaATP-actin, which polymerizes in a quasireversible fashion, profilin does not
promote assembly at the barbed ends and has only a G-actin-sequestering function; 3) plant profilins do not accelerate nucleotide
exchange on G-actin, yet they promote assembly at the barbed end. The enhancement of nucleotide exchange by profilin is therefore
not involved in its promotion of actin assembly, and the productive growth of filaments from profilin-actin complex requires
the coupling of ATP hydrolysis to profilin-actin assembly, a condition fulfilled by Mg-actin, and not by Ca-actin.
Solvent polarity and the local environment have profound effects on the emission spectral properties of fluorophores. The effects of solvent polarity are one origin of the Stokes shift, which is one of the earliest observations in fluorescence. Emission spectra are easily measured, resulting in numerous publications on emission spectra of fluorophores in different solvents, and when bound to proteins, membranes, and nucleic acids. One common use of solvent effects is to determine the polarity of the probe binding site on the macromolecule. This is accomplished by comparison of the emission spectra and/or quantum yields when the flu-orophore is bound to the macromolecule or dissolved in solvents of different polarity. However, there are many additional instances where solvent effects are used. Suppose a fluorescent ligand binds to a protein or membrane. Binding is usually accompanied by spectral shift or change in quantum yield due to the different environment for the bound ligand. Alternatively, the ligand may induce a spectral shift in the intrinsic or extrinsic protein fluorescence. In either case the spectral changes can be used to measure the extent of binding. The effects of solvent and environment on fluorescence spectra are complex, and are due to several factors in addition to solvent polarity. The factors that affect fluorescence emission spectra and quantum yields include:
On starvation, Dictyostelium cells aggregate to form multicellular fruiting bodies containing spores that germinate when transferred to nutrient-rich medium. This developmental cycle correlates with the extent of actin phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells but high in viable mature spores. Here we describe high-resolution crystal structures of pY53-actin and unphosphorylated actin in complexes with gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate group on Tyr-53 makes hydrogen-bonding interactions with residues of the DNase I-binding loop (D-loop) of actin, resulting in a more stable conformation of the D-loop than in the unphosphorylated structures. A more rigidly folded D-loop may explain some of the previously described properties of pY53-actin, including its increased critical concentration for polymerization, reduced rates of nucleation and pointed end elongation, and weak affinity for DNase I. We show here that phosphorylation of Tyr-53 inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide exchange on actin. The structure of profilin–Dictyostelium-actin is strikingly similar to previously determined structures of profilin–β-actin and profilin–α-actin. By comparing this representative set of profilin–actin structures with other structures of actin, we highlight the effects of profilin on the actin conformation. In the profilin–actin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4.7° rotation of the two major domains of actin relative to each other. As a result, the nucleotide cleft becomes moderately more open in the profilin–actin complex, probably explaining the stimulation of nucleotide exchange on actin by profilin.
• actin phosphorylation
• profilin–actin structure
• pY53-actin structure
• Dictyostelium discoideum actin
• gelsolin–actin structure
We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.
We have reexamined the interaction of purified platelet profilin with actin and present evidence that simple sequestration of actin monomers in a 1:1 complex with profilin cannot explain many of the effects of profilin on actin assembly. Three different methods to assess binding of profilin to actin show that the complex with platelet actin has a dissociation constant in the range of 1 to 5 microM. The value for muscle actin is similar. When bound to actin, profilin increases the rate constant for dissociation of ATP from actin by 1,000-fold and also increases the rate of dissociation of Ca2+ bound to actin. Kinetic simulation showed that the profilin exchanges between actin monomers on a subsecond time scale that allows it to catalyze nucleotide exchange. On the other hand, polymerization assays give disparate results that are inconsistent with the binding assays and each other: profilin has different effects on elongation at the two ends of actin filaments; profilin inhibits the elongation of platelet actin much more strongly than muscle actin; and simple formation of 1:1 complexes of actin with profilin cannot account for the strong inhibition of spontaneous polymerization. We suggest that the in vitro effects on actin polymerization may be explained by a complex mechanism that includes weak capping of filament ends and catalytic poisoning of nucleation. Although platelets contain only 1 profilin for every 5-10 actin molecules, these complex reactions may allow substoichiometric profilin to have an important influence on actin assembly. We also confirm the observation of I. Lassing and U. Lindberg (1985. Nature [Lond.] 318:472-474) that polyphosphoinositides inhibit the effects of profilin on actin polymerization, so lipid metabolism must also be taken into account when considering the functions of profilin in a cell.
In buffer containing 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 5 mM imidazole, pH 7.5, 0.1 mM CaCl2, 0.2 mM dithiothreitol, 0.01% NaN3, and 0.2 mM ATP, the KD for the formation of the 1:1 complex between Acanthamoeba actin and Acanthamoeba profilin was about 5 microM. When the actin was modified by addition of a pyrenyl group to cysteine 374, the KD increased to about 40 microM but the critical concentration (0.16 microM) was unchanged. The very much lower affinity of profilin for modified actin explains the anomalous critical concentrations curves obtained for 5-10% pyrenyl-labeled actin in the presence of profilin and the apparently weak inhibition by profilin of the rate of filament elongation when polymerization is quantified by the increase in fluorescence of pyrenyl-labeled actin. Light-scattering assays of the polymerization of unmodified actin in the absence and presence of profilin gave a similar value for the KD (about 5-10 microM) when determined by the increase in the apparent critical concentration of F-actin at steady state at all concentrations of actin up to 20 microM and by the inhibition of the initial rates of polymerization of actin nucleated by either F-actin or covalently cross-linked actin dimer. In the same buffer, but with ADP instead of ATP, the critical concentration of actin was higher (4.9 microM) and the KD of the profilin-actin complex was lower for both unmodified (1-2 microM) and 100% pyrenyl-labeled actin (4.9 microM).
Actin purified by a new, simple, and rapid purification procedure activated the ATPase activity of both heavy meromyosin and
Subfragment 1 of heavy meromyosin, and this activation was not inhibited by the removal of Ca2+. Preparations of tropomyosin-troponin inhibited (by 85%) both the acto-heavy meromyosin and acto-Subfragment 1 ATPases in
the absence of, but not in the presence of, Ca2+. This inhibition was shown to result from binding of the tropomyosin-troponin complex solely to actin and in a ratio of about
1 mole of tropomyosin-troponin to 7 moles of actin.
The fluorescent nucleotides epsilon ADP and epsilon ATP were used to study the binding and hydrolysis mechanisms of subfragment 1 (S-1) and acto-subfragment 1 from striated and smooth muscle. The quenching of the enhanced fluorescence emission of bound nucleotide by acrylamide analyzed either by the Stern-Volmer method or by fluorescence lifetime measurements showed the presence of two bound nucleotide states for 1-N6-ethenoadenosine triphosphate (epsilon ATP), 1-N6-ethenoadenosine diphosphate (epsilon ADP), and epsilon ADP-vanadate complexes with S-1. The equilibrium constant relating the two bound nucleotide states was close to unity. Transient kinetic studies showed two first-order transitions with rate constants of approximately 500 and 100 s-1 for both epsilon ATP and epsilon ADP and striated muscle S-1 and 300 and 30 s-1, respectively, for smooth muscle S-1. The hydrolysis of [gamma-32P] epsilon ATP yielded a transient phase of small amplitude (less than 0.2 mol/site) with a rate constant of 5-10 s-1. Consequently, the hydrolysis of the substrate is a step in the mechanism which is distinct from the two conformational changes induced by the binding of epsilon ATP. An essentially symmetric reaction mechanism is proposed in which two structural changes accompany substrate binding and the reversal of these steps occurs in product release. epsilon ATP dissociates acto-S-1 as effectively as ATP. For smooth muscle acto-S-1, dissociation proceeds in two steps, each accompanied by enhancement of fluorescence emission. A symmetric reaction scheme is proposed for the acto-S-1 epsilon ATPase cycle. The very similar kinetic properties of the reactions of epsilon ATP and ATP with S-1 and acto-S-1 suggest that two ATP intermediate states also occur in the ATPase reaction mechanism.
The effect of profilin, a G-actin binding protein, on the mechanism of exchange of the tightly bound metal ion and nucleotide
on G-actin, has been investigated. 1) In low ionic strength buffer, profilin increases the rates of Ca and Mg dissociation from G-actin 250- and 50-fold, respectively. On the profilin-actin complex as well as on G-actin alone, nucleotide
exchange is dependent on the concentration of divalent metal ion and is kinetically limited, at low concentration of metal
ion, by the dissociation of the metal ion. 2) Under physiological ionic conditions, nucleotide exchange on G-actin is 1 order
of magnitude faster than at low ionic strength. The rate of MgATP dissociation is increased by profilin from 0.05 s to 2 s, the rate of MgADP dissociation is increased from 0.2 s to 24 s. The dependences of the exchange rates on profilin concentration are consistent with a high affinity (5 × 106 to 107M) of profilin for ATP-G-actin, and a 20-fold lower affinity for ADP-Gactin. Profilin binding to actin lowers the affinity
of metal-nucleotide by about 1 order of magnitude. These results restrain the possible roles of profilin in actin assembly
in vivo.
We determined the structures of Acanthamoeba profilin I and profilin II by x-ray crystallography at resolutions of 2.0 and 2.8 A, respectively. The polypeptide folds and the actin-binding surfaces of the amoeba profilins are very similar to those of bovine and human profilins. The electrostatic potential surfaces of the two Acanthamoeba isoforms differ. Two areas of high positive potential on the surface of profilin II are candidate binding sites for phosphatidylinositol phosphates. The proximity of these sites to the actin binding site provides an explanation for the competition between actin and lipids for binding profilin.
ADF (actin depolymerizing factor) is an M(r) 19,000 actin-binding protein present in many vertebrate tissues and particularly abundant in neuronal cells. We have cloned human ADF and here show it to be identical in sequence to porcine destrin. Human ADF expressed in Escherichia coli behaves like native ADF from porcine brain. It binds to G-actin at pH 8 with a 1:1 stoichiometry and Kd approximately 0.2 microM, thereby sequestering monomers and preventing polymerization. It does not cosediment with F-actin at this pH, but severs actin filaments in a calcium-insensitive manner. The severing activity is only about 0.1% efficient. By contrast, at pH values below 7, ADF binds to actin filaments in a highly cooperative manner and at a 1:1 ratio to filament subunits. When the pH is raised to 8.0, the decorated filaments are rapidly severed and depolymerized.
The three-dimensional structure of bovine profilin-beta-actin has been solved to 2.55 A resolution by X-ray crystallography. There are several significant local changes in the structure of beta-actin compared with alpha-actin as well as an overall 5 degrees rotation between its two major domains. Actin molecules in the crystal are organized into ribbons through intermolecular contacts like those found in oligomeric protein assemblies. Profilin forms two extensive contacts with the actin ribbon, one of which appears to correspond to the solution contact in vitro.
Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical-chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADP-bound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi-bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.
Actin is among the most thoroughly studied of proteins. It was first identified half a century ago as the major component of thin filaments in muscle. Work in the 1960s and 1970s showed that actin is also present in nonmuscle cells as well as in plants and protozoa. The actin-based cytoskeleton appears to be ubiquitous among eukaryotes, and indeed the invention of the actin cytoskeleton may have been a key step in the earliest history of the eukaryotic lineage. A densely written summary of the most important known properties of actin fills a good-sized volume (23). But there are major discrepancies between the well-characterized in vitro behavior of purified actin and the apparent behavior of actin filaments inside of intact, living cells.
Cofilin stimulates actin filament turnover in vivo. The phenotypes of twenty yeast cofilin mutants generated by systematic mutagenesis were determined. Ten grew as well as the wild type and showed no cytoskeleton defects, seven were recessive-lethal and three were conditional-lethal and caused severe actin organization defects. Biochemical characterization of interactions between nine mutant yeast cofilins and yeast actin provided evidence that F-actin binding and depolymerization are essential cofilin functions. Locating the mutated residues on the yeast cofilin molecular structure allowed several important conclusions to be drawn. First, residues required for actin monomer binding are proximal to each other. Secondly, additional residues are required for interactions with actin filaments; these residues might bind an adjacent subunit in the actin filament. Thirdly, despite striking structural similarity, cofilin interacts with actin in a different manner from gelsolin segment-1. Fourthly, a previously unrecognized cofilin function or interaction is suggested by identification of spatially proximal residues important for cofilin function in vivo, but not for actin interactions in vitro. Finally, mutation of the cofilin N-terminus suggests that its sequence is conserved because of its critical role in actin interactions, not because it is sometimes a target for protein kinases.
Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)-twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.
Acanthamoeba actophorin is a member of ADF/cofilin family that binds both actin monomers and filaments. We used fluorescence anisotropy
to study the interaction of actin monomers with recombinant actophorin labeled with rhodamine on a cysteine substituted for
Serine-88. Labeled actophorin retains its affinity for actin and ability to reduce the low shear viscosity of actin filaments.
At physiological ionic strength, actophorin binds Mg-ADP-actin monomers (K
d = 0.1 μm) 40 times stronger than Mg-ATP-actin monomers. When bound to actin monomers, actophorin has no effect on elongation at either
end of actin filaments by Mg-ATP-actin and slightly increases the rate of elongation at both ends by Mg-ADP-actin. Thus actophorin
does not sequester actin monomers. Sedimentation equilibrium ultracentrifugation shows that actophorin and profilin compete
for binding actin monomers. Actophorin and profilin have opposite effects on the rate of exchange of nucleotide bound to actin
monomers. Despite the high affinity of actophorin for ADP-actin, physiological concentrations of profilin overcome the inhibition
of ADP exchange by actophorin. Profilin rapidly recycles ADP-actin back to the profilin-ATP-actin pool ready for elongation
of actin filaments.
We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-1 s-1) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate.
Actin-depolymerizing factor (ADF)/cofilins are essential regulators of actin filament turnover. Several ADF/cofilin isoforms are found in multicellular organisms, but their biological differences have remained unclear. Herein, we show that three ADF/cofilins exist in mouse and most likely in all other mammalian species. Northern blot and in situ hybridization analyses demonstrate that cofilin-1 is expressed in most cell types of embryos and adult mice. Cofilin-2 is expressed in muscle cells and ADF is restricted to epithelia and endothelia. Although the three mouse ADF/cofilins do not show actin isoform specificity, they all depolymerize platelet actin filaments more efficiently than muscle actin. Furthermore, these ADF/cofilins are biochemically different. The epithelial-specific ADF is the most efficient in turning over actin filaments and promotes a stronger pH-dependent actin filament disassembly than the two other isoforms. The muscle-specific cofilin-2 has a weaker actin filament depolymerization activity and displays a 5-10-fold higher affinity for ATP-actin monomers than cofilin-1 and ADF. In steady-state assays, cofilin-2 also promotes filament assembly rather than disassembly. Taken together, these data suggest that the three biochemically distinct mammalian ADF/cofilin isoforms evolved to fulfill specific requirements for actin filament dynamics in different cell types.
Twinfilin is a ubiquitous and abundant actin monomer-binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K(D) = 0.05 microM) than ATP-G-actin (K(D) = 0.47 microM) under physiological ionic conditions and forms a relatively stable (k(off) = 1.8 s(-1)) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.
The actin cytoskeleton is a complex structure that performs a wide range of cellular functions. In 2001, significant advances were made to our understanding of the structure and function of actin monomers. Many of these are likely to help us understand and distinguish between the structural models of actin microfilaments. In particular, 1) the structure of actin was resolved from crystals in the absence of cocrystallized actin binding proteins (ABPs), 2) the prokaryotic ancestral gene of actin was crystallized and its function as a bacterial cytoskeleton was revealed, and 3) the structure of the Arp2/3 complex was described for the first time. In this review we selected several ABPs (ADF/cofilin, profilin, gelsolin, thymosin beta4, DNase I, CapZ, tropomodulin, and Arp2/3) that regulate actin-driven assembly, i.e., movement that is independent of motor proteins. They were chosen because 1) they represent a family of related proteins, 2) they are widely distributed in nature, 3) an atomic structure (or at least a plausible model) is available for each of them, and 4) each is expressed in significant quantities in cells. These ABPs perform the following cellular functions: 1) they maintain the population of unassembled but assembly-ready actin monomers (profilin), 2) they regulate the state of polymerization of filaments (ADF/cofilin, profilin), 3) they bind to and block the growing ends of actin filaments (gelsolin), 4) they nucleate actin assembly (gelsolin, Arp2/3, cofilin), 5) they sever actin filaments (gelsolin, ADF/cofilin), 6) they bind to the sides of actin filaments (gelsolin, Arp2/3), and 7) they cross-link actin filaments (Arp2/3). Some of these ABPs are essential, whereas others may form regulatory ternary complexes. Some play crucial roles in human disorders, and for all of them, there are good reasons why investigations into their structures and functions should continue.
The role of profilin in the regulation of actin assembly has been reexamined. The affinity of profilin for ATP-actin appears 10-fold higher than previously thought. In the presence of ATP, the participation of the profilin-actin complex to filament elongation at the barbed end is linked to a decrease in the steady-state concentration of globular actin. This surprising effect is made possible by the involvement of the irreversible ATP hydrolysis accompanying actin polymerization. As a consequence, in the presence of thymosin β4 (Tβ4), low amounts of profilin promote extensive actin assembly off of the pool of actin-Tβ4 complex. When barbed ends are capped, profilin simply sequesters globular actin. A model is proposed for the function of profilin in actin-based motility.
Properties of human profilin I mutated in the major actin-binding site were studied and compared with wild-type profilin using β/γ-actin as interaction partner. The mutants ranged in affinity, from those that only weakly affected polymerization of actin to one that bound actin more strongly than wild-type profilin. With profilins, whose sequestering activity was low, the concentration of free actin monomers observed at steady-state of polymerization [Afree], was close to that seen with actin alone ([Acc], critical concentration of polymerization). Profilin mutants binding actin with an intermediate affinity like wild-type profilin caused a lowering of [Afree] as compared to [Acc], indicating that actin monomers and profilin:actin complexes participate in polymer formation. With a mutant profilin, which bound actin more strongly than the wild-type protein, an efficient sequestration of actin was observed, and in this case, the [Afree] at steady state was again close to [Acc], suggesting that the mutant profilin:actin had a greatly lowered ability to incorporate actin subunits at the (+)-end. The results from the kinetic and steady-state experiments presented are consonant with the idea that profilin:actin complexes are directly incorporated at the (+)-end of actively polymerizing actin filaments, while they do not support the view that profilin facilitates polymer formation.
An understanding of the actin-depolymerizing function attributed to members of the ADF/cofilin/destrin superfamily requires a structural model of these proteins in complex with actin. As a step toward defining actin-cofilin interactions, the complex of yeast cofilin with monomeric actin was predicted, starting with the actin-gelsolin segment-1 binding mode recently suggested for the actin-destrin complex. After refinement by molecular dynamics simulation, the structure of cofilin converged in a new binding mode that required only minimal changes induced in the actin-cofilin interface. The predicted complex exhibits strong interactions between the N termini of actin and cofilin, mediated by a salt bridge of cofilin Arg3 with actin Asp1. The forming of this salt bridge could be prevented by the phosphorylation of cofilin Ser4, which is believed to inhibit cofilin depolymerization activity. Recent mutagenesis studies, crosslinking experiments and peptide binding studies are consistent with the predicted model of the actin-cofilin complex. The structural homology between cofilin and gelsolin segment-1 binding to actin was confirmed experimentally by two types of competitive binding assays.
Ubiquitous among eukaryotes, the ADF/cofilins are essential proteins responsible for the high turnover rates of actin filaments in vivo. In vertebrates, ADF and cofilin are products of different genes. Both bind to F-actin cooperatively and induce a twist in the actin filament that results in the loss of the phalloidin-binding site. This conformational change may be responsible for the enhancement of the off rate of subunits at the minus end of ADF/cofilin-decorated filaments and for the weak filament-severing activity. Binding of ADF/cofilin is competitive with tropomyosin. Other regulatory mechanisms in animal cells include binding of phosphoinositides, phosphorylation by LIM kinases on a single serine, and changes in pH. Although vertebrate ADF/cofilins contain a nuclear localization sequence, they are usually concentrated in regions containing dynamic actin pools, such as the leading edge of migrating cells and neuronal growth cones. ADF/cofilins are essential for cytokinesis, phagocytosis, fluid phase endocytosis, and other cellular processes dependent upon actin dynamics.
Recent publication of the atomic structure of G-actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C., 1990, Nature 347, 37-44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno-nucleotides bound to G- and F-actin were examined in order to assess polymerization-related changes in the nucleotide phosphate site. The Mg(2+)-induced polymerization of actin quenched the fluorescence of the etheno-nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F-actin than G-actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G- and F-actin was greatest for I-, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization-related movements of charged residues in the atomic structure of G-actin are suggested. The binding of S-1 to epsilon-ATP-G-actin increased the accessibility of the etheno group to I- even over that in Mg(2+)-polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S-1 to actin. Thus, the binding of S-1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin.(ABSTRACT TRUNCATED AT 250 WORDS)
We have used polyclonal and monoclonal antibodies raised against calf thymus profilin to localize the corresponding protein in translocating, spreading, and stationary rat fibroblasts. Immunofluorescence of whole cells and immunogold labeling on ventral membranes of lysis-squirted cells showed that profilin was markedly enriched in the highly dynamic lamellipodia or pseudopodial lobes. Within these regions, a significant fraction was colocalized with dynamic actin filaments organized in actin ribs, cortical filaments, or stress fiber-like bundles, and little profilin was found in membrane areas appearing free of actin. In contrast, stress fibers of stationary cells as well as actin arcs and ring-like bundles of spreading and migrating cells showed very little label. These results are discussed in context with the proposed role of profilin in regional membrane dynamics typical for fibroblasts and are compared to previous data (Hartwig et al.: J. Cell Biol. 109:1571-1579, 1989) on profilin distribution in platelets and granulocytes.
The rotational motions of the actin from rabbit skeletal muscle and from chicken gizzard smooth muscle were measured by conventional and saturation transfer electron paramagnetic resonance (EPR) spectroscopy using maleimide spin-label rigidly bound at Cys-374. The conventional EPR spectra indicate a slight difference in the polarity of the environment of the label and in the rotational mobility of the monomeric gizzard actin compared to its skeletal muscle counterpart. These differences disappear upon polymerization. The EPR spectra of the two actins in their F form and in their complexes with heavy meromyosin (HMM) did not reveal any difference in the rotational dynamic properties that might be correlated with the known differences in the activation of myosin ATPase activity by smooth and skeletal muscle actin.
Our results agree with earlier EPR studies on skeletal muscle actin in showing that polymerization stops the nanosecond rotational motion of actin monomers and that F-actin undergoes rotational motion having an effective correlation time of the order of 0.1 ms. However, our measurements show that complete elimination of the nanosecond motions requires prolonged incubation of F-actin, suggesting that the slow formation of interfilamental cross-links in concentrated F-actin solutions contributes to this process.
We have also used the EPR spectroscopy to study the interaction between HMM and actin in the F and G form. Our results show that in the absence of salt one HMM molecule can cooperatively interact with eight monomers to produce a polymer which closely resembles F-actin in its rotational mobility but differs from the complex of F-actin with HMM. The results indicate that salt is necessary for further slowing down, in a cooperative manner, the sub-millisecond internal motion in actin polymer and for a non-cooperative change in the intramonomer conformation around Cys-374 on the binding of HMM.
There is evidence that the polymerization of actin takes place at the plasma membrane, and that profilactin (profilin/actin complex), the unpolymerized form of actin found in extracts of many non-muscle cells, serves as the immediate precursor. Both isolated profilin and profilactin interact with detergent when analysed by charge shift electrophoresis, indicating that they have amphipathic properties and may be able to interact directly with the plasma membrane. We demonstrate here that isolated profilin, as well as the profilactin complex, interacts with anionic phospholipids. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was found to be the most active phospholipid, causing a rapid and efficient dissociation of profilactin with a concomitant polymerization of the actin in appropriate conditions. These and other observations suggest the possibility of a relationship between the induction of actin filament formation and the increased activity in the phosphatidylinositol cycle seen as a result of ligand-receptor interactions in various systems.
To obtain information of the disposition of α-toxin when bound to the acetylcholine receptor (AChR), we evaluated the accessibility of solutes to fluorescein isothiocyanate (FIITC) conjugated to α-toxin (siamensis 3) at lysine 23 (FITC-toxin) by measuring the rate constants for iodide quenching of the fluorescence of fluorescein free in solution and FITC-toxin free in solution and bound to AChR. Relative to the free fluorescein, we observed a 55% reduction in the quenching rate constant for the unbound FITC-toxin and 80% reduction for the AChR-bound FITC-toxin. It is tempting to interpret a decrease in the quenching rate constant due to an increase in the masking of the labeling fluorophore, which in our case would then be indicative of masking of fluorescein conjugated to the free toxin and masking of FITC-toxin, in the region of lysine 23, when bound to AChR. However, elementary considerations indicate that the quenching rate depends not only on geometrical masking factors but also on the translational and rotational mobilities of the labeled molecules as well as orientational constraints. To evaluate these effects we have established quantitative relations between the rate of fluorescence quenching, the degree of masking of fluorophore, translational and rotational rates, and orientational constraints of the labeled macromolecules, using recent formulations for the rate of reaction between asymmetric molecules. These relations predict that the decrease in quenching constant observed for the labeled FITC-toxin as well as the AChR-bound FITC-toxin is largely due to differences in translational and rotational rates and orientational constraints and not to significant increases in geometrical masking. Our theoretical formulation shows that the quenching rate can be decreased by a factor of 2-5 merely by immobilizing a fluorophore on the surface of a large protein without any significant increase in geometrical masking.
Cofilin, an actin-binding protein isolated from porcine brain that reacts with actin in a 1:1 molar ratio [Nishida, E., Maekawa, S., & Sakai, H. (1984) Biochemistry 23, 5307-5313], decreases the rate of exchange of ATP bound to G-actin with 1,N6-ethenoadenosine 5'-triphosphate in solution. From analyses of the dependence of the exchange rate on the cofilin concentration under different KCl concentrations, dissociation constants (KD) for the cofilin-actin binding at 0, 50, and 140 mM KCl were determined to be 0.12, 0.15, and 0.25 microM, respectively. In contrast to cofilin, profilin isolated from porcine brain increases the rate of exchange of G-actin-bound ATP, like Acanthamoeba profilin. The kinetic analyses gave KD values for the profilin-actin binding of 1.1 and 1.5 microM, respectively, at 50 and 200 mM KCl.
Intrinsic optical density, Folin, and Biuret color development have been carefully studied as methods of determining actin concentration in solution. It appears that the Lowry (Folin) method is the most sensitive and reliable method as standardized by Kjeldahl analysis. Intrinsic optical density is also found to be a reliable method and the extinction coefficients of F and G actin at 280 and 290 nm are determined. The Biuret reaction is found to be the least reliable of the three methods for determining the concentration of actin in solution.
The complete amino-acid sequence of actin of rabbit skeletal muscle was determined. The actin polypeptide chain is composed of 374 residues, including one residue of the unusual amino acid N(r)-methyl histidine, and has a calculated molecular weight of 41,785. The sequence of actin was determined by isolating the peptides produced by cleavage of the protein with cyanogen bromide, determining the sequence of these peptides, and establishing their order within the molecule. This study represents the first complete determination of the aminoacid sequence of a myofibrillar protein. Comparison of this sequence with peptides from actins isolated from different sources indicates that the sequence of actin is highly conserved.
It was found that the fluorescence of 1,N6-ethenoadenosine triphosphate (ε-ATP) bound to myosin subfragment-1 (S-1) is resistant to quenching by acrylamide, while free ε-ATP is effectively quenched. Thus in the presence of acrylamide the bound ε-ATP is still highly fluorescent, while free ε-ATP is much less fluorescent. The Stern-Volmer constants of bound and free ε-ATP are 6.83 and 57.86 M−1, respectively. Therefore it is easy to distinguish spectro-scopically the nucleotide-ligated S-1 from nucleotide-free S-1. Moreover acrylamide does not alter the S-1-Mg2+-ε-ATPase behavior.
Cofilin, a 21 000 molecular weight protein of porcine brain, reacts stoichiometrically with actin in a 1:1 molar ratio. Upon binding of cofilin, the fluorescence of pyrene-labeled actin under polymerizing conditions is changed into the monomer form, irrespective of whether cofilin is added to actin before or after polymerization. Cofilin decreases the viscosity of actin filaments but increases the light-scattering intensity of the filaments. The centrifugation assay and the DNase I inhibition assay demonstrate that cofilin binds to actin filaments in a 1:1 molar ratio of cofilin to actin monomer in the filament and that cofilin increases the monomeric actin to a limited extent (up to 1.1-1.5 microM monomer) in the presence of physiological concentrations of Mg2+ and KCl. Cofilin is also able to bind to monomeric actin, as demonstrated by gel filtration. Electron microscopy showed that actin filaments are shortened and slightly thickened in the presence of cofilin. No bundle formation was observed in the presence of various concentrations of cofilin. The gel point assay using an actin cross-linking protein and the nucleation assay also suggested that cofilin shortens the actin filaments and hence increases the filament number. Cofilin blocks the binding of tropomyosin to actin filaments. Tropomyosin is dissociated from actin filaments by the binding of cofilin to actin filaments. Cofilin was found to inhibit the superprecipitation of actin-myosin mixtures as well as the actin-activated myosin ATPase. All these results suggest that cofilin is a new type of actin-associated protein.
A sevenfold molar excess of Acanthamoeba profilin, a 12 000-dalton protein that inhibits actin polymerization, increases the rate of exchange of ATP bound to G-actin with ATP in solution about 17-fold, i.e., from 7.7 x 10(-4) to 1.3 x 10(-2) S-1, at 25 degrees C, 0.033 mM Ca2+, and 0.1 mM ATP, pH 7.5. Detailed analysis of the equilibrium isotope-exchange data shows that profilin and actin form a 1:1 complex with KD = 4.7 x 10(-5) M and that the binding of profilin to actin is rapid and reversible. The actin-profilin complex binds 1 mol of ATP/mol, as does G-actin. Profilin does not interact with ATP or Ca2+.
Profilin is a ubiquitous eukaryotic protein that binds to both cytosolic actin and the phospholipid phospha-tidylinositol-4,5-bisphosphate. These dual competitive binding capabilities of profilin suggest that profilin serves as a link between the phosphatidyl inositol cycle and actin polymerization, and thus profilin may be an essential component in the signaling pathway leading to cytoskeletal rearrangement.
The refined three-dimensional solution structure of human profilin I has been determined using multidimensional heteronuclear NMR spectroscopy. Twenty structures were selected to represent the solution conformational ensemble. This ensemble of structures has root-mean-square distance deviations from the mean structure of 0.58 Å for the backbone atoms and 0.98 Å for all non-hydrogen atoms. Comparison of the solution structure of human profilin to the crystal structure of bovine profilin reveals that, although profilin adopts essentially identical conformations in both states, the solution structure is more compact than the crystal structure. Interestingly, the regions that show the most structural diversity are located at or near the actin-binding site of profilin. We suggest that structural differences are reflective of dynamical properties of profilin that facilitate favorable interactions with actin. The global folding pattern of human profilin also closely resembles that of Acanthamoeba profilin I, reflective of the 22% sequence identity and ∼45% sequence similarity between these two proteins.
Actophorin from Acanthamoeba castellanii severs actin filaments and sequesters actin monomers. Here we report that actophorin binds ADP-bound monomers with higher affinity than ATP-bound monomers. Actophorin is therefore much less efficient at severing actin filaments in the presence of ADP compared to ATP, particularly taking account of the higher critical concentration in ADP. Monomer binding is also reduced in the presence of 25 mM inorganic phosphate (which is assumed to form ADP.Pi-actin). These findings are discussed in the light of observations on the nucleotide specificity of other monomer binding proteins and related to the role of actin in lamellar protrusion and cell locomotion.
Profilin regulates the behavior of the eukaryotic microfilament system through its interaction with non-filamentous actin. It also binds several ligands, including poly(l-proline) and the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Bovine profilin crystals (space group C2; a = 69·15 Å, b = 34·59 Å, c = 52·49 Å; α = γ = 90°, β = 92·56°) were grown from a mixture of poly(ethylene glycol) 400 and ammonium sulfate. X-ray diffraction data were collected on an imaging plate scanner at the DORIS storage ring (DESY, Hamburg), and were phased by molecular replacement, using a search model derived from the 2·55 Å structure of profilin complexed to β-actin. The refined model of bovine profilin has a crystallographic R -factor of 16·5% in the resolution range 6·0 to 2·0 Å and includes 128 water molecules, several of which form hydrogen bonds to stabilize unconventional turns.
The role of profilin in the regulation of actin assembly has been reexamined. The affinity of profilin for ATP-actin appears 10-fold higher than previously thought. In the presence of ATP, the participation of the profilin-actin complex to filament elongation at the barbed end is linked to a decrease in the steady-state concentration of globular actin. This surprising effect is made possible by the involvement of the irreversible ATP hydrolysis accompanying actin polymerization. As a consequence, in the presence of thymosin beta 4 (T beta 4), low amounts of profilin promote extensive actin assembly off of the pool of actin-T beta 4 complex. When barbed ends are capped, profilin simply sequesters globular actin. A model is proposed for the function of profilin in actin-based motility.
The structure of an "open state" of crystalline profilin:beta-actin has been solved to 2.65 A by X-ray crystallography. The open-state crystals, in 1.8 M potassium phosphate, have an expanded unit cell dimension in the c direction of 185.7 A compared with 171.9 A in the previously solved ammonium sulphate-stabilized "tight-state" structure. The unit cell change between the open and the tight states is accompanied by large subdomain movements in actin. Furthermore, the nucleotide in the open state is significantly more exposed to solvent, and local conformational changes in the hydrophobic pocket surrounding cysteine 374 occur during the transition to the tight state. Significant changes were observed at the N terminus and in the DNase-I binding loop. Neither the structure of profilin nor its contact with beta-actin are affected by the changes in the unit cell. Applying osmotic pressure to profilin:beta-actin crystals brings about a collapse of the unit cell comparable with that seen in the open to tight-state transition, enabling an estimate of the work required to cause this transformation of beta-actin in the crystals. The slight difference in energy between the open and collapsed states explains the extreme sensitivity of profilin:beta-actin crystals to changes in chemical and thermal environment.
Previous crystallographic investigations have shown that actin can undergo large conformational changes, even when complexed to the same actin binding protein. We have conducted a formal analysis of domain motions in actin, using the four available crystal structures, to classify the mechanism as either hinge or shear and to quantify the magnitude of these changes. We demonstrate that actin consists of two rigid cores, a semi-rigid domain and three conformationally variable extended loops. Confirming predictions about the nature of the domain rotation in actin based on its structural similarity to hexokinase, we show, using an algorithm previously used only to identify protein hinges, that residues at the interface between the two rigid cores undergo a shear between alternative conformations of actin. Rotations of less than 7 degrees in the torsion angles of five residues in the polypeptides that connect the rigid cores enable one actin conformation to be transformed into another. Because these torsion angle changes are small, the interface between the domains is maintained. In addition, we show that actin secondary structure elements, including those outside the rigid cores, are conformationally invariant among the four crystal structures, even when actin is complexed to different actin binding proteins. Finally, we demonstrate that the current F-actin models are inconsistent with the principles of actin conformational change identified here.
Three methods, fluorescence anisotropy of rhodamine-labeled profilin, intrinsic fluorescence and nucleotide exchange, give the same affinity, Kd = 0.1 microM, for Acanthamoeba profilins binding amoeba actin monomers with bound Mg-ATP. Replacement of serine 38 with cysteine created a unique site where labeling with rhodamine did not alter the affinity of profilin for actin. The affinity for rabbit skeletal muscle actin is about 4-fold lower. The affinity for both actins is 5-8-fold lower with ADP bound to actin rather than ATP. Pyrenyliodoacetamide labeling of cysteine 374 of muscle actin reduces the affinity for profilin 10-fold. The affinity of profilin for nucleotide-free actin is approximately 3-fold higher than for Mg-ATP-actin and approximately 24-fold higher than for Mg-ADP-actin. As a result, profilin binding reduces the affinity of actin 3-fold for Mg-ATP and 24-fold for Mg-ADP. Mg-ATP dissociates 8 times faster from actin-profilin than from actin and binds actin-profilin 3 times faster than actin. Mg-ADP dissociates 14 times faster from actin-profilin than from actin and binds actin-profilin half as fast as actin. Thus, profilin promotes the exchange of ADP for ATP. These properties allow profilin to bind a high proportion of unpolymerized ATP-actin in the cell, suppressing spontaneous nucleation but allowing free barbed ends to elongate at more than 500 subunits/second.
The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 microM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins. The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to the Kd of 2.0 microM for the interaction between yeast profilin and pyrene-labeled yeast actin. The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface. The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar to the reported affinity of Acanthamoeba profilin for poly-L-proline. Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin. The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure.
We have investigated the effects of profilin on nucleotide binding to actin and on steady state actin polymerization. The rate constants for the dissociation of ATP and ADP from monomeric Mg-actin at physiological conditions are 0.003 and 0.009 s-1, respectively. Profilin increases these dissociation rate constants to 0.08 s-1 for MgATP-actin and 1.4 s-1 for MgADP-actin. Thus, profilin can increase the rate of exchange of actin-bound ADP for ATP by 140-fold. The affinity of profilin for monomeric actin is found to be similar for MgATP-actin and MgADP-actin. Continuous sonication was used to allow study of solutions having sustained high filament end concentrations. During sonication at steady state, F-actin depolymerizes toward the critical concentration of ADP-actin [Pantaloni, D., et al. (1984)J. Biol. Chem. 259, 6274-6283], our analysis indicates that under these conditions a significant number of filaments contain terminal ADP-actin subunits. Addition of profilin to this system increases the polymer concentration and increases the steady state ATPase activity during sonication. These data are explained by the fast exchange of ATP for ADP on the profilin-ADP-actin complex, resulting in rapid ATP-actin regeneration. An important function of profilin may be to provide the growing ends of filaments with ATP-actin during periods when the monomer cycling rate exceeds the intrinsic nucleotide exchange rate of monomeric actin.
Thymosin-beta(4) (Tbeta(4)) binds actin monomers stoichiometrically and maintains the bulk of the actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers. The K(d) of the actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation. At 25 degrees C, the free energy of Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange.
Cellular movements are powered by the assembly and disassembly of actin filaments. Actin dynamics are controlled by Arp2/3 complex, the Wiskott-Aldrich syndrome protein (WASp) and the related Scar protein, capping protein, profilin, and the actin-depolymerizing factor (ADF, also known as cofilin). Recently, using an assay that both reveals the kinetics of overall reactions and allows visualization of actin filaments, we showed how these proteins co-operate in the assembly of branched actin filament networks. Here, we investigated how they work together to disassemble the networks.
Actin filament branches formed by polymerization of ATP-actin in the presence of activated Arp2/3 complex were found to be metastable, dissociating from the mother filament with a half time of 500 seconds. The ADF/cofilin protein actophorin reduced the half time for both dissociation of gamma-phosphate from ADP-Pi-actin filaments and debranching to 30 seconds. Branches were stabilized by phalloidin, which inhibits phosphate dissociation from ADP-Pi-filaments, and by BeF3, which forms a stable complex with ADP and actin. Arp2/3 complex capped pointed ends of ATP-actin filaments with higher affinity (Kd approximately 40 nM) than those of ADP-actin filaments (Kd approximately 1 microM), explaining why phosphate dissociation from ADP-Pi-filaments liberates branches. Capping protein prevented annealing of short filaments after debranching and, with profilin, allowed filaments to depolymerize at the pointed ends.
The low affinity of Arp2/3 complex for the pointed ends of ADP-actin makes actin filament branches transient. By accelerating phosphate dissociation, ADF/cofilin promotes debranching. Barbed-end capping proteins and profilin allow dissociated branches to depolymerize from their free pointed ends.