Fig 5 - available from: Nature Communications
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Crystal structure of full-length FasR in complex with DNA. Electron density map (sigmaA-weighted 2mF obs -DF calc Fourier) is shown (magenta mesh), overlaid on the final refined model, showing the protein dimer (protomers in light blue and pale green) and the DNA double helix (orange) in cartoon representation. The map was carved around the atomic model with a border of 3 Å to improve the clarity. Helices α3 are seen fitting within the DNA major groove as expected.
Source publication
Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal struc...
Contexts in source publication
Context 1
... mutants showed significant functional effects, uncoupling ligand-binding and DNA association (Fig. 3d), with a clearer effect observed in the case of FasR F123A . To further dissect the underlying mechanisms, DNA-binding affinities were analyzed, comparing wild-type vs FasR L98A and FasR F123A mutants ( Supplementary Fig. 5). Dissociation constants were quantitated from EMSA data, all in the nanomolar range (Table 1). ...
Context 2
... FasR-DNA 3D structure confirms the allosteric mechanism. The crystal structure of full-length FasR was solved by cocrystallisation with a 25-bp double-stranded oligonucleotide bearing the native FasR-binding sequence motif (Fig. 5). A number of crystals and cryo-protections methods were tested, consistently producing strongly anisotropic X-ray diffraction data, reaching 3.85 Å resolution in the best direction (Supplementary Table 1). A form of crystal disorder affected the position of the DNA double helix (details in Methods), occupying equivalent positions in ...
Citations
... 20,21 A key part of MftR's regulatory mechanism involves binding to a conserved Omft site upstream of mftA matching the palindrome sequence pattern 'T-N2-GGCA-N5-TGCC-N2-A'. 15 Regarding ligand binding, TetR proteins, 22,23 including MftR, are important regulators that play critical roles in modulating bacterial processes and sensitivity to drugs by interacting with various small molecules, 24,25 including long-chain fatty acyl-CoAs. [26][27][28] However, the ligand-binding domains of acyl-CoA-binding proteins exhibit relatively low sequence homology and are regulated by different acyl-CoA chain lengths. This sequence variability likely underlies structural differences that contribute to distinct regulatory functions. ...
Mycobacterium tuberculosis, the pathogen of the deadly disease tuberculosis, depends on the redox cofactor mycofactocin (MFT) to adapt to and survive under hypoxic conditions. MftR is a TetR family transcription regulator that binds upstream of the MFT gene cluster and controls MFT synthesis. To elucidate the structural basis underlying MftR regulation, we determined the crystal structure of Mycobacterium tuberculosis MftR (TB‐MftR). The structure revealed an interconnected hydrogen bond network in the α1‐α2‐α3 helices of helix‐turn‐helix (HTH) DNA‐binding domain that is essential for nucleic acid interactions. The ligand‐binding domain contains a hydrophobic cavity enclosing long‐chain fatty acyl‐CoAs like the key regulatory ligand oleoyl‐CoA. Despite variations in ligand‐binding modes, comparative analyses suggest regulatory mechanisms are largely conserved across TetR family acyl‐CoA sensors. By elucidating the intricate structural mechanisms governing DNA and ligand binding by TB‐MftR, our study enhances understanding of the regulatory roles of this transcription factor under hypoxic conditions, providing insights that could inform future research into Mycobacterium tuberculosis pathogenesis.
... The usage of strain as a probe for protein function is justified because on functional timescales proteins behave largely as elastic materials [7,8], which explains why elastic paradigms have been so successful in protein biophysics during the past decades [9]. However, despite recent applications to diverse protein structures [10][11][12], the relationship between strain patterns and rotation or displacement descriptions traditionally associated to protein function remains unaddressed. Here, we establish this relationship, and show that distinct strain patterns correspond to distinct functions performed by one same protein. ...
One of the tenets of molecular biology is that dynamic transitions between three-dimensional structures determine the function of proteins. Therefore, it seems only natural that evolutionary analysis of proteins, presently based mainly on their primary sequence, needs to shift its focus toward their function as assessed by corresponding structural transitions. This can be facilitated by recent progress in cryogenic electron microscopy that provides atomic structures of multiple conformational states for proteins and protein assemblies isolated from evolutionarily related species. In this work, we study evolutionary conservation of multiprotein assembly function by using mechanical strain as a quantitative footprint of structural transitions. We adopt the formalism of finite strain theory, developed in condensed matter physics, and apply it, as a case study, to a classical multiprotein assembly, the ATP synthase. Protein strain analysis provides a precise characterization of rotation domains that agrees with the present biophysical knowledge. In addition, we obtain a strain distribution on the protein structure associated with functional transitions. By analyzing in detail the strain patterns of the chains responsible for ATP synthesis across distinct species, we show that they are evolutionarily conserved for the same functional transition. Such conservation is not revealed by displacement or rotation patterns. Furthermore, within each functional transition, we can identify conserved strain patterns for ATP synthases isolated from different organisms. The observed strain conservation across evolutionary distant species indicates that strain should be essential in future structure-based evolutionary studies of protein function.
... The usage of strain as a probe for protein function is justified because on func-tional timescales proteins behave largely as elastic materials [7,8], which explains why elastic paradigms have been so successful in protein biophysics during the last decades [9]. However, despite recent applications to diverse protein structures [10][11][12], the relationship between strain patterns and rotation or displacement descriptions traditionally associated to protein function remains unaddressed. Here, we establish this relationship, and show that distinct strain patterns correspond to distinct functions performed by one same protein. ...
One of the tenets of molecular biology is that dynamic transitions between three-dimensional structures determine the function of proteins. Therefore, it seems only natural that evolutionary analysis of proteins, presently based mainly on their primary sequence, needs to shift its focus towards their function as assessed by corresponding structural transitions. This can be facilitated by recent progress in cryogenic electron microscopy that provides atomic structures of multiple conformational states for proteins and protein assemblies isolated from evolutionarily related species. In this work, we study evolutionary conservation of multi-protein assembly function by using mechanical strain as a quantitative footprint of structural transitions. We adopt the formalism of finite strain analysis, developed in condensed matter physics, and apply it, as a case study, to a classical multi-protein assembly, the ATP synthase. Our Protein Strain Analysis (PSA) provides a precise characterization of rotation domains that agrees with the present biophysical knowledge. In addition, we obtain a strain distribution on the protein structure associated with functional transitions. By analyzing in detail, the strain patterns of the chains responsible for ATP synthesis across distinct species, we show that they are evolutionarily conserved for the same functional transition. Such conservation is not revealed by displacement or rotation patterns. Furthermore, within each functional transition, we can identify conserved strain patterns for ATP synthases isolated from different organisms. The observed strain conservation across evolutionary distant species indicates that strain should be essential in future structure-based evolutionary studies of protein function.
... The transposon 10 (Tn10) of Escherichia coli is the prototype of TetR regulators, which regulates the expression of the tetracycline efflux pump in Gram-negative bacteria (Orth et al., 2000). However, TetR regulators are widely distributed among bacteria and control a broad range of processes, including fatty acid biosynthesis in Mtb (Lara et al., 2020). ...
Mycobacteria, like other microorganisms, survive under different environmental variations by expressing an efficient adaptive response, oriented by regulatory elements, such as transcriptional repressors of the TetR family. These repressors in mycobacteria also appear to be related to cholesterol metabolism. In this study, we have evaluated the effect of a fatty acid (oleic–palmitic–stearic)/cholesterol mixture on some phenotypic and genotypic characteristics of a tetR-mutant strain (BCG_2177c mutated gene) of M. bovis BCG, a homologous of Rv2160A of M. tuberculosis. In order to accomplish this, we have analyzed the global gene expression of this strain by RNA-seq and evaluated its neutral-lipid storage capacity and potential to infect macrophages. We have also determined the macrophage response by measuring some pro- and anti-inflammatory cytokine expressions. In comparison with wild-type microorganisms, we showed that the mutation in the BCG_2177c gene did not affect the growth of M. bovis BCG in the presence of lipids but it probably modified the structure/composition of its cell envelope. Compared to with dextrose, an overexpression of the transcriptome of the wild-type and mutant strains was observed when these mycobacteria were cultured in lipids, mainly at the exponential phase. Twelve putative intracellular redox balance maintenance genes and four others coding for putative transcriptional factors (including WhiB6 and three TetR-like) were the main elements repeatedly overexpressed when cultured in the presence of lipids. These genes belonged to the central part of what we called the “genetic lipid signature” for M. bovis BCG. We have also found that all these mycobacteria genotypic changes affected the outcome of BCG-infected macrophages, being the mutant strain most adapted to persist longer inside the host. This high persistence result was also confirmed when mutant-infected macrophages showed overexpression of the anti-inflammatory cytokine TGF-β versus pro-inflammatory cytokines. In summary, the lack of this TetR-like repressor expression, within a lipid environment, may help mycobacteria overcome intracellular redox stress and survive longer inside their host.
... The above structural comparison suggests that 2,4-DAPG mechanically triggers a closed to open conformational transition of PhlH. It is intriguing that the ligand-free form of PhlH exhibits a closed, DNA-binding competent configuration, which is in sharp contrast with the previous notion that the vast majority of available TetR-family crystal structures including TetR, with no effector bound, correspond to the open conformation (25)(26)(27). For those TetR-family regulators, the presence of cognate operator DNA rather than ligand dissociation induces an open to closed conformational switching. ...
... For most TetR-family regulators, the apo-form structures usually adopt open, DNA-binding incompetent conformation, which most closely resembles the ligand-induced conformation (27). Therefore, the allosteric mechanism of TetR regulators is typically illustrated by comparing the DNA-bound and ligand-bound structures of TetR-family proteins. ...
The production of secondary metabolites is a major mechanism used by beneficial rhizobacteria to antagonize plant pathogens. These bacteria have evolved to coordinate the production of different secondary metabolites due to the heavy metabolic burden imposed by secondary metabolism. However, for most secondary metabolites produced by bacteria, it is not known how their biosynthesis is coordinated. Here, we showed that PhlH from the rhizobacterium Pseudomonas fluorescens is a TetR-family regulator coordinating the expression of enzymes related to the biosynthesis of several secondary metabolites, including 2,4-diacetylphloroglucinol (2,4-DAPG), mupirocin, and pyoverdine. We present structures of PhlH in both its apo form and 2,4-DAPG-bound form and elucidate its ligand-recognizing and allosteric switching mechanisms. Moreover, we found that dissociation of 2,4-DAPG from the ligand-binding domain (LBD) of PhlH was sufficient to allosterically trigger a pendulum-like movement of the DNA-binding domains (DBD) within the PhlH dimer, leading to a closed-to-open conformational transition. Finally, molecular dynamics simulations confirmed that two distinct conformational states were stabilized by specific hydrogen bonding interactions and that disruption of these hydrogen bonds had profound effects on the conformational transition. Our findings not only reveal a well-conserved route of allosteric signal transduction in TetR-family regulators, but also provide novel mechanistic insights into bacterial metabolic co-regulation.
... FasR regulates FAS-I by activation of the fas-acpS cluster, and MabR is an activator of the FAS-II gene cluster consisting of fabD-acpM-kasA-kasB, responsible for the biosynthesis of the longer mero-chain of mycolic acids (6,7). Both transcription factors (TFs) have been shown to coordinate target promoter binding in response to long-chain acyl-CoA binding in vitro and upon uptake of exogenous fatty acids (1,7,8). Mycolic acids are not limited to pathogenic mycobacterial species, rather they are ubiquitous among all mycobacteria, including nonpathogenic environmental mycobacteria and also other related genera of the suborder Corynebacterineae (1). ...
... The transcriptional activator of the FAS-II operon MabR similarly binds to long-chain acyl-CoAs between C 18 and C 24 to activate expression of the FAS-II operon members (fabD-acpM-kasA-kasB-accD6) (1,6). Additionally, FasR is the regulatory activator of the fasI-acpS operon and responds to long-chain acyl-CoAs >C 16 -CoA to inhibit DNA binding and transcriptional activation (7,8). This presents a regulatory system wherein FAS-I expression is down-regulated in response to its output (C 16 -to C 24 -CoA), and both FAS-II operon members and desA1/desA2 are up-regulated in response to their long-chain acyl-CoA substrates (C 18 -to C 24 -CoA and C 16 -to C 24 -CoA, respectively). ...
Significance
Our studies show that the mycolic acid desaturase regulator (MadR) acts as a molecular switch, controlling the desaturation and biosynthesis of mycolic acids, key lipids of the cell envelopes of mycobacteria. MadR works by a distinct mechanism wherein it binds various acyl-coenzyme As (aceyl-CoAs), but only saturated acyl-CoAs relieve DNA binding and repression. This suggests a unique mechanism that involves sensing of acyl-CoA pools as a checkpoint for coordinating mycolic acid remodeling and biosynthesis in response to cell surface perturbation. Our findings further our understanding of how mycobacteria control cell wall composition in response to stress across various environments ranging from soil to an intracellular niche in infected macrophages, with implications for understanding strategies for pathogenesis in the tubercle bacillus.
... Despite the loose association of MFT to cholesterol catabolism, we did not observe DNA release by MftR in the presence of cholesterol ( Figure 4A Figure 4C), and the number of binding sites (~0.6 sites per monomer MftR) were obtained from the nonlinear one-site model to the normalized fitting curve. The Kd value is comparable to known mycobacterial TFRs that are activated by oleoyl-CoA (39,40). To establish the specific acyl-CoA(s) that activate MftR, we measured the Kd's for myristoyl-, palmitoyl-, and steroyl-CoA's and found the values to be within the 2-4 M range ( Figure 4C). ...
... For the putative acyl binding pocket residues Phe65, Phe96, and Ile114, mutations to alanine led to no or modest change to the dissociation constant for oleoyl-CoA (Table 1). This is consistent with other TFR proteins where single residue changes to the acyl binding pocket resulted in little to no change in their dissociation constant to acyl-CoA's (39). However, we cannot rule out the possibility that Phe65, Phe96, and Ile114 do not participate in acyl-CoA binding. ...
Mycofactocin (MFT) is a ribosomally-synthesized and post-translationally-modified redox cofactor found in pathogenic mycobacteria. While MFT biosynthetic proteins have been extensively characterized, the physiological conditions under which MFT biosynthesis is required are not well understood. To gain insights into the mechanisms of regulation of MFT expression in Mycobacterium smegmatis mc 2155, we investigated the DNA-binding and ligand-binding activities of the putative TetR-like transcription regulator, MftR. In this study, we demonstrated that MftR binds to the mft promoter region. We used DNase I footprinting to identify the 27 bp palindromic operator located 5’ to mftA , and found it to be highly conserved in M. tuberculosis , M. bovis , M. ulcerans , and M. marinum . To determine under which conditions the mft biosynthetic gene cluster (BGC) is induced, we screened for effectors of MftR. As a result, we found that MftR binds to long-chain acyl-CoAs with low micromolar affinities. To demonstrate that oleoyl-CoA induces the mft BGC in vivo , we re-engineered a fluorescent protein reporter system to express a MftA-mCherry fusion protein. Using this mCherry fluorescent readout, we show that the mft BGC is upregulated in M. smegmatis mc 2155 when oleic acid is supplemented to the media. These results suggest that MftR controls expression of the mft BGC and that MFT production is induced by long chain acyl-CoAs. Since MFT-dependent dehydrogenases are known to colocalize with acyl-ACP/CoA modifying enzymes, these results suggest that MFT might be critical for fatty acid metabolism or cell wall reorganization.
... 6O6O is here schematically blurred to highlight regions of higher flexibility in TFRs, constituting a multi-state conformational ensemble. DNA selects the proper conformation and stabilizes the DNA-bound form (rightmost panel, PDB 6O6N), achieving the proper distance between DBD helices that insert into the major groove (Lara et al. 2020) regulators that control iron homeostasis in bacteria, as well as other metals (zinc, manganese, nickel, etc.). Fur, among several other OCSs, is an excellent illustration of this allosteric coupling mechanism. ...
... TFRs are homodimeric OCSs, most of which act as repressors, inducing the expression of target genes via ligand-bindingdependent DNA dissociation. Recent work with FasR, a TFR from Mycobacterium tuberculosis, further confirms how these OCSs' allostery relies on strong dynamic modulation, in the form of order/disorder transitions that link the signal sensing to the output DNA-binding sites (Lara et al. 2020). M. tuberculosis FasR is unusual in that it acts as a transcriptional activator of fas I and acpS genes encoding fatty acid biosynthesis enzymes, key actors in the lipid metabolism and cell wall homeostasis of this pathogen. ...
... The hydrophobic spine is interrupted precisely by the ligand-binding cavity, such that only in the ligand-bound condition is the spine completed by the ligand itself (Fig. 1c). The allosteric mechanism at play in TFRs appears to be based on a disordered (flexible, no ligand bound) to ordered transition, with the latter stabilized in a DNA-incompetent conformation, as observed in M. tuberculosis FasR (Lara et al. 2020) and TetR (Kamionka et al. 2004;Reichheld et al. 2009 Stimuli are usually environmental (yellow sphere), yet intracellular cues can also be perceived by intracytoplasmic sensor domains, where the allosteric site is located. Conformational rearrangements are coupled to the orthosteric site, in this case located at the HK catalytic reaction center. ...
Bacteria sense intracellular and environmental signals using an array of proteins as antennas. The information is transmitted from such sensory modules to other protein domains that act as output effectors. Sensor and effector can be part of the same polypeptide or instead be separate diffusible proteins that interact specifically. The output effector modules regulate physiologic responses, allowing the cells to adapt to the varying conditions. These biological machineries are known as signal transduction systems (STSs). Despite the captivating architectural diversity exhibited by STS proteins, a universal feature is their allosteric regulation: signal binding at one site modifies the activity at a physically distant site. Allostery requires protein plasticity, precisely encoded within their 3D structures, and implicating programmed molecular motions. This review summarizes how STS proteins connect stimuli to specific responses by exploiting allostery and protein plasticity. Illustrative examples spanning a wide variety of protein folds will focus on one- and two-component systems (TCSs). The former encompass the entire transmission route within a single polypeptide, whereas TCSs have evolved as separate diffusible proteins that interact specifically, sometimes including additional intermediary proteins in the pathway. Irrespective of their structural diversity, STS proteins are able to modulate their own molecular motions, which can be relatively slow, rigid-body movements, all the way to fast fluctuations in the form of macromolecular flexibility, thus spanning a continuous protein dynamics spectrum. In sum, STSs rely on allostery to steer information transmission, going from simple two-state switching to rich multi-state conformational order/disorder transitions.
... However, there were families that recognized fatty acids, fatty acid-CoA or other CoA derivatives. Representative examples are AlkX, which recognizes different long-chain fatty acids and regulates alkane biodegradation (Liang et al. 2016), and FasR, which binds a series of fatty acid-CoAs and regulates fatty acid biosynthesis (Lara et al. 2020). In addition, there is evidence for the specific recognition of additional ligands. ...
Bacteria have evolved many different signal transduction systems that sense signals and generate a variety of responses. Generally, most abundant are transcriptional regulators, sensor histidine kinases and chemoreceptors. Typically, these systems recognize their signal molecules with dedicated ligand-binding domains (LBDs), which, in turn, generate a molecular stimulus that modulates the activity of the output module. There are an enormous number of different LBDs that recognize a similarly diverse set of signals. To give a global perspective of the signals that interact with transcriptional regulators, sensor kinases and chemoreceptors, we manually retrieved information on the protein-ligand interaction from about 1,200 publications and 3D structures. The resulting 811 proteins were classified according to the Pfam family into 127 groups. These data permit a delineation of the signal profiles of individual LBD families as well as distinguishing between families that recognize signals in a promiscuous manner and those that possess a well-defined ligand range. A major bottleneck in the field is the fact that the signal input of many signaling systems is unknown. The signal repertoire reported here will help the scientific community design experimental strategies to identify the signaling molecules for uncharacterised sensor proteins.
... Along this line of thinking, we recently solved the crystal structures of M. tuberculosis FasR in complex with DNA and the acyl effector ligands, which provided insights into the molecular sensory and transmission mechanisms of this regulatory protein (Lara et al., 2020). This knowledge could lead now to perform structure-guided drug discovery strategies to identify antimycobacterial molecules with novel mechanisms of action. ...
Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is the world’s leading cause of death from an infectious disease. One of the main features of this pathogen is the complex and dynamic lipid composition of the cell envelope, which adapts to the variable host environment and defines the fate of infection by actively interacting with and modulating immune responses. However, while much has been learned about the enzymes of the numerous lipid pathways, little knowledge is available regarding the proteins and metabolic signals regulating lipid metabolism during M. tuberculosis infection. In this work, we constructed and characterized a FasR-deficient mutant in M. tuberculosis and demonstrated that FasR positively regulates fas and acpS expression. Lipidomic analysis of the wild type and mutant strains revealed complete rearrangement of most lipid components of the cell envelope, with phospholipids, mycolic acids, sulfolipids, and phthiocerol dimycocerosates relative abundance severely altered. As a consequence, replication of the mutant strain was impaired in macrophages leading to reduced virulence in a mouse model of infection. Moreover, we show that the fasR mutant resides in acidified cellular compartments, suggesting that the lipid perturbation caused by the mutation prevented M. tuberculosis inhibition of phagolysosome maturation. This study identified FasR as a novel factor involved in regulation of mycobacterial virulence and provides evidence for the essential role that modulation of lipid homeostasis plays in the outcome of M. tuberculosis infection.
