Figure 5 - uploaded by Vu To
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Overlay of extracted region of two SOLEXSY frequency-labeled data points, NH (black) and ND (gray), collected at a mixing time (t mix ) of 1000 ms. Peaks of the same residue are resolved by the deuterium shift of 0.7 ppm.
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The type 1 Human Immunodeficiency Virus (HIV-1) Transactivator of transcription (Tat) is a small RNA-binding protein essential for viral gene expression and replication. It has also been shown to bind to a large number of human proteins and to modulate many different cellular activities. We have used NMR spectroscopy and hydrogen exchange chemistry...
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Context 1
... The results indicate that a majority of the resonances in Tat are within the random coil range and there are no regions with more than three consecutive resonances in the α-helix or β-sheet chemical shift ranges. The 3 J HNHα coupling constants (see Figure S5 of the Supporting Information) also fall in the random coil range of 5.9−7.7 Hz. 75 These results are in agreement with a prediction done on the CSI 3.0 server 76 indicating a random coil structure and fully flexible backbone for HIV-1 full-length Tat. Thus, the second exon product (73− 101) is intrinsically disordered, and its addition to the protein does not appear to induce any folding in the first exon product (1−72). ...
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... Exchange rates and protection factors for full-length Tat were extracted from CLEANEX 68,97 and SOLEXSY 50 experiments that probe exchange on different time scales. Figure 5 shows sample spectra at one mixing time of the axial peaks and cross-peaks in a SOLEXSY experiment of Tat dissolved in 50% D 2 O. Figure 6 compares hydrogen exchange rates and protection factors measured by CLEANEX (empty bars) and SOLEXSY (filled bars) that in general show excellent agreement. The rates of hydrogen exchange are much faster in the His tag region (residues 1−20) compared to the rest of the protein (Figure 6), giving rise to the lowest protection factors in the protein. ...
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The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is...
Citations
... 11 Nuclear magnetic resonance opened the veil on the structural propensity of Tat protein suggesting that the cysteine-rich region tends to fold into α-helixes in contrast to the basic domain with extended or β-sheet conformation. 18 Comparison analysis of X-ray and nuclear magnetic resonance studies suggests that different fragments of Tat protein can employ different folding mechanisms. 18 This flexibility enables Tat to adopt diverse conformations upon interaction with its physiological partners, thus greatly extending its multifunctionality. ...
... 18 Comparison analysis of X-ray and nuclear magnetic resonance studies suggests that different fragments of Tat protein can employ different folding mechanisms. 18 This flexibility enables Tat to adopt diverse conformations upon interaction with its physiological partners, thus greatly extending its multifunctionality. ...
Tat (transactivator of transcription) regulates transcription from the HIV provirus. It plays a crucial role in disease progression, supporting efficient replication of the viral genome. Tat also modulates many functions in the host genome via its interaction with chromatin and proteins. Many of the functions of Tat are associated with its basic domain rich in arginine and lysine residues. It is still unknown why the basic domain exhibits so many diverse functions. However, the highly charged basic domain, coupled with the overall structural flexibility of Tat protein itself, makes the basic domain a key player in binding to or associating with cellular and viral components. In addition, the basic domain undergoes diverse posttranslational modifications, which further expand and modulate its functions. Here, we review the current knowledge of Tat basic domain and its versatile role in the interaction between the virus and the host cell.
... Alpha-synuclein (Eliezer et al., 2001) and tau (Bibow et al., 2011;Künze et al., 2012), implicated in PD (Parkinson's diseases) and AD (Alzheimer's disease) respectively, are also IDPs. Furthermore, several viral strains including the well-known AIDS-causing HIV-1 produce IDPs (Chi et al., 2007;Feuerstein et al., 2012;Kim et al., 2009b;Liang et al., 2007;Reingewertz et al., 2009;To et al., 2016). Clearly, there is an immediate and strong need to acquire very thorough knowledge not only on the normal functionality of IDPs but also on their pathologic connection to above diseases since it has become apparent that the classical globular protein based approach is unlikely to provide us with sufficient information that can be used for developing effective weaponry against IDP-associated diseases. ...
... The most obvious characteristic of IDPs is that they do not possess spatially-disposed active pockets, a fact that brings us to a simple but profound question of how then these long malleable stretches of amino acids (sometimes hundreds of amino acids) can bind their targets. Targets of IDPs are not just proteins, but can be nucleic acids (Thapar et al., 2004;To et al., 2016;Wells et al., 2008), lipids, metals, and small molecules (Follis et al., 2008;Metallo, 2010). Efforts were made recently to classify IDPs into several subfamilies (van der Lee et al., 2014). ...
Intrinsically disordered proteins (IDPs) are highly unorthodox proteins that do not form three-dimensional structures under physiological conditions. The discovery of IDPs has destroyed the classical structure-function paradigm in protein science, 3-D structure = function, because IDPs even without well-folded 3-D structures are still capable of performing important biological functions and furthermore are associated with fatal diseases such as cancers, neurodegenerative diseases and viral pandemics. Pre-structured motifs (PreSMos) refer to transient local secondary structural elements present in the target-unbound state of IDPs. During the last two decades PreSMos have been steadily acknowledged as the critical determinants for target binding in dozens of IDPs. To date, the PreSMo concept provides the most convincing structural rationale explaining the IDP-target binding behavior at an atomic resolution. Here we present a brief developmental history of PreSMos and describe their common characteristics. We also provide a list of newly discovered PreSMos along with their functional relevance.
... As has become increasingly common in the foldedprotein community, utilization of the heteronuclear NOE as a reporter of backbone flexibility, without downstream modelling, has gained wide acceptance [37,47]. In addition, while spectral density mapping [48,49] has never overtaken Lipari-Szabo analysis in the cooperatively folded protein community, it has emerged as a rigorous means to connect spin relaxation and conformational dynamics in IDPs [50,51]. Collection of massive spin-relaxation data sets spanning magnetic field strengths from 9.4 to 23.5 T enabled direct spectral density mapping that demonstrated a wide range of dynamic timescales sampled by the disordered Engrailed transcription factor [52]. ...
The prevalence of intrinsically disordered protein regions, particularly in eukaryotic proteins, and their clear functional advantages for signaling and gene regulation have created an imperative for high-resolution structural and mechanistic studies. NMR spectroscopy has played a central role in enhancing not only our understanding of the intrinsically disordered native state, but also how that state contributes to biological function. While pathological functions associated with protein aggregation are well established, it has recently become clear that disordered regions also mediate functionally advantageous assembly into high-order structures that promote the formation of membrane-less sub-cellular compartments and even hydrogels. Across the range of functional assembly states accessed by disordered regions, post-translational modifications and regulatory macromolecular interactions, which can also be investigated by NMR spectroscopy, feature prominently. Here we will explore the many ways in which NMR has advanced our understanding of the physical-chemical phase space occupied by disordered protein regions and provide prospectus for the future role of NMR in this emerging and exciting field.
Viruses infect all kingdoms of life; their genomes vary from DNA to RNA and in size from 2kB to 1 MB or more. Viruses frequently employ disordered proteins, that is, protein products of virus genes that do not themselves fold into independent three-dimensional structures, but rather, constitute a versatile molecular toolkit to accomplish a range of functions necessary for viral infection, assembly, and proliferation. Interestingly, disordered proteins have been discovered in almost all viruses so far studied, whether the viral genome consists of DNA or RNA, and whatever the configuration of the viral capsid or other outer covering. In this review, I present a wide-ranging set of stories illustrating the range of functions of IDPs in viruses. The field is rapidly expanding, and I have not tried to include everything. What is included is meant to be a survey of the variety of tasks that viruses accomplish using disordered proteins.
Intrinsically disordered proteins and regions (IDPs/IDRs) make up a large part of viral proteomes, but their real prevalence across the global plant virome is still murky, partly because of their massive diversity. Here, we propose an evolutionary quantitative proteomic approach to foray into genomic signatures that are preserved in the amino acid sequences of orthologous IDRs. Markedly, we found that relatively abundant IDP varies substantially in viral species among and within plant virus families, including according to genome size, partition or replication strategies. We also demonstrate that most encoded proteomic modules of the plant virome contain multiple disordered features that are phylogenomically preserved, and can be correlated to genomic, bio-physical and evolutionary strategies. Furthermore, our focused interactome-wide analysis highlights lines of evidence indicating that various IDPs with similar evolutionary signatures modulate viral multifunctionality. Moreover, estimated fractions of IDR in the vicinity of pivotal evolutionary structural domains embedded in interaction modules are strongly enriched with affinity binding functional annotations and relate to vector-borne virus transmission modes. Importantly, molecular recognition features (MoRFs) are abundantly widespread in IDRs of viral hallmark modules and their binding partners. Finally, we propose a coarse-grained conceptual framework in which evolutionary proteome-wide IDP/IDRs patterns can be, rather, reliably exploited to elucidate their foundational fine-tuning role in plant virus transmission mechanisms. While opening unexplored avenues for consistently predicting virus–host functions for many new or uncharacterized viruses based on their proteomic repertoire, other considerations advocating further structural IDP research in Plant Virology are thoroughly discussed in light of viral modular evolution.
Pre-Structured Motifs (PreSMos) are transient secondary structures observed in many intrinsically disordered proteins (IDPs) and serve as protein target-binding hot spots. The prefix “pre” highlights that PreSMos exist a priori in the target-unbound state of IDPs as the active pockets of globular proteins pre-exist before target binding. Therefore, a PreSMo is an “active site” of an IDP; it is not a spatial pocket, but rather a secondary structural motif. The classical and perhaps the most effective approach to understand the function of a protein has been to determine and investigate its structure. Ironically or by definition IDPs do not possess structure (here structure refers to tertiary structure only). Are IDPs then entirely structureless? The PreSMos provide us with an atomic-resolution answer to this question. For target binding, IDPs do not rely on the spatial pockets afforded by tertiary or higher structures. Instead, they utilize the PreSMos possessing particular conformations that highly presage the target-bound conformations. PreSMos are recognized or captured by targets via conformational selection (CS) before their conformations eventually become stabilized via structural induction into more ordered bound structures. Using PreSMos, a number of, if not all, IDPs can bind targets following a sequential pathway of CS followed by an induced fit (IF). This chapter presents several important PreSMos implicated in cancers, neurodegenerative diseases, and other diseases along with discussions on their conformational details that mediate target binding, a structural rationale for unstructured proteins.
Human immunodeficiency virus type-1 (HIV-1) transactivator of transcription (Tat) is an intrinsically disordered protein that exerts multiple functions, including activation of HIV-1 replication and induction of T-cell apoptosis and cytokine secretion via zinc binding and cellular uptake by endocytosis. However, the effects of zinc and endosomal low pH on the structure of isolated Tat protein are poorly understood. Here, we purified a monomeric zinc-bound Tat and studied its structure and acid denaturation by circular dichroism, NMR, and small-angle X-ray scattering. We found that at pH 7, the zinc-bound Tat was in a pre-molten globule state; it exhibited largely disordered conformations with residual helices and was slightly more compact than the fully unfolded states that were observed at pH 4 or in the zinc-free form. Moreover, acid-induced unfolding transitions in secondary structure and molecular size occurred at different pH ranges, indicating the presence of an expanded and helical intermediate at pH ∼6. Taken together, the extent of structural disorder in the intrinsically disordered Tat protein is highly sensitive to zinc and pH, suggesting that zinc binding and pH affect Tat structures and thereby control the versatile functions of Tat.
The pancreatic and duodenal homeobox 1 (Pdx1) is an essential pancreatic transcription factor. The C-terminal intrinsically disordered domain of Pdx1 (Pdx1-C) has a heavily biased amino acid composition; most notably, 18 of 83 residues are proline, including a hexa-proline cluster near the middle of the chain. For these reasons, Pdx1-C is an attractive target for structure characterization, given the availability of suitable methods. To determine the solution ensembles of disordered proteins, we have developed a suite of 13C direct-detect NMR experiments that provide high spectral quality, even in the presence of strong proline enrichment. Here, we have extended our suite of NMR experiments to include four new pulse programs designed to record backbone residual dipolar couplings (RDCs) in a 13C,15N-CON detection format. Using our NMR strategy, in combination with small angle x-ray scattering (SAXS) measurements and Monte Carlo simulations, we have determined that Pdx1-C is extended in solution, with a radius of gyration and internal scaling similar to that of an excluded volume polymer, and a subtle tendency toward a collapsed structure to the N-terminal side of the hexa-proline sequence. This structure leaves Pdx1-C exposed for interaction with trans-regulatory co-factors that contribute with Pdx1 to transcription control in the cell.
