A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Dujardin, Marie; Madan, Vanesa; Montserret, Roland; Ahuja, Puneet; Huvent, Isabelle; Launay, Hélène; Leroy, Arnaud; Bartenschlager, Ralf; Pénin, François; Lippens, Guy; Hanoulle, Xavier

doi:10.1074/jbc.m115.644419

Cited by 23 publications

(42 citation statements)

References 72 publications

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“…CypA binding (64). Moreover, we have shown that the CypAbinding region in NS5A-D2 (residues 306 -323) is also involved in the direct interaction with NS5B (65).…”

Section: Allosteric Regulation Of the Hcv Polymerasementioning

confidence: 80%

NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B

Bessa¹,

Launay²,

Dujardin³

et al. 2017

Journal of Biological Chemistry

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Non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase that catalyzes replication of the hepatitis C virus (HCV) RNA genome and therefore is central for its life cycle. NS5B interacts with the intrinsically disordered domain 2 of NS5A (NS5A-D2), another essential multifunctional HCV protein that is required for RNA replication. As a result, these two proteins represent important targets for anti-HCV chemotherapies. Despite this importance and the existence of NS5B crystal structures, our understanding of the conformational and dynamic behavior of NS5B in solution and its relationship with NS5A-D2 remains incomplete. To address these points, we report the first detailed NMR spectroscopic study of HCV NS5B lacking its membrane anchor (NS5B). Analysis of constructs with selective isotope labeling of the δ1 methyl groups of isoleucine side chains demonstrates that, in solution, NS5B is highly dynamic but predominantly adopts a closed conformation. The addition of NS5A-D2 leads to spectral changes indicative of binding to both allosteric thumb sites I and II of NS5B and induces long-range perturbations that affect the RNA-binding properties of the polymerase. We compared these modifications with the short- and long-range effects triggered in NS5B upon binding of filibuvir, an allosteric inhibitor. We demonstrate that filibuvir-bound NS5B is strongly impaired in the binding of both NS5A-D2 and RNA. NS5A-D2 induces conformational and functional perturbations in NS5B similar to those triggered by filibuvir. Thus, our work highlights NS5A-D2 as an allosteric regulator of the HCV polymerase and provides new insight into the dynamics of NS5B in solution.

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“…CypA binding (64). Moreover, we have shown that the CypAbinding region in NS5A-D2 (residues 306 -323) is also involved in the direct interaction with NS5B (65).…”

Section: Allosteric Regulation Of the Hcv Polymerasementioning

confidence: 80%

NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B

Bessa¹,

Launay²,

Dujardin³

et al. 2017

Journal of Biological Chemistry

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“…Proline is a special residue because it is rigid and pre-bent, thus suites very well for the β-turn format. Furthermore, two or three Pro residues present in an nascent amino acid sequence could act as a specific switch or the hinge [61,62] and thus, might regulate the structure and/or activity of the protein [63][64][65]. Verweij et al [55] hypothesized that because the P27 residue was conserved among all the functional UL49.5 inhibitors, it could be responsible for the TAP inhibition.…”

Section: Structure and Dynamics Of Ul495 In Popc Membranementioning

confidence: 99%

Structure determination of UL49.5 transmembrane protein from bovine herpesvirus 1 by NMR spectroscopy and molecular dynamics

Karska

Graul

Sikorska

et al. 2019

Biochimica et Biophysica Acta (BBA) - Biomembranes

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A B S T R A C TThe transporter associated with antigen processing (TAP) directly participates in the immune response as a key component of the cytosolic peptide to major histocompatibility complex (MHC) class I protein loading machinery. This makes TAP an important target for viruses avoiding recognition by CD8+ T lymphocytes. Its activity can be suppressed by the UL49.5 protein produced by bovine herpesvirus 1, although the mechanism of this inhibition has not been understood so far.Therefore, the main goal of our study was to investigate the 3D structure of bovine herpesvirus 1 -encoded UL49.5 protein. The final structure of the inhibitor was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane mimetic environments. In NMR studies, UL49.5 was represented by two fragments: the extracellular region (residues 1-35) and the transmembraneintracellular fragment (residues 36-75), displaying various functions during viral invasion. After the empirical structure determination, a molecular docking procedure was used to predict the complex of UL49.5 with the TAP heterodimer.Our results revealed that UL49.5 adopted a highly flexible membrane-proximal helical structure in the extracellular part. In the transmembrane region, we observed two short α-helices. Furthermore, the cytoplasmic part had an unordered structure. Finally, we propose three different orientations of UL49.5 in the complex with TAP. Our studies provide, for the first time, the experimental structural information on UL49.5 and structurebased insight in its mechanism of action which might be helpful in designing new drugs against viral infections.

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“…For example, stabilizing the transient helix with which the transactivation domain (TAD) of the tumor suppressor p53 binds to the repressor oncoprotein MDM2 leads to stronger binding and concomitant effects on target gene expression and cell cycle regulation (Zondlo et al, 2006 ; Li et al, 2010 ; Borcherds et al, 2014 ). In the disordered domain D2 of hepatitis C virus (HCV) protein NS5A, a short structural motif dubbed the PW turn was identified whose disruption abolished HCV replication and affected interaction with the host protein cyclophilin A (Dujardin et al, 2015 ). These and other examples underline the importance of pre-formed structure in IDP binding and indicate that it typically has a functional role.…”

Section: Possible Advantages Of Intrinsic Disordermentioning

confidence: 99%

Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment

Mollica

Bessa

Hanoulle

et al. 2016

Front. Mol. Biosci.

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125

128

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In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs). In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an associated function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiological partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed, e.g., in the “fly-casting” hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit), are subjects of intense debate. Experimentally, these questions remain difficult to address. Here, we review experimental studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theoretical concepts and numerical methods that can be applied to describe these interactions at the atomic level. The available literature suggests that the kinetic and thermodynamic parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes observed experimentally. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context.

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A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Cited by 23 publications

References 72 publications

NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B

NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B

Structure determination of UL49.5 transmembrane protein from bovine herpesvirus 1 by NMR spectroscopy and molecular dynamics

Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment

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