Dataset Information

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation.

ABSTRACT: Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein's interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (?250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191-447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (?-helical and ?-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.

SUBMITTER: Solyom Z

PROVIDER: S-EPMC4601004 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation.

Sólyom Zsófia Z Ma Peixiang P Schwarten Melanie M Bosco Michaël M Polidori Ange A Durand Grégory G Willbold Dieter D Brutscher Bernhard B

Biophysical journal 20151001 7

Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the ...[more]

PMID: 26445449

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Project description:BackgroundCurrent understanding of the molecular basis of memory consolidation points to an important function of amyloid formation by neuronal-specific isoforms of the cytoplasmic polyadenylation element binding (CPEB) protein family. In particular, CPEB is thought to promote memory persistence through formation of self-sustaining prion-like amyloid assemblies at synapses, mediated by its intrinsically disordered region (IDR) and leading to permanent physical alterations at the basis of memory persistence. Although the molecular mechanisms by which amyloid formation takes place in CPEB have been described in invertebrates, the way amyloid formation occurs in the human homolog CPEB3 (hCPEB3) remains unclear. Here, we characterize by NMR spectroscopy the atomic level conformation and ps-ms dynamics of the 426-residue IDR of hCPEB3, which has been associated with episodic memory in humans.ResultsWe show that the 426-residue N-terminal region of hCPEB3 is a dynamic, intrinsically disordered region (IDR) which lacks stable folded structures. The first 29 residues, M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical + disordered motif, and residues 86-93: P83QQPPPP93, and 166-175: P166PPPAPAPQP175 form polyproline II (PPII) helices. The (VG)5 repeat motif is completely disordered, and residues 200-250 adopt three partially populated α-helices. Residues 345-355, which comprise the nuclear localization signal (NLS), form a modestly populated α-helix which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transitions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids.ConclusionOur NMR spectroscopic analysis of hCPEB3 provides insights into the first structural transitions involved in protein-protein and protein-mRNA interactions. The atomic level understanding of these structural transitions involved in hCPEB3 aggregation is a key first step toward understanding memory persistence in humans, as well as sequence features that differentiate beneficial amyloids from pathological ones.AreasBiophysics, Structural Biology, Biochemistry & Neurosciences.

Project description:Replication of hepatitis C virus (HCV) is dependent on virus-encoded proteins and numerous cellular factors. DDX3 is a well-known host cofactor of HCV replication. In this study, we investigated the role of a DDX3-interacting protein, Y-box binding protein 1 (YB-1), in the HCV life cycle. Both YB-1 and DDX3 interacted with the viral nonstructural protein NS5A. During HCV infection, YB-1 partially colocalized with NS5A and the HCV replication intermediate double-stranded RNA (dsRNA) in HCV-infected Huh-7.5.1 cells. Despite sharing the same interacting partners, YB-1 participated in HCV RNA replication but was dispensable in steady-state HCV RNA replication, different from the action of DDX3. Moreover, knockdown of YB-1 in HCV-infected cells prevented infectious virus production and reduced the ratio of hyperphosphorylated (p58) to hypophosphorylated (p56) forms of NS5A, whereas DDX3 silencing did not affect the ratio of the p58 and p56 phosphoforms of NS5A. Interestingly, silencing of YB-1 severely reduced NS5A protein stability in NS5A-ectopically expressing, replicon-containing, and HCV-infected cells. Furthermore, mutations of serine 102 of YB-1 affected both YB-1-NS5A interaction and NS5A-stabilizing activity of YB-1, indicating that this Akt phosphorylation site of YB-1 plays an important role in stabilizing NS5A. Collectively, our results support a model in which the event of YB-1 phosphorylation-mediated interaction with NS5A results in stabilizing NS5A to sustain HCV RNA replication and infectious HCV production. Overall, our study may reveal a new aspect for the development of novel anti-HCV drugs.Chronic hepatitis C virus (HCV) infection induces liver cirrhosis and hepatocellular carcinoma. The viral nonstructural protein NS5A co-opting various cellular signaling pathways and cofactors to support viral genome replication and virion assembly is a new strategy for anti-HCV drug development. NS5A phosphorylation is believed to modulate switches between different stages of the HCV life cycle. In this study, we identified the cellular protein YB-1 as a novel NS5A-interacting protein. YB-1 is a multifunctional protein participating in oncogenesis and is an oncomarker of hepatocellular carcinoma (HCC). We found that YB-1 protects NS5A from degradation and likely regulates NS5A phosphorylation through its phosphorylation-dependent interaction with NS5A, which might be controlled by HCV-induced signaling pathways. Our observations suggest a model in which HCV modulates NS5A level and the ratio of the p58 and p56 phosphoforms for efficient viral propagation via regulation of cellular signaling inducing YB-1 phosphorylation. Our finding may provide new aspects for developing novel anti-HCV drugs.

Dataset Information

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation.

Publications

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation.

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