Amine Aladag

Transient structure and SH3 interaction sites in an intrinsically disordered fragment of the hepatitis C virus protein NS5A.

Understanding the molecular mechanisms involved in virus replication and particle assembly is of primary fundamental and biomedical importance. Intrinsic conformational disorder plays a prominent role in viral proteins and their interaction with other viral and host cell proteins via transiently populated structural elements. Here, we report on the results of an investigation of an intrinsically disordered 188-residue fragment of the hepatitis C virus non-structural protein 5A (NS5A), which contains a classical poly-proline Src homology 3 (SH3) binding motif, using sensitivity- and resolution-optimized multidimensional NMR methods, complemented by small-angle X-ray scattering data. Our study provides detailed atomic-resolution information on transient local and long-range structure, as well as fast time scale dynamics in this NS5A fragment. In addition, we could characterize two distinct interaction modes with the SH3 domain of Bin1 (bridging integrator protein 1), a pro-apoptotic tumor suppressor. Despite being largely disordered, the protein contains three regions that transiently adopt α-helical structures, partly stabilized by long-range tertiary interactions. Two of these transient α-helices form a noncanonical SH3-binding motif, which allows low-affinity SH3 binding. Our results contribute to a better understanding of the role of the NS5A protein during hepatitis C virus infection. The present work also highlights the power of NMR spectroscopy to characterize multiple binding events including short-lived transient interactions between globular and highly disordered proteins.

Interaction of nonstructural protein 5A of the hepatitis C virus with Src homology 3 domains using noncanonical binding sites.

Src homology 3 (SH3) domains are widely known for their ability to interact with other proteins using the canonical PxxP binding motif. Besides those well-characterized interaction modes, there is an increasing number of SH3 domain-containing complexes that lack this motif. Here we characterize the interaction of SH3 domains, in particular the Bin1-SH3 domain, with the intrinsically disordered part of nonstructural protein 5A of the hepatitis C virus using noncanonical binding sites in addition to its PxxP motif. These binding regions partially overlap with regions that have previously been identified as having an increased propensity to form α-helices. Remarkably, upon interaction with the Bin1-SH3 domain, the α-helical propensity decreases and a fuzzy complex is formed.

1H, 13C, and 15N resonance assignment of a 179 residue fragment of hepatitis C virus non-structural protein 5A

Non-structural protein 5A (NS5A) plays an important role in the life cycle of hepatitis C virus. This proline-rich phosphoprotein is organized into three domains. Besides its role in virus replication and virus assembly, NS5A is involved in a variety of cellular regulation processes. Recent studies on domain 2 and 3 revealed that both belong to the class of intrinsically disordered proteins as they adopt a natively unfolded state. In particular, domain 2 together with its vicinal regions is responsible for NS5A’s multiple interactions with other proteins necessary for virus persistence. The low chemical shift dispersion observed for instrinsically disordered proteins presents a challenge for NMR spectroscopy. Here we report sequential resonance assignment of a 179-residue fragment of NS5A, comprising the entire domain 2, using a set of sensitivity and resolution optimized 3D correlation experiments, as well as amino-acid-type editing in 1H-15N correlation spectra. Our assignment reveals the presence of several segments with high propensity to form α-helical structure that may be of importance to the function of this protein fragment as a versatile interaction platform.

Hepatitis C virus NS5A is able to competitively displace c-Myc from the Bin1 SH3 domain in vitro

We studied the interaction of the SH3 domain of Bin1 with a 15‐mer peptide of HCV NS5A and show its potency to competitively displace a 15‐mer human c‐Myc fragment, which is a physiological ligand of Bin1, using NMR spectroscopy. Fluorescence spectroscopy and ITC were employed to determine the affinity of Bin1 SH3 to NS5A(347–361), yielding a submicromolar affinity to NS5A. Our study compares the binding dynamics and affinities of the relevant regions for binding of c‐Myc and NS5A to Bin1 SH3. The result gives further insights into the potential role of NS5A in Bin1‐mediated apoptosis. Copyright

The non-structural protein 5A (NS5A) of hepatitis C virus interacts with the SH3 domain of human Bin1 using non-canonical binding sites

Background The hepatitis C virus (HCV) is a major human pathogen that causes severe diseases such as chronic hepatitis, liver cirrhosis and finally hepatocellular carcinoma. Although no enzymatic activity could be attributed yet to the HCV non-structural protein 5A (NS5A), it is indispensable for viral replication and particle assembly. Furthermore, it is associated with a variety of cellular pathways, although their relevance for viral pathogenesis still has to be elucidated. To fulfil its function NS5A interacts with a large number of different proteins including both viral and human ones. NS5A is organized into three domains, which are connected via two low complexity sequences (LCS). The first domain is highly conserved among different HCV genotypes and forms a well-defined globular structure [1]. The domains 2 (D2) and 3 (D3) are less conserved and intrinsically disordered. Nonetheless, three segments in LCS-I and D2 show significant propensities to adopt a-helical structures as could be shown by nuclear magnetic resonance (NMR) chemical shift and N relaxation data [2]. The LCS-II connecting D2 and D3 contains two directly neighbored class II PxxP-motifs, which are important for interactions with Src homology 3 (SH3) domains. SH3 domains mediate protein-protein interactions, often via binding to polyproline II helices. Recent studies also revealed alternative binding mechanisms, mainly involving helical motifs and positively charged amino acid residues. The SH3 domain of the bridging integrator 1 (Bin1) is known to interact with NS5A not only via its PxxP-motifs, but also via two non-canonical binding sites, which will be further described here [3].

Analysis of the Bin1 SH3 interaction with peptides derived from the hepatitis C virus protein NS5A and c-Myc reveals that NS5A can competitively displace c-Myc in vitro

Background Severe liver damage like cirrhosis and hepatocellular carcinoma (HCC) can be caused by manifestation of the hepatitis C virus (HCV) infection. Constitutively activated c-Myc oncogene has been shown to contribute to the establishment of HCV-mediated HCC. Interestingly, only one of many isoforms of the tumor suppressor protein Bin1 (bridging integrator 1), Bin1+12A, contains an internal, canonical SH3 binding motif that recognizes its own SH3 domain. This leads to the inability of Bin1+12A to interact with c-Myc. The expression of the Bin1+12A isoform is a main phenotype in malignant melanoma cells. We suggest that also other mechanisms that disturb the interaction of Bin1 and c-Myc might have severe consequences since the latter is tightly regulated in healthy cells. The HCV nonstructural protein 5A (NS5A) plays a key role in virus replication and assembly. NS5A plays an intercepting role in several cellular pathways, which are linked to cell growth, cell cycle control, cell survival, cellular stress response, apoptosis as well as HCC. It is known that NS5A contains a highly conserved canonical, polyproline (PxxP) SH3-binding motif, which is located between its D2 and D3 domains. This PxxP motif was described to interact with the SH3 domain of Bin1. In addition to a biophysical analysis of the canonical binding between Bin1 SH3 and the PxxP motif of NS5A [1], we identified two additional low-affinity binding sites for noncanonical SH3 binding on NS5A [2]. The hypothesis underlying the work presented here is that viral NS5A is able to sequester cellular Bin1 from c-Myc.