title: VAPC, an human endogenous inhibitor for hepatitis C virus (HCV) infection, is intrinsically unstructured but forms a "fuzzy complex" with HCV NS5B.
abstract: Nearly 200 million people are infected by hepatitis C virus (HCV) worldwide. For replicating the HCV genome, the membrane-associated machinery needs to be formed by both HCV non-structural proteins (including NS5B) and human host factors such as VAPB. Recently, the 99-residue VAPC, a splicing variant of VAPB, was demonstrated to inhibit HCV replication via binding to NS5B, thus acting as an endogenous inhibitor of HCV infection. So far, the structure of VAPC remains unknown, and its interaction with NS5B has not been biophysically characterized. In this study, we conducted extensive CD and NMR investigations on VAPC which led to several striking findings: 1) although the N-terminal 70 residues are identical in VAPC and VAPB, they constitute the characteristic β-barrel MSP fold in VAPB, while VAPC is entirely unstructured in solution, only with helical-like conformations weakly populated. 2) VAPC is indeed capable of binding to NS5B, with an average dissociation constant (Kd) of ∼20 µM. Intriguingly, VAPC remains dynamic even in the complex, suggesting that the VAPC-NS5B is a "fuzzy complex". 3) NMR mapping revealed that the major binding region for NS5B is located over the C-terminal half of VAPC, which is composed of three discrete clusters, of which only the first contains the region identical in VAPC and VAPB. The second region containing ∼12 residues appears to play a key role in binding since mutation of 4 residues within this region leads to almost complete loss of the binding activity. 4) A 14-residue mimetic, VAPC-14 containing the second region, only has a ∼3-fold reduction of the affinity. Our study not only provides critical insights into how a human factor mediates the formation of the HCV replication machinery, but also leads to design of VAPC-14 which may be further used to explore the function of VAPC and to develop anti-HCV molecules.
authors: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore.
authors: Shaveta
authors: S
authors: Goyal
authors: Garvita
authors: G
authors: Gupta
authors: Haina
authors: H
authors: Qin
authors: Megha Haridas
authors: MH
authors: Upadya
authors: Yee Joo
authors: YJ
authors: Tan
authors: Vincent T K
authors: VT
authors: Chow
authors: Jianxing
authors: J
authors: Song
mesh_terms: D000595:Amino Acid Sequence; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D010446:Peptide Fragments; D033921:Vesicular Transport Proteins; D017361:Viral Nonstructural Proteins; C496748:VAPB protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase