FC0068
Vesicle associated membrane protein C (VAPC)  -  Hepatitis C virus nonstructural protein NS5B

Biological function
The formation of the HCV replication machinery has been shown to require interactions of viral nonstructural NS5A/NS5B to host proteins including human vesicle-associated membrane protein-associated protein (VAP) subtypes A and B. NS5A is a critical component of HCV replication, and is additionally involved in the modulation of cell signaling pathways, interferon response, pathogenesis, and apoptosis regulation. Although NS5B functions as an RNA-dependent RNA polymerase, it appears also to engage in protein-protein interactions critical for forming the replication machinery. VAPC, a splicing variant of VAPA/VAPB, was recently identified to block the HCV RNA replication as well as HCV propagation.

Structural evidence
VAPC was characterized to be highly disordered in solution, only with helical-like conformations weakly populated over several segments. The HSQC spectral dispersions of VAPC did not significantly increase even upon binding to NS5B, implying that VAPC remained largely flexible even in the complex. Even at a molar ratio of 1:2.5, the HSQC spectral dispersions still remained largely unchanged. This implies that like the full-length VAPC, VAPC-43 also remains largely flexible without any tight tertiary packing in the complex with NS5B. The chemical shift differences indicate that, three discrete regions appears to be critical for binding with NS5B, i.e. the first one centered at Ile62, second over Arg73- Ala82, and third centered at Glu95.

Biochemical evidence
VAPC was indeed capable of binding to NS5B with three discrete regions, with an average dissociation constant (K_D) of 20 mM. Site directed mutagenesis of four residues in second region demonstrates that they are critical for binding with NS5B. Kd value of VAPC-14 is 49.13 mM, only 3-fold larger than that for VAPC-43 (18.29 mM), suggesting that deletion of the first and third binding regions only resulted in a 3-fold reduction of the binding affinity to NS5B.

Mechanism category
tethering

Significance
The fuzziness of the VAPC-NS5B complex results in discrete multi-binding site scenario for which the binding affinity of each separate site is relatively low, and also many residues are not significantly engaged in binding to NS5B and consequently remain flexible.