FC0064
Nonstructural protein 5A (NS5A)  -  Src homology 3 domain

Biological function
Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) is involved in a variety of viral and cellular processes. NS5A is indispensable for viral replication and particle assembly.

Domain organization/sequence features
The domain structure of NS5A comprises an N-terminal region contains an amphipathic helix, anchoring NS5A to the membranes of the endoplasmic reticulum, followed by a well-folded zinc-binding domain (D1). The C-terminal part of NS5A, comprising domains D2 and D3, is intrinsically disordered. The three domains are linked by low-complexity sequences LCS-1 and LCS-2. LCS-2 contains a proline-rich region comprising two class II PxxP motifs (PP2.1 and PP2.2), out of which PP2.2 motif is highly conserved among different HCV genotypes and can interact with a variety of SH3 domains of the Src kinase family, including Fyn, Lyn, Lck, and Hck, as well as the SH3 domains of the adaptor proteins Grb2 and Bin1. In addition to thecanonical PxxP binding mode, SH3 domains also interact with protein partners via other, noncanonical binding modes. An increasing number of complexes between SH3 domains and peptides that lack the canonical PxxP motif.

Structural evidence
NMR results reveal that in addition to the canonical SH3 binding to the PxxP motif, NS5A has two additional low-affinity binding sites for non-canonical SH3 binding, mainly through electrostatic interactions. Besides the interaction with SH3 domains mediated by a canonical PxxP motif located in the LCS-2 region of NS5A (binding site B3), two additional peptide regions (B1 and B2) were identified in NS5A that interact with the four SH3 domains mentioned above. Each SH3 domain can bind to only a single NS5A interaction site, and that the different binding modes are mutually exclusive as they all compete for the same binding pocket on the SH3 domain. Two low-affinity binding regions B1 and B2 partly overlap with peptide segments that were previously identified as having increased α-helical propensities. The population of α-helical structure decreases upon binding: the SSP scores in segment B1 are reduced to almost zero, while for B2, they are significantly reduced. The hetNOE values of NS5A residues in the binding regions decrease upon interaction with SH3, in agreement with a more flexible structural ensemble.

Biochemical evidence
The low-affinity NS5A binding sites were studied using a shorter NS5A construct, NS5A(191−340), that lacks the high-affinity SH3 binding site (PxxP motif) and a long construct NS5A(191-369). A global fit of the titration curves assuming a kinetic model with two independent binding sites, B1 and B2, yields a K¹_d of 100 ± 50μM for the B1 region and a K²_d of 240 ± 50 μM for the B2 region. The B1 site has a 2−3-fold higher binding affinity for the Bin1- SH3 domain than the B2 site. Low- and high-affinity NS5A binding affects the same binding pocket on the surface of the SH3 domain.

Structure/Mechanism
Albeit transiently formed α-helices are not the recognition motifs for SH3 binding they might serve as preformed structural elements for interactions with other proteins.

Mechanism category
Tethering

Significance
The fuzzy nature of the complex adds an entropic contribution to the binding free energy that otherwise seems to be governed by electrostatic interactions between the positively charged residues of NS5A in the binding region and the negatively charged interaction surface of Bin1-SH3. The additional SH3 binding modes may be important for the regulation of molecular interaction and phosphorylation events that NS5A undergoes during the viral life cycle.