Abstract
Implicated in numerous human diseases, intrinsically disordered proteins (IDPs) are dynamic ensembles of interconverting conformers that often contain many proline residues. Whether and how proline conformation regulates the functional aspects of IDPs remains an open question, however. Here, we studied the disordered domain 2 of nonstructural protein 5A (NS5A-D2) of hepatitis C virus (HCV). NS5A-D2 comprises a short structural motif (PW-turn) embedded in a proline-rich sequence, whose interaction with the human prolyl isomerase cyclophilin A (CypA) is essential for viral RNA replication. Using NMR, we show here that the PW-turn motif exists in a conformational equilibrium between folded and disordered states. We found that the fraction of conformers in the NS5A-D2 ensemble that adopt the structured motif is allosterically modulated both by the cis/trans isomerization of the surrounding prolines that are CypA substrates and by substitutions conferring resistance to cyclophilin inhibitor. Moreover, we noted that this fraction is directly correlated with HCV RNA replication efficiency. We conclude that CypA can fine-tune the dynamic ensemble of the disordered NS5A-D2, thereby regulating viral RNA replication efficiency.
Highlights
Implicated in numerous human diseases, intrinsically disordered proteins (IDPs) are dynamic ensembles of interconverting conformers that often contain many proline residues
The local r.m.s. deviation values suggest that beyond the core of the PW-turn (i.e. 310PAWA313), where the ring of Pro310 and the aromatic side chain of tryptophan 312 (Trp312) are engaged in a CH/ interaction [61], the residues at its C terminus adopt a certain degree of order (Fig. 2b)
RNA replication, a central step in the hepatitis C virus (HCV) life cycle, requires the formation of a replication complex that includes the viral NS5B and NS5A proteins associated to membrane structures (DMVs) [7, 74]
Summary
The 1H,15N-HSQC NMR spectrum of NS5A-D2 of the HCV JFH1 strain (genotype 2a) (Fig. 1b) displays a narrow 1H chemical shift dispersion that confirms the high level of intrinsic disorder in this domain (Fig. S1a) [18]. The secondary structure propensity analysis, based on the 13C chemical shifts, indicates the presence of several residual ␣-helices and extended regions in the N-terminal half (Fig. 1c) This is similar to the observations made on NS5A-D2 from HCV strains HC-J4 and Con (both genotype 1b) [30, 59]. To investigate the functional role of the PW-turn motif in the JFH1 strain, we introduced the analogous mutation, A311G, in NS5A-D2 We found that this mutation efficiently disrupts the PW-turn in NS5A-D2 because in the different NMR spectra acquired on this NS5A-D2 mutant, the proton amide and 13C␣ resonances of Trp312 and Ala313 move toward their expected frequencies for a fully disordered region
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