Allosteric Inhibitors Have Distinct Effects, but Also Common Modes of Action, in the HCV Polymerase

Abstract

Recently discovered information alters the interpretation of our paper published previously in Biophysical Journal. In the study, we used molecular dynamics (MD) simulations to understand the mechanisms that govern allosteric inhibition of the RNA polymerase from hepatitis C virus (HCV). Our findings revealed that allosteric inhibitors alter the conformations adopted by the enzyme in a manner that allows their inhibitory effects to be readily understood. Their primary modes of action were found to be: 1) restricting enzyme fluctuations to nonfunctional regions of conformational space, 2) destabilizing the enzyme to the extent that it is unable to stably adopt functional conformations, and 3) blocking RNA template access to the enzyme's active site.

NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B

“…These conformations are mainly characterized by the extent of opening of the polymerase, following rotation of the thumb subdomain with respect to palm and fingers. For example, 1) a crystal structure of NS5B has revealed the presence of two conformations (30), one being more open than the other; 2) NS5B has been shown to be able to bind a circular RNA template (92), which implies opening of its encircled closed active site; and 3) molecular simulations have suggested that NS5B may sample a conformational space in which the intersubdomain angle and the RNA channel width can vary significantly (36). Recently, Deredge et al have shown using hydrogen/deuterium exchange followed by MS that fingers, palm, and, to a lesser extent, the core of the thumb are highly exchange-protected regions of apo-NS5B (42).…”

“…7f; see supplemental Fig. S7), which has been shown to reduce functional dynamics in NS5B (36,41,42).…”

“…In the NS5B L30S mutant, which is largely inactive (93), the fingertips loop ⌳1 is displaced from the thumb (94) and becomes more flexible and sensitive to proteolysis (44). This polymerase mutant has been suggested to be more open (31,79) or alternatively to sample both open and closed conformations more rapidly (36). Second, we studied the spectrum of a truncated polymerase (NS5B ⌬60 ) (supplemental Fig.…”

“…Among the residues for which no NMR resonances were observed, Ile-405, -412, and -413 have been associated with the same functional region in a molecular dynamics study of NS5B (36) and are all located in direct proximity of the ␤-loop, whereas Ile-560 belongs to the C-terminal linker of the polymerase. Both of these structural motifs have been shown to be regulatory elements and are required for the inhibitory activity of thumb site II inhibitors (95).…”

“…NS5B is targeted by two types of DAAs (19): nucleoside/nucleotide inhibitors that act as substrate analogues at the active site of the polymerase during the enzymatic reaction and nonnucleoside inhibitors (NNIs, also termed allosteric inhibitors) that bind away from the active site (36). At least four NNIbinding sites have been identified on NS5B: thumb site I (NNI-1), thumb site II (NNI-2), palm site I (NNI-3), and palm site II (NNI-4) (37).…”