Structural and Regulatory Elements of HCV NS5B Polymerase – β-Loop and C-Terminal Tail – Are Required for Activity of Allosteric Thumb Site II Inhibitors
Figure 6
Binding of thumb domain NNIs to NS5B results in appearance of a new transition with Tm3.
(A) Apo Δ21 thermal unfolding profile (black line) shown with fit to a non-two-state unfolding model (red) with two transitions: major with Tm1 (dark blue line) and leading shoulder with Tm2 (light blue line); (B) Thermal unfolding of Δ21 bound to palm site I shows the same profile as observed for apo Δ21. Melting profile of apo Δ21 bound to palm site II inhibitor is similar to the one observed for Δ21-palm site I complex and apo protein (not shown); (C) Thermal unfolding of Δ21 bound to thumb site I inhibitor with fit to a non-two-state model (red line) with two transitions: major with Tm1 (dark blue line) and a new transition with Tm3 (green line); (D) Unfolding profile of Δ21 bound to thumb site II inhibitor GS-9669 shows three transitions. Tm3 is analogous to the midpoint of second transition observed with thumb site I. (E) and (F) Melting profiles for Δ39 and Δ47 bound to thumb site II inhibitor. (G) Average Tm3 for the third transition in constructs with C-terminal tail truncations. Value of Tm3 remains constant with standard deviation better than 0.5°C, but ΔTm3 increases with increasing C-terminal truncations. (H) Comparison of changes in ΔTm1 and ΔTm3 for GS-9669 bound NS5B truncation mutants. Although ΔTm3 increases, ΔTm1 (st dev 0.1°C) decreases with tail truncation, in agreement with destabilization of polymerase.