Figure 1.
HCV NS5B polymerase nonnucleoside inhibitors binding sites and NS5B constructs used in studies.
(A) Thumb site I and thumb site II are located on the thumb domain (green); palm site I and palm site II are at the interface of the three domains, thumb, palm (blue) and fingers (red). GS-9669 inhibitor bound in the thumb site II pocket is shown in stick representation (grey, description of crystal structure of NS5B bound to thumb site II inhibitor GS-9669 will be published elsewhere). The active site is indicated by the cyan circle. The other main structural features shown are the C-terminal tail residues (magenta) which contact the β-loop (yellow). (B) 2D representation of domain structure of polymerase and C-terminal truncation sites Δ21, Δ39, Δ47, Δ55, as well as the β-loop deletion mutant Δ21-Δ8 (deleted residues shown in yellow) and LWF triple A mutant F550A/W551A/L553A. Δ55 is a tag free construct and all others contain C6-His. (C) Location of the mutations relative to the tertiary protein structure. (D) Close-up view of interface between LWF motif (magenta, stick representation) and β-loop (yellow) which is dominated by hydrophobic contacts on the surface of the protein.
Figure 2.
Structures of HCV NS5B active site nucleotide inhibitor and NNIs targeting four allosteric sites used in this study.
Figure 3.
Model of HCV NS5B bound to duplex RNA in elongation mode.
Front (A) and side view (90° rotation with cutaway through the finger domain) (B) of NS5B bound to primer/template RNA (dark grey). Side view shows how the β-loop wraps around RNA as it exits the polymerase. Front (C) and side (D) view of the overlay of NS5B Δ21 structure (fingers-pink, palm-light blue, thumb-light green, PDB entry 1C2P) with the model. Overlay shows that the transition from closed inactive polymerase to elongating enzyme requires only small expansion of the finger (red), palm (blue) and thumb (green) domains and major displacement of the β-loop (yellow) and C-terminal tail (magenta). Black circle in (C) denotes the position of double stranded RNA in the model.
Figure 4.
IC50 values for inhibition of RdRp activity of NS5B by Nuc (3′-d′CTP) and NNIs.
Numbers represent mean and standard deviation of IC50 values determined for each inhibitor against the set of NS5B constructs. Heatmap is colored according to IC50 fold shift relative to Δ21 to show changes in inhibition profile for given NNI across NS5B mutant constructs. Thumb site II inhibitors begin to show significant loss of inhibitory potency as interactions between β-loop and C-terminal residues are disrupted by mutations in the interface (Δ21-AAA, truncations past Δ39 and Δ21-Δ39 mutations). Palm site I inhibitor is affected as well, which is explained by disruption of the inhibitor's interaction with the β-loop and C-terminal residues. Inhibition by thumb site I remains for the most part unaffected by NS5B mutations whereas inhibition by palm site II NNI is reduced due to decrease in binding to NS5B with truncated β-loop and/or C-terminal.
Table 1.
Equilibrium dissociation constants (KD) for binding of NNIs to Δ21 and fold-shifts in KD for association to Δ55 and Δ21-Δ8 determined by SPR.
Figure 5.
Effect of mutations in structural motifs (C-terminus, β-loop) of NS5B on protein stability and inhibitor binding.
(A) Representative DSF melting profile for apo NS5B mutant constructs at 5 µM protein concentration are shown. Trend in stability is: Δ21 > Δ39 >> Δ21–AAA ≈ Δ47 > Δ55 > Δ21-Δ8. (B) DSF average Tm and Tm shifts relative to apo Δ21 (ΔTm) for mutant constructs. (C) Direct binding of NNIs to NS5B constructs determined by upshift of NS5B Tm in the presence of 25 µM concentration of thumb site II inhibitor. Values represent an average of two or more independent experiments (each run in triplicate) with standard deviations within 0.5 to 1.0°C, unless mentioned otherwise. Heatmap is colored according to the magnitude of ΔTm elicited by inhibitor binding. Thumb site II inhibitors show >2°C Tm upshift for all constructs in agreement with SPR data obtained for selected constructs.
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.