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 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.