HIV-1 subtype C proteases: overexpression, structural, kinetic and thermodynamic characterisation

Abstract

According to UNAIDS, there are ~36.9 million people infected with HIV-1 in the world. Of those, 25.8 million live in sub-Saharan Africa and 6.8 million in South Africa. HIV-1 subtype C accounts for over 95% of HIV infections in South Africa. HIV-1 retrovirus acquires mutations rapidly because of the viral reverse transcriptase. Naturally occurring polymorphisms distinguishing wild type C-SA PR from other proteases make it less susceptible to inhibitors. E35D↑G↑S is a C-SA PR variant with a double insertion in the flap region of the protease. The insertions and background mutations may decrease susceptibility to inhibitors as well as alter kinetic parameters due to increased flap flexibility. This study intended to characterise the effect of the mutations and insertions in E35D↑G↑S on structural, kinetic activity and drug susceptibility. Chemically-synthesised E35D↑G↑S autocatalyses rapidly, impeding further characterisation. There was no detectable overexpression of the E35D↑G↑S protease in Escherichia coli BL21 (DE3)pLysS and Rosetta 2® cells. If the protease is catalytically enhanced, attributed cytotoxicity may prevent overexpression of the protein. Increased autocatalytic activity could also prevent crystallisation. Inactive E35D↑G↑S D25A did not overexpress either, indicating that codon harmonisation with the expression host ought to be performed. C-SA PR was shown to be a predominantly beta-sheeted protein using circular dichroism spectroscopy. The KM of the fluorogenic substrate resembling the capsid/ p2 cleavage site for C-SA PR was 22.02 ±4.09 μM. The specific activity, catalytic turnover and catalytic efficiency of the wild-type C-SA PR protease were found to be 35.68 ±1.06 μmole.min-1.mg-1, 12.79 ±0.38 s-1 and 1.17 ±0.055 s-1.μM-1, respectively. The thermodynamics of binding of atazanavir, ritonavir and darunavir to C-SA PR were determined using isothermal titration calorimetry. The binding of atazanavir and ritonavir to C-SA PR is entropically driven and enthalpically opposed. However, the binding of darunavir to C-SA PR was found to be both entropically and enthalpically favourable. The dissociation constants of the inhibitors in the absence of substrate (Kd) are in the pico-molar range and increased by approximately one order of magnitude when saturating concentrations of substrate were introduced. Atazanavir, ritonavir and darunavir have dissociation constants (Kd) of 160.56 ±54.59 pM, 113.34 ±46.47 pM and 10.24 ±6.02 pM, respectively. Darunavir binds significantly tighter. Keywords: C-SA PR, E35D↑G↑S, insertion mutations, protease, autocatalysis, ITC.