School of Molecular & Cell Biology (ETDs)

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Browsing School of Molecular & Cell Biology (ETDs) by Author "Achilonu, Ikechukwu"

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    Biochemical and biophysical analysis of a hinge region protease variant in HIV-1 subtype C
    (University of the Witwatersrand, Johannesburg, 2023-10) Mokhantso, Tshele; Achilonu, Ikechukwu; Sayed, Yasien
    HIV-1 protease plays a crucial role in the maturation of the virus by cleaving gag and gag-pol polyproteins. Understanding the structural and functional consequences of mutations in this enzyme is essential for developing effective anti-HIV drugs, especially in the face of emerging drug-resistant variants. This study focused on the N37T↑V+10•D25A mutant, a novel HIV-1 subtype C protease variant harbouring an insertion (↑V) and a substitution (N→T) at position 37, along with 10 naturally occurring polymorphisms. Mutations occurring distal to the active site have long been thought to contribute to drug resistance, with this in mind the study aimed to assess the impact these mutations have on the structure, stability, dynamics and drug binding of HIV-1 protease. The N37T↑V+10•D25A mutant and wild-type (WT•D25A) HIV-1 protease were overexpressed and purified from inclusion bodies formed in Escherichia coli cells using ion-exchange chromatography. Far-UV CD and SE-HPLC analysis showed that N37T↑V+10•D25A exhibited a predominantly β-sheet secondary structure (218 nm trough) and had a homodimeric size of ~23 kDa, respectively, both similar to WT•D25A. Assessment of the local tertiary structure by intrinsic tryptophan fluorescence indicated that the protease retained its tertiary structure in the presence of mutations, with partial exposure of Trp residues (Trp 6, Trp 6', Trp 42/43, and Trp 42'/43’). Overall, the insertion and substitution mutations did not significantly alter the overall structure of HIV-1 protease. However, the conformational stability of N37T↑V+10•D25A was found to be reduced compared to WT•D25A as determined by urea-induced equilibrium unfolding and thermal unfolding experiments. When denatured using urea and temperature, the mutant exhibited a two-state mechanism of unfolding without stable intermediates during the folding and unfolding process. Thermal unfolding experiment determined the melting temperature of N37T↑V+10•D25A as 58 ± 1.2 °C, which is lower than that of the wild type of 62 ± 0.9 °C. This suggests a potential decrease in dimer stability due to the mutations present in N37T↑V+10•D25A. Isothermal titration calorimetry with acetyl pepstatin as a model inhibitor was employed to examine the impact of mutations on drug binding. The enthalpy (ΔH) for WT•D25A and N37T↑V+10•D25A were 35.94 and 36.02 kJ/mol, respectively. The entropy (ΔS) for WT•D25A and N37T↑V+10•D25A was found to be 256.7 and 261.6 J.mol.K, respectively. These differences in thermodynamic parameters between the WT•D25A and N37T↑V+10•D25A proteases, may indicate altered drug-protease interactions. Induced fit molecular docking predicted the binding strengths of both WT•D25A and N37T↑V+10•D25A with nine protease inhibitors (atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir and tipranavir), revealing that specific background mutations, such as P40S present in N37T↑V+10•D25A, significantly decreased the binding energy of the HIV-1 protease to these inhibitors. Molecular dynamics simulations provided insights into the structural dynamics of N37T↑V+10•D25A, showing reduced stability and increased dynamics, particularly in the flaps and hinges of the protease. An increase in flap tip curling and involvement of the cantilever tips were observed to be related to the flap opening mechanism of HIV-1 protease. Interactions between the HIV-1 protease and inhibitors were examined and the radius of gyration and solvent accessible surface area were calculated to evaluate protein compactness and solvent accessibility of the bound inhibitors, respectively. In general, the N37T↑V+10•D25A mutant exhibited decreased compactness when bound to inhibitors, which correlated with the increased solvent exposure of PIs when bound to N37T↑V+10•D25A. This may contribute to the drug resistance mechanisms of the protease, as inhibitors would have difficulty binding the active site and exhibit weaker binding. During the N37T↑V+10•D25A and PI interactions, there was a decrease in hydrogen interactions, which form the basis for protease inhibitor drug-design and these were replaced by water-bridge interactions, which are weaker and can be easily broken. In conclusion, this study provides a comprehensive characterisation of the N37T↑V+10•D25A mutant of HIV-1 protease, shedding light on its structural alterations, conformational stability, drug binding properties, and dynamic behaviour. These findings contribute to our understanding of drug resistance mechanisms in HIV-1 protease and offer valuable insights for the design of more effective inhibitors to combat HIV-1.