Enhanced Stability of Monomer Fold Correlates with Extreme Drug Resistance of HIV-1 Protease

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• 作者：John M. Louis ; J贸zsef T枚zs茅r ; Julien Roche ; Krisztina Mat煤z ; Annie Aniana ; Jane M. Sayer
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：October 29, 2013
• 年：2013
• 卷：52
• 期：43
• 页码：7678-7688
• 全文大小：535K
• 年卷期：v.52,no.43(October 29, 2013)
• ISSN：1520-4995

文摘

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased K_d and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20_T26A and dimeric PR20 exhibit T_m values 6鈥?.5 掳C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the T_m of monomeric PR_T26A by 8 掳C, similar to PR20_T26A. However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR_D25N in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the 螖T_m on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.