Comparing the Accumulation of Active- and Nonactive-Site Mutations in the HIV-1 Protease

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• 作者：José ; C. Clemente ; Rebecca E. Moose ; Reena Hemrajani ; Lisa R. S. Whitford ; Lakshmanan Govindasamy ; Robbie Reutzel ; Robert McKenna ; Mavis Agbandje-McKenna ; Maureen M. Goodenow ; and Ben M. Dunn
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：September 28, 2004
• 年：2004
• 卷：43
• 期：38
• 页码：12141 - 12151
• 全文大小：753K
• 年卷期：v.43,no.38(September 28, 2004)
• ISSN：1520-4995

文摘

Protease inhibitor resistance still poses one of the greatest challenges in treating HIV. Tobetter design inhibitors able to target resistant proteases, a deeper understanding is needed of the effectsof accumulating mutations and the contributions of active- and nonactive-site mutations to the resistance.We have engineered a series of variants containing the nonactive-site mutations M46I and I54V and theactive-site mutation I84V. These mutations were added to a protease clone (V6) isolated from a pediatricpatient on ritonavir therapy. This variant possessed the ritonavir-resistance-associated mutations in theactive-site (V32I and V82A) and nonactive-site mutations (K20R, L33F, M36I, L63P, A71V, and L90M).The I84V mutation had the greatest effect on decreasing catalytic efficiency, 10-fold when compared tothe pretherapy clone LAI. The decrease in catalytic efficiency was partially recovered by the addition ofmutations M46I and I54V. The M46I and I54V were just as effective at decreasing inhibitor binding asthe I84V mutation when compared to V6 and LAI. The V6^54/84 variant showed over 1000-fold decreasein inhibitor-binding strength to ritonavir, indinavir, and nelfinavir when compared to LAI and V6. Crystal-structure analysis of the V6^54/84 variant bound to ritonavir and indinavir shows structural changes in the80's loops and active site, which lead to an enlarged binding cavity when compared to pretherapy structuresin the Protein Data Bank. Structural changes are also seen in the 10's and 30's loops, which suggestpossible changes in the dynamics of flap opening and closing.