Defective Hydrophobic Sliding Mechanism and Active Site Expansion in HIV-1 Protease Drug Resistant Variant Gly48Thr/Leu89Met: Mechanisms for the Loss of Saquinavir Binding Potency

详细信息    查看全文

• 作者：Nathan E. Goldfarb ; Meray Ohanessian ; Shyamasri Biswas ; T. Dwight McGee ; Jr. ; Brian P. Mahon ; David A. Ostrov ; Jose Garcia ; Yan Tang ; Robert McKenna ; Adrian Roitberg ; Ben M. Dunn
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：January 20, 2015
• 年：2015
• 卷：54
• 期：2
• 页码：422-433
• 全文大小：502K
• ISSN：1520-4995

文摘

HIV drug resistance continues to emerge; consequently, there is an urgent need to develop next generation antiretroviral therapeutics.1 Here we report on the structural and kinetic effects of an HIV protease drug resistant variant with the double mutations Gly48Thr and Leu89Met (PR_G48T/L89M), without the stabilizing mutations Gln7Lys, Leu33Ile, and Leu63Ile. Kinetic analyses reveal that PR_G48T/L89M and PR_WT share nearly identical Michaelis鈥揗enten parameters; however, PR_G48T/L89M exhibits weaker binding for IDV (41-fold), SQV (18-fold), APV (15-fold), and NFV (9-fold) relative to PR_WT. A 1.9 脜 resolution crystal structure was solved for PR_G48T/L89M bound with saquinavir (PR_{G48T/L89M-SQV}) and compared to the crystal structure of PR_WT bound with saquinavir (PR_WT-SQV). PR_{G48T/L89M-SQV} has an enlarged active site resulting in the loss of a hydrogen bond in the S3 subsite from Gly48 to P3 of SQV, as well as less favorable hydrophobic packing interactions between P1 Phe of SQV and the S1 subsite. PR_{G48T/L89M-SQV} assumes a more open conformation relative to PR_WT-SQV, as illustrated by the downward displacement of the fulcrum and elbows and weaker interatomic flap interactions. We also show that the Leu89Met mutation disrupts the hydrophobic sliding mechanism by causing a redistribution of van der Waals interactions in the hydrophobic core in PR_{G48T/L89M-SQV}. Our mechanism for PR_{G48T/L89M-SQV} drug resistance proposes that a defective hydrophobic sliding mechanism results in modified conformational dynamics of the protease. As a consequence, the protease is unable to achieve a fully closed conformation that results in an expanded active site and weaker inhibitor binding.