title: 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.
abstract: 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 (PRG48T/L89M), without the stabilizing mutations Gln7Lys, Leu33Ile, and Leu63Ile. Kinetic analyses reveal that PRG48T/L89M and PRWT share nearly identical Michaelis-Menten parameters; however, PRG48T/L89M exhibits weaker binding for IDV (41-fold), SQV (18-fold), APV (15-fold), and NFV (9-fold) relative to PRWT. A 1.9 Å resolution crystal structure was solved for PRG48T/L89M bound with saquinavir (PRG48T/L89M-SQV) and compared to the crystal structure of PRWT bound with saquinavir (PRWT-SQV). PRG48T/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. PRG48T/L89M-SQV assumes a more open conformation relative to PRWT-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 PRG48T/L89M-SQV. Our mechanism for PRG48T/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.
authors.affiliation: Department of Biochemistry and Molecular Biology, ‡Department of Chemistry, and §Departments of Pathology, Immunology, and Laboratory Medicine, University of Florida , Gainesville, Florida 32601-0245, United States.
authors.forename: Nathan E
authors.initials: NE
authors.lastname: Goldfarb
authors.forename: Meray
authors.initials: M
authors.lastname: Ohanessian
authors.forename: Shyamasri
authors.initials: S
authors.lastname: Biswas
authors.forename: T Dwight
authors.initials: TD
authors.lastname: McGee
authors.forename: Brian P
authors.initials: BP
authors.lastname: Mahon
authors.forename: David A
authors.initials: DA
authors.lastname: Ostrov
authors.forename: Jose
authors.initials: J
authors.lastname: Garcia
authors.forename: Yan
authors.initials: Y
authors.lastname: Tang
authors.forename: Robert
authors.initials: R
authors.lastname: McKenna
authors.forename: Adrian
authors.initials: A
authors.lastname: Roitberg
authors.forename: Ben M
authors.initials: BM
authors.lastname: Dunn
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D024882:Drug Resistance, Viral; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D016333:HIV Protease / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D019258:Saquinavir / Q000494:pharmacology*
chemical_list: D017320:HIV Protease Inhibitors; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1; D019258:Saquinavir