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Structural Studies of a Rationally Selected Multi-Drug Resistant HIV-1 Protease Reveal Synergistic Effect of Distal Mutations on Flap Dynamics

Journal Article · · PLoS ONE
 [1];  [2];  [3];  [1];  [1];  [4]
  1. Georgia State Univ., Atlanta, GA (United States)
  2. National Inst. of Health (NIH), Bethesda, MD (United States)
  3. National Inst. of Health (NIH), Bethesda, MD (United States); Iowa State Univ., Ames, IA (United States)
  4. Univ. of Pittsburgh, PA (United States)
+ Show Author Affiliations
We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker than PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir.
Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Grant/Contract Number:
W-31109-ENG-38
OSTI ID:
1337185
Journal Information:
PLoS ONE, Journal Name: PLoS ONE Journal Issue: 12 Vol. 11; ISSN 1932-6203
Publisher:
Public Library of ScienceCopyright Statement
Country of Publication:
United States
Language:
ENGLISH

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An in silico pharmacological approach toward the discovery of potent inhibitors to combat drug resistance HIV‐1 protease variants journal December 2018
Protein engineering: the potential of remote mutations journal March 2019
Exploring the drug resistance mechanism of active site, non-active site mutations and their cooperative effects in CRF01_AE HIV-1 protease: molecular dynamics simulations and free energy calculations journal January 2019
Exploring the flap dynamics of the South African HIV subtype C protease in presence of FDA-approved inhibitors: MD study journal September 2018
Highly drug‐resistant HIV‐1 protease reveals decreased intra‐subunit interactions due to clusters of mutations journal January 2020
Exploring the drug resistance mechanism of active site, non-active site mutations and their cooperative effects in CRF01_AE HIV-1 protease: molecular dynamics simulations and free energy calculations text January 2019
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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
HIV
NMR spectroscopy
antimicrobial resistance
crystal structure
crystallization
dimers (chemical physics)
hydrogen bonding
proteases