Proton transfer and drug binding details revealed in neutron diffraction studies of wild-type and drug resistant HIV-1 protease

Andrey Kovalevsky, Oksana Gerlits, Kaira Beltran, Kevin L. Weiss, David A. Keen, Matthew P. Blakeley, John M. Louis, Irene T. Weber

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

4 Scopus citations

Abstract

HIV-1 protease is an essential therapeutic target for the design and development of antiviral inhibitors to treat AIDS. We used room temperature neutron crystallography to accurately determine hydrogen atom positions in several protease complexes with clinical drugs, amprenavir and darunavir. Hydrogen bonding interactions were carefully mapped to provide an unprecedented picture of drug binding to the protease target. We demonstrate that hydrogen atom positions within the enzyme catalytic site can be altered by introducing drug resistant mutations and by protonating surface residues that trigger proton transfer reactions between the catalytic Asp residues and the hydroxyl group of darunavir. When protein perdeuteration is not feasible, we validate the use of initial H/D exchange with unfolded protein and partial deuteration in pure D2O with hydrogenous glycerol to maximize deuterium incorporation into the protein, with no detrimental effects on the growth of quality crystals suitable for neutron diffraction experiments.

Original languageEnglish
Title of host publicationNeutron Crystallography in Structural Biology
PublisherAcademic Press Inc.
Pages257-279
Number of pages23
DOIs
StatePublished - 2020

Publication series

NameMethods in Enzymology
Volume634
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Funding

The neutron diffraction experiments were performed on IMAGINE beamline at Oak Ridge National Laboratory's High Flux Isotope Reactor sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences (BES), U.S. Department of Energy (DOE), and on beamline LADI-III at the Institut Laue-Langevin. Protein deuteration, purification and crystallization were carried out at ORNL's Center for Structural Molecular Biology (CSMB) supported by the DOE Office of Biological and Environmental Research (BER), using facilities supported by the Scientific User Facilities Division (DOE BES). This work was supported by the NIH and by the DOE BES. The neutron diffraction experiments were performed on IMAGINE beamline at Oak Ridge National Laboratory's High Flux Isotope Reactor sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences (BES), U.S. Department of Energy (DOE), and on beamline LADI-III at the Institut Laue-Langevin. Protein deuteration, purification and crystallization were carried out at ORNL's Center for Structural Molecular Biology (CSMB) supported by the DOE Office of Biological and Environmental Research (BER), using facilities supported by the Scientific User Facilities Division (DOE BES). This work was supported by the NIH and by the DOE BES.

Keywords

  • Acquired immunodeficiency syndrome
  • Clinical protease inhibitor
  • HIV-1 protease
  • Human immunodeficiency virus type 1
  • Hydrogen bond
  • Neutron crystallography
  • Proton transfer
  • Protonation state
  • Structure-assisted drug design

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