Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K

Gerlits, Oksana O.; Keen, David A.; Blakeley, Matthew P.; Louis, John M.; Weber, Irene T.; Kovalevskyi, Andrii Y.

doi:10.1021/acs.jmedchem.6b01767

Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K

Journal Article · Tue Feb 14 00:00:00 EST 2017 · Journal of Medicinal Chemistry

DOI:https://doi.org/10.1021/acs.jmedchem.6b01767· OSTI ID:1361359

Gerlits, Oksana O. ^[1]; Keen, David A. ^[2]; Blakeley, Matthew P. ^[3]; Louis, John M. ^[4]; Weber, Irene T. ^[5]; Kovalevskyi, Andrii Y. ^[6]

Univ. of Tennessee, Knoxville, TN (United States). Joint Inst. of Biological Sciences
Science and Technology Facilities Council (STFC), Oxford (United Kingdom). Rutherford Appleton Lab., ISIS Neutron Source
Inst. Laue-Langevin (ILL), Grenoble (France)
National Inst. of Health (NIH), Bethesda, MD (United States)
Georgia State Univ., Atlanta, GA (United States)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D+ ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly alter the drug–enzyme interactions. This is in contrast to the observations in previous 100 K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100 K.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1361359

Journal Information:: Journal of Medicinal Chemistry, Journal Name: Journal of Medicinal Chemistry Journal Issue: 5 Vol. 60; ISSN 0022-2623

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

References (52)

Conformational flexibility of the catalytic Asp dyad in HIV-1 protease: An ab initio study on the free enzyme Piana, Stefano; Carloni, Paolo Proteins: Structure, Function, and Genetics, Vol. 39, Issue 1 https://doi.org/10.1002/(SICI)1097-0134(20000401)39:1<26::AID-PROT3>3.0.CO;2-N	journal	April 2000
Enhancing Protein Backbone Binding-A Fruitful Concept for Combating Drug-Resistant HIV Ghosh, Arun K.; Anderson, David D.; Weber, Irene T. Angewandte Chemie International Edition, Vol. 51, Issue 8 https://doi.org/10.1002/anie.201102762	journal	January 2012
Long-Range Electrostatics-Induced Two-Proton Transfer Captured by Neutron Crystallography in an Enzyme Catalytic Site Gerlits, Oksana; Wymore, Troy; Das, Amit Angewandte Chemie International Edition, Vol. 55, Issue 16 https://doi.org/10.1002/anie.201509989	journal	March 2016
Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors Tie, Yunfeng; Wang, Yuan-Fang; Boross, Peter I. Protein Science, Vol. 21, Issue 3 https://doi.org/10.1002/pro.2019	journal	January 2012
Structural implications of drug-resistant mutants of HIV-1 protease: High-resolution crystal structures of the mutant protease/substrate analogue complexes Mahalingam, Bhuvaneshwari; Louis, John M.; Hung, Jason Proteins: Structure, Function, and Genetics, Vol. 43, Issue 4 https://doi.org/10.1002/prot.1057	journal	January 2001
Structural and Dynamical Aspects of HIV-1 Protease and Its Role in Drug Resistance Meher, Biswa Ranjan; Patel, Seema Dynamics of Proteins and Nucleic Acids https://doi.org/10.1016/B978-0-12-411636-8.00008-0	book	January 2013
[16] SHELXL: High-resolution refinement Sheldrick, George M.; Schneider, Thomas R. Methods in Enzymology https://doi.org/10.1016/S0076-6879(97)77018-6	book	January 1997
HIV-I protease: Maturation, enzyme specificity, and drug resistance Louis, John M.; Webert, Irene T.; Tözsér, József HIV-1: Molecular Biology and Pathogenesis https://doi.org/10.1016/S1054-3589(00)49025-3	book	January 2000
Ultra-high Resolution Crystal Structure of HIV-1 Protease Mutant Reveals Two Binding Sites for Clinical Inhibitor TMC114 Kovalevsky, Andrey Y.; Liu, Fengling; Leshchenko, Sofiya Journal of Molecular Biology, Vol. 363, Issue 1 https://doi.org/10.1016/j.jmb.2006.08.007	journal	October 2006
Effect of Flap Mutations on Structure of HIV-1 Protease and Inhibition by Saquinavir and Darunavir Liu, Fengling; Kovalevsky, Andrey Y.; Tie, Yunfeng Journal of Molecular Biology, Vol. 381, Issue 1 https://doi.org/10.1016/j.jmb.2008.05.062	journal	August 2008
Conformational variation of an extreme drug resistant mutant of HIV protease Shen, Chen-Hsiang; Chang, Yu-Chung; Agniswamy, Johnson Journal of Molecular Graphics and Modelling, Vol. 62 https://doi.org/10.1016/j.jmgm.2015.09.006	journal	November 2015
Hydrophobic Sliding: A Possible Mechanism for Drug Resistance in Human Immunodeficiency Virus Type 1 Protease Foulkes-Murzycki, Jennifer E.; Scott, Walter Robert Peter; Schiffer, Celia A. Structure, Vol. 15, Issue 2 https://doi.org/10.1016/j.str.2007.01.006	journal	February 2007
Crystal Cryocooling Distorts Conformational Heterogeneity in a Model Michaelis Complex of DHFR Keedy, Daniel A.; van den Bedem, Henry; Sivak, David A. Structure, Vol. 22, Issue 6 https://doi.org/10.1016/j.str.2014.04.016	journal	June 2014
Follow the Protons: A Low-Barrier Hydrogen Bond Unifies the Mechanisms of the Aspartic Proteases Northrop, Dexter B. Accounts of Chemical Research, Vol. 34, Issue 10 https://doi.org/10.1021/ar000184m	journal	October 2001
Human immunodeficiency virus-1 protease. 2. Use of pH rate studies and solvent kinetic isotope effects to elucidate details of chemical mechanism Hyland, Lawrence J.; Tomaszek, Thaddeus A.; Meek, Thomas D. Biochemistry, Vol. 30, Issue 34 https://doi.org/10.1021/bi00098a024	journal	August 1991
HIV-1 Protease with 20 Mutations Exhibits Extreme Resistance to Clinical Inhibitors through Coordinated Structural Rearrangements Agniswamy, Johnson; Shen, Chen-Hsiang; Aniana, Annie Biochemistry, Vol. 51, Issue 13 https://doi.org/10.1021/bi2018317	journal	March 2012
Elucidating a Relationship between Conformational Sampling and Drug Resistance in HIV-1 Protease de Vera, Ian Mitchelle S.; Smith, Adam N.; Dancel, Maria Cristina A. Biochemistry, Vol. 52, Issue 19 https://doi.org/10.1021/bi400109d	journal	May 2013
Influence of Flanking Sequences on the Dimer Stability of Human Immunodeficiency Virus Type 1 Protease ^† Wondrak, Ewald M.; Louis, John M. Biochemistry, Vol. 35, Issue 39 https://doi.org/10.1021/bi960984y	journal	January 1996
Differential Flap Dynamics in Wild-Type and a Drug Resistant Variant of HIV-1 Protease Revealed by Molecular Dynamics and NMR Relaxation Cai, Yufeng; Kurt Yilmaz, Nese; Myint, Wazo Journal of Chemical Theory and Computation, Vol. 8, Issue 10 https://doi.org/10.1021/ct300076y	journal	May 2012
Drug Resistance Mutations Alter Dynamics of Inhibitor-Bound HIV-1 Protease Cai, Yufeng; Myint, Wazo; Paulsen, Janet L. Journal of Chemical Theory and Computation, Vol. 10, Issue 8 https://doi.org/10.1021/ct4010454	journal	June 2014
The Low-Barrier Double-Well Potential of the O ^δ ¹ −H−O ^δ ¹ Hydrogen Bond in Unbound HIV Protease: A QM/MM Characterization Porter, Melissa A.; Molina, Pablo A. Journal of Chemical Theory and Computation, Vol. 2, Issue 6 https://doi.org/10.1021/ct600200s	journal	August 2006
Crystal structure of HIV-1 protease in complex with VX-478, a potent and orally bioavailable inhibitor of the enzyme Kim, E. E.; Baker, C. T.; Dwyer, M. D. Journal of the American Chemical Society, Vol. 117, Issue 3 https://doi.org/10.1021/ja00108a056	journal	January 1995
Joint X-ray/Neutron Crystallographic Study of HIV-1 Protease with Clinical Inhibitor Amprenavir: Insights for Drug Design Weber, Irene T.; Waltman, Mary Jo; Mustyakimov, Marat Journal of Medicinal Chemistry, Vol. 56, Issue 13 https://doi.org/10.1021/jm400684f	journal	June 2013
Twenty-Six Years of Anti-HIV Drug Discovery: Where Do We Stand and Where Do We Go? Mehellou, Youcef; De Clercq, Erik Journal of Medicinal Chemistry, Vol. 53, Issue 2 https://doi.org/10.1021/jm900492g	journal	January 2010
Reaction Mechanism of HIV-1 Protease by Hybrid Car-Parrinello/Classical MD Simulations Piana, Stefano; Bucher, Denis; Carloni, Paolo The Journal of Physical Chemistry B, Vol. 108, Issue 30 https://doi.org/10.1021/jp037651c	journal	July 2004
Understanding the HIV-1 Protease Reactivity with DFT: What Do We Gain from Recent Functionals? Garrec, J.; Sautet, P.; Fleurat-Lessard, P. The Journal of Physical Chemistry B, Vol. 115, Issue 26 https://doi.org/10.1021/jp200565w	journal	June 2011
pH-REMD Simulations Indicate That the Catalytic Aspartates of HIV-1 Protease Exist Primarily in a Monoprotonated State McGee, T. Dwight; Edwards, Jesse; Roitberg, Adrian E. The Journal of Physical Chemistry B, Vol. 118, Issue 44 https://doi.org/10.1021/jp504011c	journal	October 2014
Direct detection of CH/π interactions in proteins Plevin, Michael J.; Bryce, David L.; Boisbouvier, Jérôme Nature Chemistry, Vol. 2, Issue 6 https://doi.org/10.1038/nchem.650	journal	May 2010
Ionization state of the catalytic dyad Asp25/25′ in the HIV-1 protease: NMR studies of site-specifically 13C labelled HIV-1 protease prepared by total chemical synthesis Torbeev, Vladimir Yu.; Kent, Stephen B. H. Organic & Biomolecular Chemistry, Vol. 10, Issue 30 https://doi.org/10.1039/c2ob25569c	journal	January 2012
Structure of HIV-1 protease in complex with potent inhibitor KNI-272 determined by high-resolution X-ray and neutron crystallography Adachi, M.; Ohhara, T.; Kurihara, K. Proceedings of the National Academy of Sciences, Vol. 106, Issue 12 https://doi.org/10.1073/pnas.0809400106	journal	March 2009
Protein conformational dynamics in the mechanism of HIV-1 protease catalysis Torbeev, V. Y.; Raghuraman, H.; Hamelberg, D. Proceedings of the National Academy of Sciences, Vol. 108, Issue 52 https://doi.org/10.1073/pnas.1111202108	journal	December 2011
Accessing protein conformational ensembles using room-temperature X-ray crystallography Fraser, J. S.; van den Bedem, H.; Samelson, A. J. Proceedings of the National Academy of Sciences, Vol. 108, Issue 39 https://doi.org/10.1073/pnas.1111325108	journal	September 2011
Molecular dynamics simulations of HIV-1 protease with peptide substrate Harrison, Robert W.; Weber, Irene T. "Protein Engineering, Design and Selection", Vol. 7, Issue 11 https://doi.org/10.1093/protein/7.11.1353	journal	January 1994
Global indicators of X-ray data quality Weiss, Manfred S. Journal of Applied Crystallography, Vol. 34, Issue 2 https://doi.org/10.1107/S0021889800018227	journal	April 2001
LAUEGEN , an X-windows-based program for the processing of Laue diffraction data Campbell, J. W. Journal of Applied Crystallography, Vol. 28, Issue 2 https://doi.org/10.1107/S002188989400991X	journal	April 1995
LAUEGEN version 6.0 and INTLDM Campbell, J. W.; Hao, Q.; Harding, M. M. Journal of Applied Crystallography, Vol. 31, Issue 3 https://doi.org/10.1107/S0021889897016683	journal	June 1998
LSCALE – the new normalization, scaling and absorption correction program in the Daresbury Laue software suite Arzt, Steffi; Campbell, John W.; Harding, Marjorie M. Journal of Applied Crystallography, Vol. 32, Issue 3 https://doi.org/10.1107/S0021889898015350	journal	June 1999
HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8 https://doi.org/10.1107/S0907444906019949	journal	July 2006
Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules Adams, Paul D.; Mustyakimov, Marat; Afonine, Pavel V. Acta Crystallographica Section D Biological Crystallography, Vol. 65, Issue 6 https://doi.org/10.1107/S0907444909011548	journal	May 2009
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Neutron macromolecular crystallography with LADI-III Blakeley, Matthew P.; Teixeira, Susana C. M.; Petit-Haertlein, Isabelle Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 11 https://doi.org/10.1107/S0907444910019797	journal	October 2010
The influence of temperature on lysozyme crystals. Structure and dynamics of protein and water Kurinov, I. V.; Harrison, R. W. Acta Crystallographica Section D Biological Crystallography, Vol. 51, Issue 1 https://doi.org/10.1107/S0907444994009261	journal	January 1995
Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential Blakeley, Matthew P.; Hasnain, Samar S.; Antonyuk, Svetlana V. IUCrJ, Vol. 2, Issue 4 https://doi.org/10.1107/S2052252515011239	journal	June 2015
Drug resistance in HIV-1 protease: Flexibility-assisted mechanism of compensatory mutations Piana, Stefano; Carloni, Paolo; Rothlisberger, Ursula Protein Science, Vol. 11, Issue 10 https://doi.org/10.1110/ps.0206702	journal	October 2002
Crystal structure of an in vivo HIV-1 protease mutant in complex with saquinavir: Insights into the mechanisms of drug resistance Hong, Lin; Zhang, Xuenjun C.; Hartsuck, Jean A. Protein Science, Vol. 9, Issue 10 https://doi.org/10.1110/ps.9.10.1898	journal	January 2000
Amprenavir complexes with HIV-1 protease and its drug-resistant mutants altering hydrophobic clusters: HIV protease mutants altering hydrophobic clusters Shen, Chen-Hsiang; Wang, Yuan-Fang; Kovalevsky, Andrey Y. FEBS Journal, Vol. 277, Issue 18 https://doi.org/10.1111/j.1742-4658.2010.07771.x	journal	August 2010
Structural and Thermodynamic Basis for the Binding of TMC114, a Next-Generation Human Immunodeficiency Virus Type 1 Protease Inhibitor King, N. M.; Prabu-Jeyabalan, M.; Nalivaika, E. A. Journal of Virology, Vol. 78, Issue 21 https://doi.org/10.1128/JVI.78.21.12012-12021.2004	journal	October 2004
INHIBITORS OF HIV-1 PROTEASE: A Major Success of Structure-Assisted Drug Design Wlodawer, Alexander; Vondrasek, Jiri Annual Review of Biophysics and Biomolecular Structure, Vol. 27, Issue 1 https://doi.org/10.1146/annurev.biophys.27.1.249	journal	June 1998
HIV-1 Protease: Structural Perspectives on Drug Resistance Weber, Irene; Agniswamy, Johnson Viruses, Vol. 1, Issue 3 https://doi.org/10.3390/v1031110	journal	December 2009
Current and Novel Inhibitors of HIV Protease Pokorná, Jana; Machala, Ladislav; Řezáčová, Pavlína Viruses, Vol. 1, Issue 3 https://doi.org/10.3390/v1031209	journal	December 2009
Molecular Basis for Drug Resistance in HIV-1 Protease Ali, Akbar; Bandaranayake, Rajintha M.; Cai, Yufeng Viruses, Vol. 2, Issue 11 https://doi.org/10.3390/v2112509	journal	November 2010
Highly resistant HIV-1 proteases and strategies for their inhibition Weber, Irene T.; Kneller, Daniel W.; Wong-Sam, Andres Future Medicinal Chemistry, Vol. 7, Issue 8 https://doi.org/10.4155/fmc.15.44	journal	June 2015

Cited By (8)

Molecular motion regulates the activity of the Mitochondrial Serine Protease HtrA2 Merski, Matthew; Moreira, Cátia; Abreu, Rui MV Cell Death & Disease, Vol. 8, Issue 10 https://doi.org/10.1038/cddis.2017.487	journal	October 2017
Neutron Macromolecular Crystallography Blakeley, Matthew Paul; Fisher, Stuart John Encyclopedia of Biophysics https://doi.org/10.1007/978-3-642-35943-9_658-1	book	January 2020
Neutron macromolecular crystallography Blakeley, Matthew P.; Podjarny, Alberto D. Emerging Topics in Life Sciences, Vol. 2, Issue 1 https://doi.org/10.1042/etls20170083	journal	February 2018
Productive reorientation of a bound oxime reactivator revealed in room temperature X-ray structures of native and VX-inhibited human acetylcholinesterase Gerlits, Oksana; Kong, Xiaotian; Cheng, Xiaolin Journal of Biological Chemistry, Vol. 294, Issue 27 https://doi.org/10.1074/jbc.ra119.008725	journal	May 2019
Membrane-protein crystals for neutron diffraction Sørensen, Thomas Lykke-Møller; Hjorth-Jensen, Samuel John; Oksanen, Esko Acta Crystallographica Section D Structural Biology, Vol. 74, Issue 12 https://doi.org/10.1107/s2059798318012561	journal	November 2018
Membrane-protein crystals for neutron diffraction Sørensen, Thomas Lykke-Møller; Hjorth-Jensen, Samuel John; Oksanen, Esko International Union of Crystallography (IUCr) https://doi.org/10.1107/s2059798318012561/fs5168sup1.pdf	text	January 2018
Neutron scattering in the biological sciences: progress and prospects Ashkar, Rana; Bilheux, Hassina Z.; Bordallo, Heliosa Acta Crystallographica Section D Structural Biology, Vol. 74, Issue 12 https://doi.org/10.1107/s2059798318017503	journal	December 2018
Highly drug‐resistant HIV‐1 protease reveals decreased intra‐subunit interactions due to clusters of mutations Kneller, Daniel W.; Agniswamy, Johnson; Harrison, Robert W. The FEBS Journal, Vol. 287, Issue 15 https://doi.org/10.1111/febs.15207	journal	January 2020

Similar Records

Structural studies of antiviral inhibitor with HIV-1 protease bearing drug resistant substitutions of V32I, I47V and V82I

Journal Article · Sat May 11 20:00:00 EDT 2019 · Biochemical and Biophysical Research Communications · OSTI ID:1531006

Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors

Journal Article · Thu Mar 15 00:00:00 EDT 2012 · Protein Sci. · OSTI ID:1034955

Amprenavir complexes with HIV-1 protease and its drug-resistant mutants altering hydrophobic clusters

Journal Article · Fri Oct 22 00:00:00 EDT 2010 · FEBS J. · OSTI ID:1002740

Related Subjects

60 APPLIED LIFE SCIENCES

Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K

Citation Formats

References (52)

Cited By (8)

Similar Records

Related Subjects