Molecular basis for substrate recognition and drug resistance from 1.1 to 1.6 A resolution crystal structures of HIV-1 protease mutants with substrate analogs

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) protease processes and cleaves the Gag and Gag-Pol polyproteins, allowing viral maturation, and therefore is an important target for antiviral therapy. Ligand binding occurs when the flaps open, allowing access to the active site. This flexibility in flap geometry makes trapping and crystallizing structural intermediates in substrate binding challenging. In this study, we report two crystal structures of two HIV-1 protease variants bound with their corresponding nucleocapsid-p1 variant. One of the flaps in each of these structures exhibits an unusual “intermediate” conformation. Analysis of the flap-intermediate and flap-closed crystal structures reveals that the intermonomer flap movements may be asynchronous and that the flap which wraps over the P3 to P1 (P3-P1) residues of the substrate might close first. This is consistent with our hypothesis that the P3-P1 region is crucial for substrate recognition. The intermediate conformation is conserved in both the wild-type and drug-resistant variants. The structural differences between the variants are evident only when the flaps are closed. Thus, a plausible structural model for the adaptability of HIV-1 protease to recognize substrates in the presence of drug-resistant mutations has been proposed.

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Combatting Drug Resistance: Lessons from the viral proteases of HIV and HCV

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314098830 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C116-C116

Author(s):

Celia Schiffer

Keyword(s):

Drug Resistance ◽

Hepatitis C ◽

Biological Function ◽

Substrate Recognition ◽

Atomic Resolution ◽

Inhibitor Binding ◽

Detailed Understanding ◽

Viral Proteases ◽

Hiv 1 ◽

Substrate Envelope

Drug resistance negatively impacts the lives of millions of patients and costs our society billions of dollars by limiting the longevity of many of our most potent drugs. Drug resistance can be caused by a change in the balance of molecular recognition events that selectively weakens inhibitor binding but maintains the biological function of the target. To reduce the likelihood of drug resistance, a detailed understanding of the target's function is necessary. Both structure at atomic resolution and evolutionarily constraints on its variation is required. "Resilient" targets are less susceptible to drug resistance due to their key location in a particular pathway. This rationale was derived through crystallographic studies elucidating substrate recognition and drug resistance in HIV-1 protease and Hepatitis C (HCV) NS3/4A protease. Both are key therapeutic targets and are potentially "resilient" targets where resistant mutations occur outside of the substrate binding site. To reduce the probability of drug resistance inhibitors should be designed to fit within what we define as the "substrate envelope". These principals are likely more generally applicable to other quickly evolving diseases where drug resistance is quickly evolving. http://www.umassmed.edu/schifferlab/index.aspx

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Molecular Basis of HIV-1 Protease Drug Resistance: Structural Analysis of Mutant Proteases Complexed with Cyclic Urea Inhibitors†

Biochemistry ◽

10.1021/bi962234u ◽

1997 ◽

Vol 36 (7) ◽

pp. 1573-1580 ◽

Cited By ~ 77

Author(s):

Paul J. Ala ◽

Edward E. Huston ◽

Ronald M. Klabe ◽

Denise D. McCabe ◽

Jodie L. Duke ◽

...

Keyword(s):

Drug Resistance ◽

Structural Analysis ◽

Molecular Basis ◽

Hiv 1

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Molecular Basis of HIV-1 Protease Drug Resistance: Structural Analysis of Mutant Proteases Complexed with Cyclic Urea Inhibitors

Biochemistry ◽

10.1021/bi9750044 ◽

1997 ◽

Vol 36 (21) ◽

pp. 6556-6556 ◽

Cited By ~ 3

Author(s):

Paul J. Ala ◽

Edward E. Huston ◽

Ronald M. Klabe ◽

Denise D. McCabe ◽

Jodie L. Duke ◽

...

Keyword(s):

Drug Resistance ◽

Structural Analysis ◽

Molecular Basis ◽

Hiv 1

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Computational study of protein specificity: The molecular basis of HIV-1 protease drug resistance

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.251265598 ◽

2001 ◽

Vol 98 (26) ◽

pp. 14937-14942 ◽

Cited By ~ 167

Author(s):

W. Wang ◽

P. A. Kollman

Keyword(s):

Drug Resistance ◽

Molecular Basis ◽

Computational Study ◽

Protein Specificity ◽

Hiv 1

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A representation of a possible intermediate step during substrate recognition of HIV-1 protease: crystal structures of substrate bound enzyme exhibiting a novel flap conformation

Acta Crystallographica Section A Foundations of Crystallography ◽

10.1107/s0108767305091634 ◽

2005 ◽

Vol 61 (a1) ◽

pp. c196-c196

Author(s):

M. Prabu-Jeyabalan ◽

E. A. Nalivaika ◽

C. A. Schiffer

Keyword(s):

Crystal Structures ◽

Substrate Recognition ◽

Intermediate Step ◽

Hiv 1

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Molecular Basis for Reduced Cleavage Activity and Drug Resistance in D30N HIV-1 protease

10.1101/2021.02.28.433284 ◽

2021 ◽

Author(s):

Subhash Chandra Bihani ◽

Madhusoodan V Hosur

Keyword(s):

Drug Resistance ◽

Molecular Basis ◽

Viral Fitness ◽

Hiv Protease ◽

Cross Resistance ◽

Coulombic Interaction ◽

Cleavage Activity ◽

Hiv 1 ◽

Paediatric Formulation ◽

Haart Therapy

Nelfinavir is one of the FDA approved HIV-1 protease inhibitors and is a part of HAART therapy for the treatment of HIV-AIDS. Nelfinavir was the first HIV-1 protease inhibitor to be approved as a Paediatric formulation. The application of HAART had resulted into significant improvement in the life of AIDS patients. However, emergence of drug resistance in HIV-1 protease limited the use of many of these drugs including nelfinavir. A unique mutation observed frequently in patients treated with nelfinavir is D30N as it is selected exclusively by nelfinavir. It imparts very high resistance to nelfinavir but unlike other primary mutations does not give cross resistance to the majority of other drugs. D30N mutation also significantly reduces cleavage activity of HIV-1 protease and affects the viral fitness. Here, we have determined structures of D30N HIV-1 protease in unliganded form and in complex with the drug nelfinavir. These structures provide rationale for rduced cleavage activity and molecular basis of resistance induced by D30N mutation. The loss of coulombic interaction part of a crucial hydrogen bond between the drug and the enzyme, is a likely explanation for reduced affinity and drug resistance towards nelfinavir. The decreased catalytic activity of D30N HIV protease, due to altered interaction with substrates and reduced stability of folding core may be the reasons for reduced replicative capacity of the HIV harboring D30N HIV-1 protease.

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