Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

Mittal, Sakshi; Cai, Yufeng; Nalam, M.N.L.; Bolon, Daniel N.; Schiffer, Celia A.

doi:10.1021/ja2095766

Cited by 69 publications

(88 citation statements)

References 30 publications

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“…HIV-1 protease is a highly dynamic protein, and conformational dynamics, especially around the active site, is crucial to substrate binding and enzymatic activity (32)(33)(34)(35)(36). Although crystal structures provide key insights, alteration of dynamic behavior, not captured by static structures, is emerging as an additional contribution to mechanisms of drug resistance (37,38).…”

Section: Discussionmentioning

confidence: 99%

Structural basis and distal effects of Gag substrate coevolution in drug resistance to HIV-1 protease

Özen

Lin

Yilmaz

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Drug resistance mutations in response to HIV-1 protease inhibitors are selected not only in the drug target but elsewhere in the viral genome, especially at the protease cleavage sites in the precursor protein Gag. To understand the molecular basis of this proteasesubstrate coevolution, we solved the crystal structures of drug resistant I50V/A71V HIV-1 protease with p1-p6 substrates bearing coevolved mutations. Analyses of the protease-substrate interactions reveal that compensatory coevolved mutations in the substrate do not restore interactions lost due to protease mutations, but instead establish other interactions that are not restricted to the site of mutation. Mutation of a substrate residue has distal effects on other residues' interactions as well, including through the induction of a conformational change in the protease. Additionally, molecular dynamics simulations suggest that restoration of active site dynamics is an additional constraint in the selection of coevolved mutations. Hence, protease-substrate coevolution permits mutational, structural, and dynamic changes via molecular mechanisms that involve distal effects contributing to drug resistance.HIV-1 protease | drug resistance | coevolution | crystallography | active site dynamics

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Section: Discussionmentioning

confidence: 99%

Structural basis and distal effects of Gag substrate coevolution in drug resistance to HIV-1 protease

Özen

Lin

Yilmaz

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Slow exchange is expected here due to low solvent accessibility and a complex network of interactions around the active-site core. The flap tips (residues at positions [49][50][51][52][53] are the regions of the proteases that display the fastest deuterium incorporation. The flaps are completely solvent-exposed and highly mobile.…”

Section: Dynamics Of the Hiv-1 Proteasesmentioning

confidence: 99%

“…Polymorphic sites of the subtype B protease (PDB ID: 2PC0) [13] and the C-SA protease (PDB ID: 3U71) [37]. The flexible flaps of the protease (residues at positions [46][47][48][49][50][51][52][53][54] are coloured cyan. The fulcrum region (residues at positions 10-23), hinge region (residues at positions 35-42 and 57-61) and cantilever region (residues at positions 62-78) are implicated in flap opening.…”

Section: Introductionmentioning

confidence: 99%

Amide hydrogen exchange in HIV–1 subtype B and C proteases – insights into reduced drug susceptibility and dimer stability

et al. 2014

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Since its identification, HIV has continued to have a detrimental impact on the lives of millions of people throughout the world. The protease of HIV is a major target in antiviral treatment. The South African HIV–1 subtype C (C–SA) protease displays weaker binding affinity for some clinically approved protease inhibitors in comparison with the HIV–1 subtype B protease. The heavy HIV burden in sub‐Saharan Africa, where subtype C HIV–1 predominates, makes this disparity a topic of great interest. In light of this, the enzyme activity and affinity of protease inhibitors for the subtype B and C–SA proteases were determined. The relative vitality, indicating the selective advantage of polymorphisms, of the C–SA protease relative to the subtype B protease in the presence of ritonavir and darunavir was four‐ and tenfold greater, respectively. Dynamic differences that contribute to the reduced drug susceptibility of the C–SA protease were investigated by performing hydrogen–deuterium exchange/mass spectrometry (HDX/MS) on unbound subtype B and C–SA proteases. The reduced propensity to form the E35–R57 salt bridge, and alterations in the hydrophobic core of the C–SA protease, are proposed to affect the anchoring of the flexible flaps, resulting in an increased proportion of the fully open flap conformation. HDX/MS data suggested that the N–terminus of both proteases is less stable than the C–terminus of the proteases, thus explaining the increased efficacy of dimerization inhibitors targeted toward the C–terminus of HIV proteases. As far as we are aware, this is the first report on assessment of HIV protease dynamics using HDX/MS.

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“…Previous studies of protease inhibitor (PI) resistance mutations in HIV-1 PR suggested that some PR mutations contribute to the flexibility of the hydrophobic core of the enzyme, a mechanism that has been called hydrophobic sliding (33,34). In this mechanism, some of the resistance mutations that reduce the susceptibility of PR to PIs allow some of the hydrophobic core residues in PR to exchange interactions, allowing the residues (and the enzyme) more conformational flexibility, which in turn affects the processing of the cleavage sites in the HIV-1 polyproteins and reduces the binding of PIs that prevent these essential cleavages (33,34).…”

Section: Discussionmentioning

confidence: 99%

Analysis of the Zidovudine Resistance Mutations T215Y, M41L, and L210W in HIV-1 Reverse Transcriptase

Boyer

Das

Arnold

et al. 2015

Antimicrob Agents Chemother

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bAlthough anti-human immunodeficiency virus type 1 (HIV-1) therapies have become more sophisticated and more effective, drug resistance continues to be a major problem. Zidovudine (azidothymidine; AZT) was the first nucleoside reverse transcriptase (RT) inhibitor (NRTI) approved for the treatment of HIV-1 infections and is still being used, particularly in the developing world. This drug targets the conversion of single-stranded RNA to double-stranded DNA by HIV-1 RT. However, resistance to the drug quickly appeared both in viruses replicating in cells in culture and in patients undergoing AZT monotherapy. The primary resistance pathway selects for mutations of T215 that change the threonine to either a tyrosine or a phenylalanine (T215Y/ F); this resistance pathway involves an ATP-dependent excision mechanism. The pseudo-sugar ring of AZT lacks a 3= OH; RT incorporates AZT monophosphate (AZTMP), which blocks the end of the viral DNA primer. AZT-resistant forms of HIV-1 RT use ATP in an excision reaction to unblock the 3= end of the primer strand, allowing its extension by RT. The T215Y AZT resistance mutation is often accompanied by two other mutations, M41L and L210W. In this study, the roles of these mutations, in combination with T215Y, were examined to determine whether they affect polymerization and excision by HIV-1 RT. The M41L mutation appears to help restore the DNA polymerization activity of RT containing the T215Y mutation and also enhances AZTMP excision. The L210W mutation plays a similar role, but it enhances excision by RTs that carry the T215Y mutation when ATP is present at a low concentration. Generally speaking, most mutations in HIV-1 reverse transcriptase (RT) have a negative impact on the enzyme; the primary mutations that cause zidovudine (AZT) resistance are not exceptions. In some cases, secondary mutations are selected in viruses that are replicating in patients because these mutations help to compensate for the deleterious effects of primary resistance mutations. In other cases, the most important effect of a secondary mutation is to cause a further decrease in the susceptibility of the virus to the inhibitor. In this report, we have analyzed the effects of the secondary mutations M41L and L210W, in the presence of the primary mutation T215Y, on both ATP-dependent AZTMP excision and polymerase activities of HIV-1 RT to determine whether these secondary mutations enhance the excision of AZTMP by RT and/or act as compensatory mutations that partially restore the reduced polymerase activity of the T215Y mutant.During the replication of HIV-1, the single-stranded RNA genome is converted into double-stranded DNA by HIV-1 RT. This conversion requires both enzymatic activities of RT: a DNA polymerase that can copy either an RNA or a DNA template and an RNase H that cleaves RNA if, and only if, it is part of an RNA/DNA duplex. Like many DNA polymerases, RT requires both a template and a primer; nucleotides are added sequentially to the 3= end of the primer strand by the polymerase activity of...

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Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

Cited by 69 publications

References 30 publications

Structural basis and distal effects of Gag substrate coevolution in drug resistance to HIV-1 protease

Structural basis and distal effects of Gag substrate coevolution in drug resistance to HIV-1 protease

Amide hydrogen exchange in HIV–1 subtype B and C proteases – insights into reduced drug susceptibility and dimer stability

Analysis of the Zidovudine Resistance Mutations T215Y, M41L, and L210W in HIV-1 Reverse Transcriptase

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