Article

Computational characterization of structural role of the non-active site mutation M36I of human immunodeficiency virus type 1 protease.

Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Journal of Molecular Biology (impact factor: 4). 08/2007; 370(3):598-607. DOI:10.1016/j.jmb.2007.04.081 pp.598-607
Source: PubMed

ABSTRACT A prominent characteristic of human immunodeficiency virus type 1 (HIV-1) is its high genetic variability, which generates diversity of the virus and often causes a serious problem of the emergence of drug-resistant mutants. Subtype B HIV-1 is dominant in advanced countries, and the mortality rate due to subtype B HIV-1 has been decreased during the past decade. In contrast, the number of patients with non-subtype B viruses is still increasing in developing countries. One of the reasons for the prevalence of non-subtype B viruses is lack of information about the biological and therapeutic differences between subtype B and non-subtype B viruses. M36I is the most frequently observed polymorphism in non-subtype B HIV-1 proteases. However, since the 36th residue is located at a non-active site of the protease and has no direct interaction with any ligands, the structural role of M36I remains unclear. Here, we performed molecular dynamics (MD) simulations of M36I protease in complex with nelfinavir and revealed the influence of the M36I mutation. The results show that M36I regulates the size of the binding cavity of the protease. The reason for the rare emergence of D30N variants in non-subtype B HIV-1 proteases was also clarified from our computational analysis.

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    Article: Flexible catalytic site conformations implicated in modulation of HIV-1 protease autoprocessing reactions.
    Liangqun Huang, Yanfei Li, Chaoping Chen
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    ABSTRACT: The HIV-1 protease is initially synthesized as part of the Gag-Pol polyprotein in the infected cell. Protease autoprocessing, by which the protease domain embedded in the precursor catalyzes essential cleavage reactions, leads to liberation of the free mature protease at the late stage of the replication cycle. To examine autoprocessing reactions in transfected mammalian cells, we previously described an assay using a fusion precursor consisting of the mature protease (PR) along with its upstream transframe region (p6*) sandwiched between GST and a small peptide epitope. In this report, we studied two autoprocessing cleavage reactions, one between p6* and PR (the proximal site) and the other in the N-terminal region of p6* (the distal site) catalyzed by the embedded protease, using our cell-based assay. A fusion precursor carrying the NL4-3 derived protease cleaved both sites, whereas a precursor with a pseudo wild type protease preferentially autoprocessed the proximal site. Mutagenesis analysis demonstrated that several residues outside the active site (Q7, L33, N37, L63, C67 and H69) contributed to the differential substrate specificity. Furthermore, the cleavage reaction at the proximal site mediated by the embedded protease in precursors carrying different protease sequences or C-terminal fusion peptides displayed varied sensitivity to inhibition by darunavir, a catalytic site inhibitor. On the other hand, polypeptides such as a GCN4 motif, GFP, or hsp70 fused to the N-terminus of p6* had a minimal effect on darunavir inhibition of either cleavage reaction. Taken together, our data suggest that several non-active site residues and the C-terminal flanking peptides regulate embedded protease activity through modulation of the catalytic site conformation. The cell-based assay provides a sensitive tool to study protease autoprocessing reactions in mammalian cells.
    Retrovirology 01/2011; 8:79. · 6.47 Impact Factor
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Keywords

36th residue
 
binding cavity
 
biological
 
computational analysis
 
direct interaction
 
generates diversity
 
genetic variability
 
human immunodeficiency virus type 1
 
M36I protease
 
molecular dynamics
 
mortality rate
 
non-subtype B HIV-1 proteases
 
non-subtype B viruses
 
prominent characteristic
 
protease
 
rare emergence
 
serious problem
 
structural role
 
subtype B
 
subtype B HIV-1
 

Hirotaka Ode