HIV-1 protease is an effective target for the design of drugs against AIDS. To help this process of drug design, three-dimensional structures have been determined of complexes between HIV-1 protease and a variety of transition-state analogue inhibitors. The true transition state, however, has not been structurally characterized. The crystal structure of the C95M/C1095A HIV-1 protease tethered dimer shows a distinctive feature in which the two flaps of the enzyme are in a ‘closed conformation’ even in the unliganded state. This unique feature has been utilized here to study the structure of HIV-1 protease complexed to an oligopeptide substrate of amino acid sequence His-Lys-Ala-Arg-Val-Leu*NPhe-Glu-Ala-Nle-Ser (where* denotes the cleavage site, and NPhe and Nle denote p-nitrophenylalanine and norleucine residues respectively). The X-ray structure of the complex refined against 2.03 Å (0.203 nm) resolution synchrotron data shows that the substrate is trapped as a tetrahedral reaction intermediate in the crystal. The hydrogen-bonding interactions between the reaction intermediate and the catalytic aspartates are different from those observed previously using transition-state analogues. The reaction intermediate did not dissociate to release the products, possibly due to the inflexibility introduced in the flaps when the enzyme is packed inside crystals.
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Research Article|
July 05 2005
Observation of a tetrahedral reaction intermediate in the HIV-1 protease–substrate complex
Mukesh Kumar;
Mukesh Kumar
1
*Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India
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Vishal Prashar;
Vishal Prashar
1
*Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India
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Smita Mahale;
Smita Mahale
†National Institute for Research in Reproductive Health, Parel, Mumbai-400074, India
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Madhusoodan V. Hosur
Madhusoodan V. Hosur
2
*Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India
2To whom correspondence should be addressed (email hosur@magnum.barc.ernet.in).
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Publisher: Portland Press Ltd
Received:
October 27 2004
Revision Received:
March 17 2005
Accepted:
March 24 2005
Accepted Manuscript online:
March 24 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 389 (2): 365–371.
Article history
Received:
October 27 2004
Revision Received:
March 17 2005
Accepted:
March 24 2005
Accepted Manuscript online:
March 24 2005
Citation
Mukesh Kumar, Vishal Prashar, Smita Mahale, Madhusoodan V. Hosur; Observation of a tetrahedral reaction intermediate in the HIV-1 protease–substrate complex. Biochem J 15 July 2005; 389 (2): 365–371. doi: https://doi.org/10.1042/BJ20041804
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