HIV Protease Complexed With A Tripeptide Inhibitor

Misty Kintzley '06 and Maggie Rosen '05

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Contents:

I. Introduction

Human immunodeficiency virus is part of the diverse family of retroviruses. The replication begins with the attachment of the virus to a new host cell and the entrance of the viral RNA through the penetrated cell membrane. Through reverse transcription, the RNA becomes double stranded DNA and enters the nucleus of the cell. Once in the nucleus, the viral DNA integrates into the cellular DNA and is translated into proteins necessary for viral assembly (Gandhi, R. et al 1999). Among the viral proteins is the HIV protease (PR), which comes from the gag-pol sequences of the HIV-1 genome. PR is required in the cutting of the gag-pol polyprotein into distinct pieces. At the cleavage sites of the Gag-Pol, protease hydrolyzes the peptide bonds through an intermolecular mechanism in its active site (Louis, J. et al 1998). This is essential for the successful proliferation of the HIV-1 virus. Once cleavage occurs the viral proteins can assemble at the cell surface and bud off as a new virion, which can infect another cell (Gandhi, R. et al 1999).

Protease inhibitors can block the cleavage action of the protease by obstructing the active site. In the presence of a protease inhibitor, the virus is misassembled and the released virion is noninfectious. As a result, protease inhibitors are one of the most potent types of HIV treatment (Louis, J. et al, 1998).

II. General Structure of HIV-1 Protease and the Tripeptide Inhibitor

HIV-1 protease is a protein consisting of two identical 99-amino acid polypetide chains fused together to form a dimer < >. The monomer chains are composed of seven beta strands and one alpha helix < >. The chains are held together to form the dimer by anitparallel beta sheets < >. Here it is complexed with a tripeptide, Glu-Asp-Leu. < >.

The tripeptide inhibitor was derived from one naturally encoded by HIV-1. The HIV-1 genome encodes a transframe region (TFR). The TFR protein has a highly conserved N-terminal domain called the transframe peptide. Its amino acid sequence is Phe-Leu-Glu-Asp-Leu-Ala-Phe. There is also a domain of another 48 amino acids that is variable among HIV-1 isolates. TFR has no specific function in HIV-1, but when there is a major deletion in the region enhanced processing of the viral Gag-Pol occurs with respect to wild type Gag-Pol. The relationship between TFP and its derivatives has been studied. The most potent inhibitor of the protease enzyme was Glu-Asp-Leu, and since the entire TFP could not be analyzed by crystallizing it with protease this tripeptide derivative was used instead. The tripeptide is charged and soluble in water < >. The first two residues, Glu, on the N-terminus, and Asp, are charged, and the third residue flanking the carboxyl terminus of Asp, Leu, is hydrophobic < >. This specific order of the tripeptide is required for its inhibiting ability (Louis, J et al, 1998) .

Reload Protease 

III. Protease Active Site

The pocket formed between the two chains is the active site of the HIV-1 protease molecule < >. Within the active site there are two regions that are essential for protease activity so it can interact with substrates. These regions are the active site triplet, Asp 25, Thr26, and Gly27 < > and the flap region < >. The flaps can exist in two conformations, semi-open and closed. When the protease has a bound substrate the conformation is closed, as shown here, and when it is in the substrate free form they are in the semi-open conformation to allow for substrate binding (Ishima, R et al, 2003) . The Asp25 residue of the active site triplet is held in place by two loops created by bonding between the Thr26 on both monomers as well as hydrogen bonding between the Thr26 on one chain and Leu24 on the other. This hydrogen bond network is referred to as the fireman's grip (Strisovsk, K et al, 2000).

Reload Protease 

The remainder of the active site is made up of part of the beta sheet and alpha helix region < >. Inhibitors can bind in the active site to prevent protease from cleaving HIV-1 proteins required for the functional virions to form. In the active site the Asp25 from chain A and B interact with each other through hydrogen bonding < >.

Reload Protease 

IV. Protease Tripeptide Binding

The interaction between the protease and this tripeptide inhibitor is mostly hydrophilic, unlike protease interaction with most other inhibitors, which is hydrophobic. The only exception is the Leu, which interacts with a pocket formed by part of each monomer chain < >. This hydrophobic pocket is composed of Gly49 and Ile50 from one beta strand of chain A and Leu23, Pro81, and Ile84 from chain B < >. The nitrogen of the Leu bonds to the carbonyl oxygen of Gly27 < >. Leu has yet another interaction within the active site with Asp29 of both monomers. One of its carboxylate oxygens is hydrogen bonded to the oxygen of Asp25 on chain A and the other carboxylate oxygen is hydrogen bonded to the oxygen of Asp25 on chain B < >. The carbonyl oxygen of Glu in the tripeptide hydrogen bonds to the main chain nitrogen of Asp29 in the active site < >. The final tripeptide residue, Asp, hydrogen bonds by its main chain nitrogen to the carbonyl oxygen of Gly48 < > and its carboxylate carbons also hydrogen bond to Asp30 < >. 

 

V. References

Gandhi, R., M.D., J. Bartlett, M.D., M. Linkinhoker, M.A. 1999 Life Cycle of HIV Infection. Johns Hopkins University Division of Infectious Diseases and AIDS Service. http://www.hopkins-aids.edu/hiv_lifecyle/hivcyle_text.html

Ishima, R., D. Torchia, S. Lynch, A. Gronenborn, and J. Louis. 2003. Solution Structure of the Mature HIV-1 Protease Monomer. The Journal of Biological Chemistry 278:42211-43319

Louis, J., F. Dyda, N. Nashed, A. Kimmel, and D. Davies. 1998. Hydrophilic Peptides Derived from the Transframe Region of Gag-Pol Inhibit the HIV-1 Protease. Biochemisty 37:2105-2110.

Strisovsky, K., U. Tessmer, J. Langner, J. Konvalinka, and H. Krausslich. 2000. Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: Rethinking the "fireman's grip" hypothesis. Protein Science 9:1631-1641.


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