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HIV-1 Protease

Miyeko Mana and David Marcey
CLU Biology Department
©David Marcey, 2001


I. Introduction
II. Structural Features
III. The Active Site
IV. Flexible Flaps
V. Substrate Recognition

VI. References

Note:  This exhibit is best viewed if the cue buttons ( ) are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.





I. Introduction

A protease is an enzyme that cleaves proteins to their component peptides. The HIV-1 Protease (PR) hydrolyzes viral polyproteins into functional protein products that are essential for viral assembly and subsequent activity. This maturation process occurs as the virion buds from the host cell. For more background see the viral life-cycle image and overview. This tutorial will focus on the structure-function relationships of PR.

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II. Structural Features

PR is a homodimer (Chain A, Chain B). Each monomer contains 99 amino acids and is identical in conformation. The position of each monomer in the active protease forms an axis of symmetry . The secondary structure of each monomer includes one a-helix, and two antiparallel b sheets .

Aliphatic residues stabilize each monomer in a hydrophobic core. Additionally, the dimer is stabilized by noncovalent interactions, hydrophobic packing of side chains and interactions involving the catalytic residues. Each monomer contains two cysteine residues, but these do not form disulfide bonds .

The active site forms at the dimer interface . It is created in a cleft between the two domains as part of a four stranded b turn . An alternate view demonstrates the position of the active site nestled in approximately the center of the molecule . (See below for further detail: III. The Active Site).

An extended turn, a b hairpin loop, of a beta sheet covers the active site . This flap remains flexible and allows for hinge-like mobility. It allows substrate access to the active site by opening and folding the tips into hydrophobic pockets thus exerting a central role in PR activity (see below: IV. Flexible Flaps).

The overall shape of PR is oblong and relatively flat (allow time to load). This surface contour illustrates where potential binding or protein interaction might occur: several binding pockets exist inside the hollow cleft. The structure at left contains a ligand (not shown here - see Substrate Binding). A comparison of liganded and unliganded PR will be presented below.)

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III. The Active Site: A Feature of Aspartyl Proteases

The Aspartyl Protease superfamily is characterized by the highly conserved catalytic triad sequence Asp-Thr-Gly . In PR, each monomer contributes one Asp-Thr-Gly triad, aa: 25(25' ) - 26(26' ) - 27(27' ). The Asp is essential to PR both catalytically and structurally. Thr26 is buried in the active site.

The carboxyl groups in Asp 25 are nearly coplanar with close contact of OD1 from each monomer ( 25:OD1, 25:OD2, 25':OD1 25':OD2) . Asp 25 from each monomer holds a water (HOH) molecule by forming hydrogen bonds .

Asp 25 (25' ) and water induce a general acid/base protein hydrolysis . Asp 25' acts as an acid as it donates a proton to the carbonyl oxygen of the substrate. Asp 25 acts as base to accepts a proton from the water molecule . A nucleophilic attack of the water molecule results in proteolysis of the subtrate . No covalent bonding is involved. Note: the substrate shown here is a nonpeptide PR inhibitor (THK) and so the cleavage reactions depicted do not represent natural substrate interactions and distances.

A network of hydrogen bonds stabilize the reaction with the following structural interactions:
(1.)
Asp25:OD1 hydrogen bonds with Gly27:NH, Ala28:NH .
(2.)
Additional water molecules form four hydrogen bonds each, relating to Thr26:O, Gly27:O, Asp29:O, and Arg87:NE . Loss off these waters results in an inactive PR, according to Molecular Simulation Modules (Okimoto, et.al).
(3.)
The 'fireman's grip' provides rigid support for catalytic stabilization . Thr26:OG1 accepts a hydrogen bond from Thr26 main chain NH on the opposing loop as well as the carbonyl O of Leu24 on the opposite loop (Rose, et.al).

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IV. Flexible Flaps

The mobile flap, residues 46-54, contains three characteristic regions: side chains that extend outward (Met46, Phe53), hydrophobic chains extending inward (Ile47, Ile54), and a glycine rich region . Ile50 remains at the tip of the turn.

A water molecule binds to Ile50 from the interior of the cleft when the protein is unliganded (Rose, et.al) . This water plays a role in the opening and closing of the flaps as well as increasing the affinity between enzyme and substrate (Okimoto, et al., 2000).

The tips of the flaps, notably the glycine rich region, are highly flexible. This is thought to be necessary for substrate binding and product release. A curling-in movement of the flap forms a hydrophobic cluster as the tips are buried against a hydrophobic wall inside the cleft. One monomer is used here to illustrate the residues involved in forming the hydrophobic cluster, although actual curling that occurs as the tips fold into the cleft is not shown in this view. The tips curl into the cleft resulting in an open space wide enough for a substrate to enter. Upon curling, the electronegative active site is exposed. Given the electronegative active site in combination with hydrophobic walls, a neutral or postively charged substrate could potentially be guided into a conformation optimal for binding (Scott and Schiffer, 2000). Notice that the curl of each monomer does not occur in a symmetric fashion given the symmetric axis.

PR undergoes substantial conformational changes as the cleft of the active sight tightens around a substrate (allow time to load the following script) . Red , white, and blue indicate negative, neutral, and positive electrostatic potential, respectively. Here, only one monomer is shown: note the change in the flap region. The top structure is a PR conformation without a ligand and the bottom structure shows a considerably more closed conformation in which PR is bound with a ligand or substrate. Together, the two flap regions of PR secure the subrate within the binding cleft. The substrate is held rigidly in place by the flaps in order to effect proper cleavage.

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V. Substrate Binding

The residues in PR cleft provide pockets into which the sidechains of the substrate extend . The substrate binds in an extended b-sheet conformation along this cleft (hydrophobic, charged) through extensive hydrogen and van der Waals bonding . Note: the substrate shown is a nonpeptide inhibitor (Indinavir) so these interactions are not depicted. Thr26 of the active site remains shielded from substrate interaction . The basis of substrate recognition is thought to involve the interaction of at least six subsites within the cleft (three subsites contributed from each monomer - Weber, et al., 1989). Hydrophobic subsites closest to the scissile bond have been shown to determine substrate specificity (Short, et al., 2000). Only two subsites are shown here. Residues Pro81, Val82, Ile84 form the binding pocket. Interestingly, the recognition subsites are identical in each monomer, yet the amino acids on each side of the scissle bond are not (Wlodawer, et al., 1993). This last view allows observation of the binding pocket (allow time to load: you are encouraged to move the image to better observe the complementarity of the substrate-inhibitor and PR).

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VI. References

Navia, M.A, Fitzgerals, P.M.D., McKeever, B.M., Leu, C., Heimbach, J.C., Herber, W.K., Sigal, I.S, Darke, P.L., Springer, J.P. Nature 1989, 337, 615-620.

Okimoto, N., Tsukui, T., Kitayama, K., Hata, M., Hoshino, T., and Tsuda, M. J. Am. Chem. Soc. 2000, 122, 5613-5622.

Rose, B.R., Craik, C.S., Stroud, R.M. Biochemistry 1998, 37, 2607-2651.

Scott, W.R.P., Schiffer, C.A. Structure 2000, 8, 1259-1265.

Short, G.F.III, Laikhter, A.L., Lodder, M., Shayo, Y., Arslan, T., Hecht, S.M. Biochemistry 2000, 39, 8768-8781.

Weber, I.T., Miller, M. Jaskolski, M., Leis, j., Skalka, A.M, Wlodawer, A. Science 1989, 243, 928-931.

Wlodawer, A., Erickson, J.W. Annu. Rev. Biochem. 1993, 62, 543-585.

Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B.K., Baldwin, E., Weber, I.T., Selk, L.M., Clawson, L., Schneider, J., Kent., S.B.H. Science 1989, 245, 616-621.

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