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Description

Proteases are one of the 3 (along with reverse transcriptases and integrases) virally encoded enzymes necessary for replication of immunodeffiency virus 1 ^[1] (HIV-1). The protease is a member of the asparctic protease which cleaves the gag and pol polyproptein from the early life cycle of the virus. This cleavage is essential for the virus maturation to form functionnal small-sized proteins so that it can infect other cells. Without these proteases the virus cannot be infective. The enzyme is a dimer composed of two identical subunits forming a tunnel with the active site inside.The mechanism of polypeptides cleavage uses a water molecule as a nucleophile simultaneously with a well-placed asparctic acid acid for hydrolysis of the scissile peptide bond.

The structure of HIV-1 protease with protein bound can't be solved as it would be cleaved before, we analyse how inhibitors bind to the active site to solve the structure.

Inhibitors like the hydroxyethylamine bind to the active site mimicking the tetrahedral transition state of the proteolytic reaction ^[2]. The inhibitor interacts with the active site by direct hydrogen bonds and indirect hydrogen bonds through water molecules.

Structure

The crystal complex of the chemically synthesized protease of human immunodeficiency virus 1 with a heptapeptide-derived inhibitor bound in the active site is a 3 chains structure which contains 203 amino acid alpha carbons.

Inhibitor structure

The sequence of the inhibitor JG-365 is ; the Ki is 0.24 nM. It's a protein with a length of 7 amino acid residues, composed of a C chain.^[3].The orientation of the inhibitor is a particular one in the protease active site, in fact its flaps are folded directly over it in order to protect it from bulk solvent.

HIV 1 protease structure

HIV-1 protease is a dimer of identical polypeptide chains : it's composed of two symmetrically related subunits, each consisting of 99 amino acid residues. Its structure is composed of A,B chains, (one in each subunit)and (8 in each subunits).

The active site of the protease is localised inside the tunnel forms by the two subunits of the protein which come together, and it consists of two conserved sequences, making it the member of the aspartyl protease family.On the top of this tunnel are two flexible flaps which move to allow proteins to enter the tunnel and thus the catalytic site : they shift from an open to a closed conformation in order to bind the target in a correct conformation for cleavage. The two Asp's are essential catalytic residues either interact with the incoming water or protonate the carbonyl to make the carbon more electrophilic for the incoming water. ^[4]

Binding site

Instead of the classic scissile bond of the substrate,the hydroxyethylamine moiety is believed to mimic a tetrahedral reaction intermediate.The bound inhibitor diastereomer has the S configuration at the hydroxyethylamine chiral carbon, and the hydroxyl group is situated between the side-chain carboxyl groups of the two active site aspartates within hydrogen bonding distance.

The binding is not symmetric between the two Asp residues : Asp-25 is a little closer than Asp-125. Another aspect of this structure is that only one substituent atom of the Asn-203 side chain makes a polar contact, plus the contact between the hydroxyl group on the tetrahedral carbon and the active site apspartates.^[5]

The monomers are directly related to inhibitor binding as this region, ,shows it.The difference between the alpha carbons upon superposition of Gly-49 and Gly-149 is 1.6 A. These loop regions correspond to the extremity of the flaps that close over the inhibitor and provide some side-chain contacts to the hydrophobic binding pockets. As shows, their position are not equivalent because of peptide bond between residues Ile-50 and Gly-51 is turned of 180° compared with Ile-150 and Gly-151 (the symmetrical residues in the other chain).In consequence a direct hydrogen bond between the extremity of the flaps is possible.

Biological and Biotechnological Relevance

HIV-1 protease has a crucial importance in drug design as inhbition of it makes the virus noninfective. It prevents formation of mature protein of the HIV virus. The most encouraging inhibtors are the hydroxyethylamine substrate-based inhibitors ^[6] which led to the discovery of the first protease inhibitor, saquinavir. But mutations coding for alteration of the active site conformation facilitates resistance to protease inhibitors. Structure comprehension of HIV protease through structural analysis is crucial to design inhibitors to slow down worldwide AIDS spreading epidemic.