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 isAc-Ser-Leu-Asn-Phe-psi[CH(OH)CH2N]-Pro-Ile-Val-OMe; 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 inhibitor is positioned in a single orientation in the protease active site with the flaps folded directly over it, protecting the inhibitor 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 and A, B chains. The subunits come together in such as way as to form a tunnel where they meet and in the inside of which is located the active site of the protease. The active site consists of two Asp-Thr-Gly conserved sequences, making it a member of the aspartyl protease family. 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. The two flexible flaps on the top of the tunnel move to allow proteins to enter the tunnel. The flaps undergo a dramatic movement, shifting from an open to a closed conformation to bind the target in an appropriate conformation for cleavage. [4]
Binding site
The hydroxyethylamine moiety, in place of the normal scissile bond of the substrate, 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 nestled between the side-chain carboxyl groups of the two active site aspartates within hydrogen bonding distance. The binding is asymmetric with Asp-25 slightly closer than Asp-125. In addition to the contact between the hydroxyl group on the tetrahedral carbon and the active site aspartates, polar contact between inhibitor and enzyme was made through only one substituent atom of the Asn-203 side chain.[5]
Monomers appear to be directly related to inhibitor binding. One of these regions is the loop 49-52 ; the difference between the alpha carbons upon superposition of Gly-49 and Gly-149 is 1.6 A. These loop regions are the tips of the flaps that close over the inhibitor and provide some side-chain contacts to the hydrophobic binding pockets. The positions of these flaps are not equivalent because the peptide bond between residues Ile 50 and Gly 51 is turned 1800 compared with that between Ile-150 and Gly-151. This provides a means for a direct hydrogen bond between the tips of the flaps.
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.
