Probing the structural basis of the catalytic activity of HIV-1 PR through total chemical protein synthesis

Abstract

Historically, total chemical synthesis had been used to prepare native proteinase from the human immunodeficiency virus (HIV-1 PR) for structural studies by X-ray crystallography. More recently, several functionally-relevant analogues of HIV-1 PR have also been obtained by total chemical synthesis. The results of structural and biochemical studies of the backbone engineered analogues put in question the established belief of the importance of an internal, tetrahedrally coordinated water molecule (water 301) in mediating catalytically important flap–substrate interaction. An enzyme analogue in which the peptide bond between residues Gly 51 and Gly 52 was replaced by a thioester moiety displayed normal enzymatic activity, while the crystal structure of its complex with the inhibitor MVT101 (solved at 2.5 Å resolution as mirror image, D-enantiomer) did not show the presence of water 301. The enzyme analogue in which the ability to donate hydrogen bonds to substrate was deleted (by substitution of Ile 50 –N(H)– by a sulfur atom) in both flaps was 2500-fold less active. By contrast, the covalent dimer form of the enzyme with the Gly 49–Ile 50 peptide bond –N(H)– atom specifically replaced by an –O– atom in one flap only retained normal enzymatic activity. The combined data from these studies strongly indicate that flap–substrate hydrogen bonds from only one flap are sufficient for full enzymatic activity of the HIV-1 PR, and raise the possibility that the retroviral enzyme may make use of only one flap in catalysis. This result may have profound implications for drug design targeted at HIV-1 PR.