Macromolecular Structure Laboratory, NCI--FCRDC, ABL--Basic Research Program,
P.O. Box B,Frederick MD 21702
The Scripps Research Institute,
10666 N.Torrey Pines Road, La Jolla, CA 92037
Historically, total chemical synthesis had been used to prepare native protease from the human immunodeficiency virus (HIV-1 PR) for structural studies of by X-ray crystallography. Several functionally-relevant analogues of HIV-1 PR have also been obtain 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. Thus: (1) An enzyme analogue in which the peptide bond between residues Gly51 and Gly52 was replaced by a thioester moiety showed normal enzymatic activity, while the crystal structure of its complex with the inhibitor MVT-101 (solved 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 Ile50 -N(H)- by a sulphur atom in both flaps) was inactive. By contrast, the covalent dimer form of the enzyme with the Gly49-Ile50 peptide bond -N(H)- atom specifically replaced by an -O- atom in one flap only retained enzymatic activity. The combined data from these studies strongly indicate that one flap is 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 would have profound implications for drug design targeted at HIV-1 PR.
This research is sponsored in part by the National Cancer Institute (DHHS) under contract with ABL and by NIH grants R01 GM 48897 and P01 GM 48870 (SBHK)