title: Solution NMR evidence that the HIV-1 protease catalytic aspartyl groups have different ionization states in the complex formed with the asymmetric drug KNI-272.
abstract: In order to improve the design of HIV-1 protease inhibitors, it is essential to understand how they interact with active site residues, particularly the catalytic Asp25 and Asp125 residues. KNI-272 is a promising, potent HIV-1 protease inhibitor (K(i) approximately 5 pM), currently undergoing phase 1 clinical trials. Because KNI-272 is asymmetric, the complex it forms with the homodimeric HIV-1 protease also lacks symmetry, and the two protease monomers can have distinct NMR spectra. Monomer specific signal assignments were obtained for amino acid residues in the drug binding site as well as for six of the eight Asp residues in the protease/KNI-272 complex. Using these assignments, the ionization states of the Asp carboxyl groups were determined from measurements of (a) the pD dependence of the chemical shifts of the Asp carboxyl carbons and (b) the H/D isotope effect upon the Asp carboxyl carbon chemical shifts. The results of these measurements indicate that the carboxyl of Asp25 is protonated while that of Asp125 is not protonated. These findings provide not only the first experimental evidence regarding the distinct protonation states of Asp25/125 in HIV-1 protease/drug complexes, but also shed light on interactions responsible for inhibitor binding that should form the basis for improved drug designs.
authors: Molecular Structural Biology Unit, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors: Y X
authors: YX
authors: Wang
authors: D I
authors: DI
authors: Freedberg
authors: T
authors: T
authors: Yamazaki
authors: P T
authors: PT
authors: Wingfield
authors: S J
authors: SJ
authors: Stahl
authors: J D
authors: JD
authors: Kaufman
authors: Y
authors: Y
authors: Kiso
authors: D A
authors: DA
authors: Torchia
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001381:Azepines; D001665:Binding Sites; D016333:HIV Protease / Q000737:chemistry* / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014508:Urea / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D001381:Azepines; D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D014508:Urea; D016333:HIV Protease; C085816:DMP 323; C077822:kynostatin 272