文摘
The HIV-1 nucleocapsid protein (NCp7) is a small, highly conserved protein with two zinc-bindingdomains that are essential for the protein's function. Molecules that bind to and inactivate NCp7 are currentlybeing evaluated as new antiviral drugs. In particular, derivatives based on a 2-mercaptobenzamide thioestertemplate have been shown to specifically eject zinc from the C-terminal zinc-binding domain (ZD2) of NCp7via acyl transfer from the thioester to a cysteine sulfur. In this study, mutational analysis of the NCp7 aminoacid sequence has been used to investigate the specificity of the interaction between ZD2 and a2-mercaptobenzamide thioester compound using UV-vis spectroscopy and mass spectrometry to monitorthe rate of metal ejection from NCp7 mutant peptides and sites of acylation, respectively. We were able toextend the previously reported mechanism of action of these thioester compounds to include a secondaryS to N intramolecular acyl transfer that occurs after the primary acyl transfer from the thioester to a cysteineside chain in the protein. Structural models of the thioester/ZD2 complex were then examined to identifythe most likely binding orientation. We determined that position x + 1 (where x is Cys36) needs to be anaromatic residue for reactivity and a hydrogen-bond donor in position x + 9 is important for optimal reactivity.A basic residue (lysine or arginine) is required at position x + 2 for the correct fold, while a lysine residueis needed for reactivity involving S to N acyl transfer. We report highly specific interactions between2-mercaptobenzamide thioester compounds and NCp7 that offer a structural basis for refining and designingnew antiretroviral therapeutics, directed toward a target that is resistant to viral mutation.