Specificity of Acyl Transfer from 2-Mercaptobenzamide Thioesters to the HIV-1 Nucleocapsid Protein

The HIV-1 nucleocapsid protein (NCp7) is a small, highly conserved protein with two zinc-binding
domains that are essential for the protein's function. Molecules that bind to and inactivate NCp7 are currently
being evaluated as new antiviral drugs. In particular, derivatives based on a 2-mercaptobenzamide thioester
template have been shown to specifically eject zinc from the C-terminal zinc-binding domain (ZD2) of NCp7
via acyl transfer from the thioester to a cysteine sulfur. In this study, mutational analysis of the NCp7 amino
acid sequence has been used to investigate the specificity of the interaction between ZD2 and a
2-mercaptobenzamide thioester compound using UV−vis spectroscopy and mass spectrometry to monitor
the rate of metal ejection from NCp7 mutant peptides and sites of acylation, respectively. We were able to
extend the previously reported mechanism of action of these thioester compounds to include a secondary
S to N intramolecular acyl transfer that occurs after the primary acyl transfer from the thioester to a cysteine
side chain in the protein. Structural models of the thioester/ZD2 complex were then examined to identify
the most likely binding orientation. We determined that position x + 1 (where x is Cys36) needs to be an
aromatic 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 residue
is needed for reactivity involving S to N acyl transfer. We report highly specific interactions between
2-mercaptobenzamide thioester compounds and NCp7 that offer a structural basis for refining and designing
new antiretroviral therapeutics, directed toward a target that is resistant to viral mutation.