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.
Miller Jenkins
Lisa M
LM
Laboratory of Cell Biology, NCI, and Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hara
Toshiaki
T
Durell
Stewart R
SR
Hayashi
Ryo
R
Inman
John K
JK
Piquemal
Jean-Philip
JP
Gresh
Nohad
N
Appella
Ettore
E
D000595:Amino Acid Sequence; D003545:Cysteine; D004952:Esters / Q000737:chemistry*; D015497:HIV-1 / Q000378:metabolism*; D008239:Lysine; D008958:Models, Molecular; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000378:metabolism
D004952:Esters; D019590:Nucleocapsid Proteins; D008239:Lysine; D003545:Cysteine