Structural characterization of the metal binding site in the cysteine-rich region of HIV-1 Tat protein.

Human immunodeficiency virus type-1 Tat protein transactivates the gene expression of retrovirus. The unique cysteine-rich region of Tat protein is biologically essential. This study characterizes the structural features of a synthetic Tat21-38 peptide covering the cysteine-rich region with Zn binding property. UV titrations confirm Tat peptide binds with two Zn2+ cations maximally per monomer, as previously reported(1). Only monomer is observed from the electrospray mass spectrum. Interestingly, a modified Ellman reaction (2) can differentiate a metal chelated thiolate of cysteine residue from a free one. Three disulfide bonds are formed in apo-Tat21-38 peptide. One Tat21-38 molecule utilize four Cys residues in coordination with the first incorporated Zn2+ cation. Five out of seven Cys residues are coordinating with two Zn cations of the complex Zn-Tat21-38 (2:1). A model of the coordination arrangement of Zn binding sites at different states is proposed.