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.
Huang
H W
HW
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China. hshuang@gate.sinica.edu.tw
Wang
K T
KT
D000595:Amino Acid Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D002942:Circular Dichroism; D003545:Cysteine; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D015696:Gene Products, tat / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D013058:Mass Spectrometry; D008961:Models, Structural; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus
D002413:Cations, Divalent; D004220:Disulfides; D015696:Gene Products, tat; D010446:Peptide Fragments; D054322:tat Gene Products, Human Immunodeficiency Virus; D004228:Dithionitrobenzoic Acid; D015032:Zinc; D003545:Cysteine