Comparison of the specificity of interaction of cellular and viral zinc-binding domains with 2-mercaptobenzamide thioesters.
The interactions of two 2-mercaptobenzamide thioester compounds with six diverse zinc-binding domains (ZBDs) have been analyzed by UV/visible spectroscopy, NMR spectroscopy, and nucleic acid binding assays. These thioester compounds serve as useful tools for probing the intrinsic chemical stability of ZBDs that exist within a variety of cellular and viral proteins. In our studies, the classical (Cys(2)His(2)) zinc finger ZBDs, the interleaved RING like ZBDs of protein kinase C delta (Cys(2)HisCys and HisCys(3)), and the carboxyl-terminal (Cys(2)HisCys) ZBD of Mouse Mammary Tumor Virus nucleocapsid protein (MMTV NCp10) were resistant to reaction with the thioester compounds. In contrast, the thioester compounds were able to efficiently eject zinc from the amino-terminal (Cys(2)HisCys) ZBD of MMTV NCp10, a Cys(2)HisCys ZBD from Friend of GATA-1 (FOG-1), and from both Cys(4) ZBDs of GATA-1. In all cases, zinc ejection led to a loss of protein structure. Interestingly, GATA-1 was resistant to reaction with the thioester compounds when bound to its target DNA sequence. The electronic and steric screening was calculated for select ZBDs to further explore their reactivity. Based on these results, it appears that both first and second zinc-coordination shell interactions within ZBDs, as well as nucleic acid binding, play important roles in determining the chemical stability and reactivity of ZBDs. These studies not only provide information regarding the relative reactivity of cysteine residues within structural ZBDs but also are crucial for the design of future therapeutic agents that selectively target ZBDs, such as those that occur in the HIV-1 nucleocapsid protein.
Jenkins
Lisa M Miller
LM
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, 30602, USA.
Durell
Stewart R
SR
Maynard
Andrew T
AT
Stahl
Stephen J
SJ
Inman
John K
JK
Appella
Ettore
E
Legault
Pascale
P
Omichinski
James G
JG
D000595:Amino Acid Sequence; D000818:Animals; D001549:Benzamides / Q000737:chemistry* / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D050982:GATA1 Transcription Factor / Q000737:chemistry / Q000378:metabolism; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D051745:Protein Kinase C-delta / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism; D014758:Viral Core Proteins / Q000737:chemistry / Q000378:metabolism
D001549:Benzamides; D002352:Carrier Proteins; D050982:GATA1 Transcription Factor; D015683:Gene Products, gag; D013438:Sulfhydryl Compounds; D014758:Viral Core Proteins; C017800:zinc-binding protein; C068662:retroviral nucleocapsid protein NCp10, Moloney murine leukemia virus; D004247:DNA; D051745:Protein Kinase C-delta