Structure and DNA binding characteristics of the three-Cys(2)His(2) domain of mouse testis zinc finger protein.
The C-terminal three-Cys(2)His(2) zinc-finger domain (TZD) of mouse testis zinc-finger protein binds to the 5'-TGTACAGTGT-3' at the Aie1 (aurora-C) promoter with high specificity. Interestingly, the primary sequence of TZD is unique, possessing two distinct linkers, TGEKP and GAAP, and distinct residues at presumed DNA binding sites at each finger, especially finger 3. A K(d) value of approximately 10(-8) M was obtained from surface plasmon resonance analysis for the TZD-DNA complex. NMR structure of the free TZD showed that each zinc finger forms a typical beta beta alpha fold. On binding to DNA, chemical shift perturbations and the R(2) transverse relaxation rate in finger 3 are significantly smaller than those in fingers 1 and 2, which indicates that the DNA binding affinity in finger 3 is weaker. Furthermore, the shift perturbations between TZD in complex with the cognate DNA and its serial mutants revealed that both ADE7 and CYT8, underlined in 5'-ATATGTACAGTGTTAT-3', are critical in specific binding, and the DNA binding in finger 3 is sequence independent. Remarkably, the shift perturbations in finger 3 on the linker mutation of TZD (GAAP mutated to TGEKP) were barely detected, which further indicates that finger 3 does not play a critical role in DNA sequence-specific recognition. The complex model showed that residues important for DNA binding are mainly located on positions -1, 2, 3, and 6 of alpha-helices in fingers 1 and 2. The DNA sequence and nonsequence-specific bindings occurring simultaneously in TZD provide valuable information for better understanding of protein-DNA recognition.
Chou
Chun-Chi
CC
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, Republic of China.
Lou
Yuan-Chao
YC
Tang
Tang K
TK
Chen
Chinpan
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D003198:Computer Simulation; D017124:Conserved Sequence; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D015032:Zinc / Q000737:chemistry; D016335:Zinc Fingers
D050505:Mutant Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C406863:Rog protein, mouse; D015032:Zinc