Conformational flexibility in T4 endonuclease VII revealed by crystallography:: Implications for substrate binding and cleavage

被引:38
作者
Raaijmakers, H
Törö, I
Birkenbihl, R
Kemper, B
Suck, D
机构
[1] European Mol Biol Lab, Struct & Computat Biol Programme, D-69117 Heidelberg, Germany
[2] Univ Cologne, Genet Inst, D-50674 Cologne, Germany
关键词
endonuclease VII; conformational flexibility; enzymatic mechanism; Mg-dependent nucleases; high-resolution X-ray structure;
D O I
10.1006/jmbi.2001.4592
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The structure of the N62D mutant of the junction-resoIving endonuclease W (EndoVII) from phage T4 has been refined at 1.3 Angstrom and a second wild-type crystal form solved and refined at 2.8 Angstrom resolution. Comparison of the mutant with the wild-type protein structure in two different crystal environments reveals considerable conformational flexibility at the dimer level affecting the substrate-binding cleft, the dimerization interface and the orientation of the C-terminal domains. The opening of the DNA-binding cleft, the orientation of the C-terminal domains relative to the central dimerization domain as well as the relative positioning of helices in the dimerization interface appear to be sensitive to the crystal packing environment. The highly unexpected rearrangement within the extended hydrophobic interface does change the contact surface area but keeps the number of hydrophobic contacts about the same and will therefore not require significant energy input. The conformational flexibility most likely is of functional significance for the broad substrate specificity of EndoVII. Binding of sulphate ions in the mutant structure and their positions relative to the active-site metal ions and residues known to be essential for catalysis allows us to propose a possible catalytic mechanism. A comparison with the active-site geometries of other magnesium-dependent nucleases, among them the homing endonuclease I-PpoI and Serratia endonuclease, shows common features, suggesting related catalytic mechanisms. (C) 2001 Academic Press.
引用
收藏
页码:311 / 323
页数:13
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