title: Modeling of the [E43S]SNase-ssDNA-Cd(2+) complex: structural insight into the action of nuclease on ssDNA.
abstract: Staphylococcal nuclease (SNase) catalyzes the hydrolysis of the phosphate backbone of DNA and RNA leaving 3'-phosphate mononucleotides and dinucleotides. SNase has been extensively used as a model protein for investigating enzymatic mechanism, thermodynamic stability, and protein folding. An unanswered question regarding enzymatic structure-function relationship is how SNase is capable of binding DNA and catalyzing the DNA hydrolysis. For understanding the mechanism of SNase-DNA interaction at the structural level, we have investigated the interactions between the E43S-mutant SNase ([E43S]SNase) and ssDNA in the presence of Cd(2+) using various NMR techniques including pulsed field gradient diffusion measurement, NMR titration and affinity measurement, chemical shift mapping, backbone dynamics, and three dimensional structural determination. [E43S]SNase retains the similar DNA-binding ability to the native SNase but loses its catalytic activity, and binding of ssDNA/Cd(2+) to [E43S]SNase induced certain degree backbone conformational exchange motion in the ssDNA and Cd(2+) binding regions, which might account for the preferential binding of DNA. Based on the NMR-derived structure of ssDNA/Cd(2+)-bound [E43S]SNase, we have built a three-dimensional model of the [E43S]SNase-ssDNA-Cd(2+) complex. The resulting model enabled the functional roles of SNase to be discussed, in particular the action of nuclease on ssDNA.
authors: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
authors: Tao
authors: T
authors: Xie
authors: Yingang
authors: Y
authors: Feng
authors: Lu
authors: L
authors: Shan
authors: Jinfeng
authors: J
authors: Wang
mesh_terms: D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D006868:Hydrolysis; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D002104:Cadmium; D008836:Micrococcal Nuclease