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 Cd2+ 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/Cd2+ to [E43S]SNase induced certain degree backbone conformational exchange motion in the ssDNA and Cd2+ binding regions, which might account for the preferential binding of DNA. Based on the NMR-derived structure of ssDNA/Cd2+-bound [E43S]SNase, we have built a three-dimensional model of the [E43S]SNase-ssDNA-Cd2+ complex. The resulting model enabled the functional roles of SNase to be discussed, in particular the action of nuclease on ssDNA. (C) 2013 Elsevier Inc. All rights reserved.