Modeling of the [E43S]SNase-ssDNA–Cd2+ complex: Structural insight into the action of nuclease on ssDNA

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.