Research Article
Nigel J Clayden, Geoffrey R Mo
Abstract
The endonuclease domain of colicin E9 binds Zn2+ in a multi-step process with a substantial increase in stability that has been suggested to result partly from changes in the dynamic properties of the protein as detected by tryptophan fluorescence. We have used NMR and molecular dynamics to investigate the effect of zinc binding on the structure and dynamics of the backbone of the colicin E9 DNase through 15N chemical shift and relaxation parameter analyses. Significant differences in the experimental chemical shifts between the metal-free and Zn2+-containing forms were limited to the region of the metal binding site, with the largest difference of 12.7 ppm seen for Val 98. Analysis of the relaxation data was carried out using the Lipari-Szabo model-free formalism. The effective rotational correlation time of 10.8 ns for the Zn2+ bound E9 DNase is similar to that of the metal-free form, 11.0 ns. The local dynamics of both the metal-free and Zn2+ bound E9 DNase, were indicative of a rigid protein. Average order parameters for the metalfree and Zn2+ bound E9 DNase are uniformly high, averaging in excess of 0.9 for all the helices and for the E9 DNase as a whole. No significant differences were seen in S2 values for residues at the metal binding site. No significant differences could be seen in the Cα backbone rmsd fluctuations between the metal bound and metal free structures and no evidence for increased deviations between calculated and experimental order parameters. We conclude that the binding of zinc only leads to changes in the protein around the binding site and that the conformation and dynamics of the rest of the E9 DNase are little affected by the presence of zinc. The dramatic effect of zinc on the stability of the protein can be understood in terms of the enthalpy changes associated with metal binding.