X-ray structures of two single-residue mutants of DNase I: H134Q and Y76A

The structures of the single-residue mutants H134Q and Y76Aof bovine pancreatic DNase I have been determined and refinedincluding data to 2.3 and 2.4 Å resolution respectively,by X-ray crystallography. H134 is an essential catalytic residue,while Y76 contributes to the binding of DNA by providing a largevan der Waals contact area that stabilizes the wide minor grooveseen in DNase I-DNA complexes. The mutant proteins, which showstrongly reduced activities of 0.001% (H134Q) and 0.3% (Y76A),were expressed in E.coli and both crystallize in space-groupC2 with almost identical unit cells. The crystal packing schemeis different from that found in wild type crystals grown undervery similar conditions, presumably due to the absence of thecarbohydrate moiety. In both mutants the conformation of theprotein is nearly identical to that of the wild type enzymeand changes are confined to surface loops involved in packing.The disruption of the hydrogen bonds between H134, E78 and Y76in both mutants leads to an increased mobility and positionalshifts in the DNA-binding loop, mainly around residue Y76. Thisin turn may further reduce DNA-binding affinity and, thus, contributeto the low activity. In contrast, symmetry contacts involvingresidues 97–108 lead to a stabilization of the flexibleloop compared to wild type DNase I.