X-ray structures of two single-residue mutants of DNase I: H134Q and Y76A.
The structures of the single-residue mutants H134Q and Y76A of bovine pancreatic DNase I have been determined and refined including data to 2.3 and 2.4 A resolution respectively, by X-ray crystallography. H134 is an essential catalytic residue, while Y76 contributes to the binding of DNA by providing a large van der Waals contact area that stabilizes the wide minor groove seen in DNase I-DNA complexes. The mutant proteins, which show strongly reduced activities of 0.001% (H134Q) and 0.3% (Y76A), were expressed in E. coli and both crystallize in space-group C2 with almost identical unit cells. The crystal packing scheme is different from that found in wild type crystals grown under very similar conditions, presumably due to the absence of the carbohydrate moiety. In both mutants the conformation of the protein is nearly identical to that of the wild type enzyme and changes are confined to surface loops involved in packing. The disruption of the hydrogen bonds between H134, E78 and Y76 in both mutants leads to an increased mobility and positional shifts in the DNA-binding loop, mainly around residue Y76. This in turn may further reduce DNA-binding affinity and, thus, contribute to the low activity. In contrast, symmetry contacts involving residues 97-108 lead to a stabilization of the flexible loop compared to wild type DNase I.
Weston
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Biological Structures and Biocomputing Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Suck
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D000818:Animals; D001665:Binding Sites; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003460:Crystallization; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D014961:X-Ray Diffraction
D011994:Recombinant Proteins; D004247:DNA; D003850:Deoxyribonuclease I