A High Resolution Structure of an Inhibitor Complex of the Extracellular Nuclease of Staphylococcus aureus

Abstract

Two isomorphously substituted derivatives of the nuclease-Ca2+-thymidine 3',5'-diphosphate complex have been prepared and used in an x-ray crystallographic study of the molecular structure. In one case, 5-iododeoxyuridine 3',5'-diphosphate was used in place of thymidine 3',5'-diphosphate (a net replacement of CH3 by I), and, in the other, Ba2+ was used in place of Ca2+. Intensities of all reflections and their Friedel pairs within the 4 A sphere were measured on this inhibitor complex and its two substituted derivatives; in addition, approximately 35% of the data between 4 A and 2 A were collected, selection being made on the basis of the peak to background ratios. These data have been used to produce an electron density map of sufficient quality to allow a complete tracing of the peptide chain except for a few residues at each terminus which presumably project into solution and are too disordered to be distinguishable from solvent. Approximately 30 residues are involved in three separated sections of helix, and about 24 residues form a three-stranded section of antiparallel β-pleated sheet. Residues 44 to 53 form a loop which is loose, highly exposed to solvent, and somewhat disordered. Residues Lys-48 and Lys-49, which are selectively vulnerable to trypsin-catalyzed hydrolysis, lie at the extremum of this loop. The most significant feature of the nuclease structure is a large pocket which serves as the inhibitor binding site. With the electron density maps now available, the lining of this pocket is revealed to be predominantly neutral or hydrophobic with the exception of several residues which specifically participate in binding the calcium ion and the nucleoside diphosphate. Of the latter, the most conspicuous are Lys-84 and Tyr-85 which form hydrogen bonds to the 3'-phosphate, the guanidinium moieties of Arg-35 and Arg-87 which form hydrogen bonds to the 5'-phosphate, and the carboxylate ions of Glu-43, Asp-21, and Asp-40 which serve as ligands to the calcium ion. Although the calcium ion is directly below the 5'-phosphate, it is not close enough for direct interaction with it. It appears that the barium ion occupies a position significantly different from that of the calcium ion.

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Affiliated Institutions

• National Postdoctoral Association US
• Duke University US
• Weizmann Institute of Science IL
• Massachusetts Institute of Technology US
• National Institutes of Health US

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