COUPLING BETWEEN TRANS CIS PROLINE ISOMERIZATION AND PROTEIN STABILITY IN STAPHYLOCOCCAL NUCLEASE/

The nucleases A produced by two strains of Staphylococcus aureus, whic h have different stabilities, differ only in the identity of the singl e amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His(124), is 1.9 kc al . mol(-1) less stable (at pH 5.5 and 20 degrees C) than the nucleas e from the V8 strain (by convention nuclease H124L), which contains Le u(124). In addition, the population of the trans conformer at the Lys( 116)-Pro(117) peptide bond, as observed by NMR spectroscopy, is differ ent for the two variants: about 15% for nuclease WT and 9% for nucleas e H124L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and H124I variants. We discovered a correlation between effe cts of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys(116)-Pro(117) pepti de bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis config uration at Lys(116)-Pro(117). This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at pro(117) of the different variants at resi due 124. We solved the X-ray structure of the cis form of the most sta ble variant, H124L, and compared it with the published high-resolution X-ray structure of the cis form of the least stable variant, WT (Hyne s TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two st ructures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the trans/cis equilibriu m of the Lys(116)-Pro(117) peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by curre nt structure determination techniques. Residue 124 is located in a hel ix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical pr opensity. A second site of conformational heterogeneity in native nucl ease is found at the His(46)-Pro(47) peptide bond, which is approximat ely 80% trans in both WT and H124L. Because proline to glycine substit utions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X- ray structures of H124L+P117G and H124L+P47G+P117G and the kinetic par ameters of H124L, H124L+P47G, H124L+P117G, and H124L+P47G+P117G. The i ndividual P117G and P47G mutations cause decreases in nuclease activit y, with k(cat) affected more than K-m, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conforma tional rearrangement described for the P117G mutant of WT (Hynes TR, H odel A, Fox RO, 1994, Biochemistry 33:5021-5030). In both P117G mutant s, the loop formed by residues 112-117 is located closer to the adjace nt loop formed by residues 77-85, and residues 115-118 adopt a type I' beta-turn conformation with the Lys(116)-Gly(117) peptide bond in the trans configuration, as compared with the parent protein in which the se residues have a type VIa beta-turn conformation with the Lys(116)-P ro(117) peptide bond in the cis configuration. Addition of the P47G mu tation appears not to cause any additional structural changes. However , the electron density for part of the loop containing this peptide bo nd was not strong enough to be interpreted.