Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease
Authors: Truckses Dagmar M. , Prehoda Kenneth E. , Miller Stephen C. , Markley John L. , Somoza John R. 
Published: February 2009

The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His124, is 1.9 kcal‐mol−1 less stable (at pH 5.5 and 20 °C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains Leu124. In addition, the population of the trans conformer at the Lys116‐Pro117 peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease 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 effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys116‐Pro117 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys116‐Pro117. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro117 of the different variants at residue 124. We solved the X‐ray structure of the cis form of the most stable variant, H124L, and compared it with the published high‐resolution X‐ray structure of the cis form of the least stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92–105). The two structures 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 equilibrium of the Lys116‐Pro117 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination techniques. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the His46‐Pro47 peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions 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 parameters of H124L, H124L+P47G, H124L+P117G, and H124L+P47G+P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kcat affected more than Km, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conformational rearrangement described for the P117G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 33:5021–5030). In both P117G mutants, the loop formed by residues 112–117 is located closer to the adjacent loop formed by residues 77–85, and residues 115–118 adopt a type I' β‐turn conformation with the Lys116‐Gly117 peptide bond in the trans configuration, as compared with the parent protein in which these residues have a typeVIa β‐turn conformation with the Lys116‐Pro117 peptide bond in the cis configuration. Addition of the P47G mutation appears not to cause any additional structural changes. However, the electron density for part of the loop containing this peptide bond was not strong enough to be interpreted.