Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease

Abstract

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 HisIz4, is 1.9 kcal.molp' less stable (at pH 5.5 and 20 "C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains LeuIZ4. In addition, the population of the trans conformer at the Ly~'"-Pro''~ peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease HI 24L. 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 HI241 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 Lys'"-Pro'17 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys"h-Pro"7. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro' l7 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H 124L, 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 transkis equilibrium of the Lys'16-Pro'17 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 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 kc,, affected more than Km, and their effects are additive. The PI 17G mutation in nuclease H124L leads to the same local conformational rearrangement described for the PI 17G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 335021-5030). In both P117G mutants, the loop formed by residues 112-1 17 is located closer to the adjacent loop formed by residues 77-...

Folding Kinetics of Staphylococcal Nuclease Studied by Tryptophan Engineering and Rapid Mixing Methods

“…In fact, a few hydrophobic residues, including Met26 (strand II) and Pro31 (turn between strands II and III), are in contact with Ile15 (strand I) in the crystal structure of H124L SNase. 47 Similarly, Phe61 located in helix H1 interacts with hydrophobic residues in helix H1 and H2, including Met65 (helix H1), Val99 (helix H2) and Leu103 (helix H2). Thus, we suggest that the the Trp15 and Trp61 side chains first encounter these hydrophobic residues upon formation of the I 1 and I 2 states, before they engage in specific contacts with the corresponding quenchers (Lys24 and Gln106, respectively).…”

Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone

“…This phenomenon may reflect a gain in stability in terms of resistance to degradation, as the Gly amino acids possess a wider conformational freedom than the original Pro residues. Thus, it is possible that the mutation of Pro to Gly favors the global stability of the protein (35)(36)(37) by generating a structure that is less susceptible to degradation. Similar stability increases have been detected in proTRH mutants assayed with Gly substitutions (Fig.…”

Crystal structure of human μ‐crystallin complexed with NADPH

“…Pro90 in monomer A adopts a cis-configuration, while in monomer B it displays a trans-configuration. In some cases, the populations of trans/cis proline isomerization can be characterized by NMR technique to prove the existence of trans/ cis equilibrium (Truckses et al 1996). Yet no crystal structure has been reported thus far exhibiting Xxx-Pro heterogeneities with both trans-and cis-proline peptide bonds.…”