Proline/Sidechain C–H/O Interactions Stabilize cis-Proline

cis-Proline amide bonds are associated with substantial changes in protein structure, dynamics, and function. Approximately 5% of all proline amide bonds are in the cis conformation, but there is an incomplete understanding of local structural effects that stabilize cis-proline. We previously identified that cis-proline in Ser-Pro sequences is stabilized by a C–H/O interaction between the side-chain Ser oxygen and the proline C–H. Herein, via bioinformatics analysis, we found that C–H/O interactions between a side-chain oxygen and Pro C–H can stabilize the cis-proline conformation at Glu-Pro, Asp-Pro, Gln-Pro, Asn-Pro, Ser-Pro, and Thr-Pro sequences. These C–H/O interactions are apparently most stabilizing at Glu-Pro sequences, which have a substantially higher than average frequency of cis-proline (7.1% of all Glu-Pro amide bonds in the PDB). DFT calculations were conducted to understand the basis and geometries of C–H/O interactions in these sequences. Computationally, these residues all exhibit close C–H/O interactions (substantially below the 2.72 Å sum of the van der Waals radii of H and O), with the closest C–H/O interactions observed with the anionic oxygens of Glu and Asp, and with closer interactions for the anionic residues than the neutral carboxamides Gln and Asn. DFT calculations revealed that C–H/O interactions also stabilize cis-proline at phosphoserine-proline and phosphothreonine-proline sequences, with closer C–H/O interactions in the dianionic forms of phosphorylated residues that predominate at physiological pH. These results also provide an explanation for the observed higher activation barrier for amide bond isomerism at phosphoserine-proline and phosphothreonine-proline sequences. Calculations suggested that C–H/O interactions mediated by these residues could also stabilize non-proline cis amide bonds, which are often functionally important when observed.