Molecular Mechanisms of the Binding and Specificity of Streptococcus Pneumoniae Sortase C Enzymes for Pilin Subunits.

Pili are elongated structures that protrude from bacteria and increase their virulence. The Streptococcus pnuenomae pilus island 1 pili are composed of three subunits, RrgA, RrgB, and RrgC, and are assembled by three class C sortase C (SrtC) enzymes: SrtC-1, SrtC-2, and SrtC-3. Pilin subunits are recognized by SrtC proteins through a pentapeptide sorting signal, and while previous studies have sought to characterize the selectivities of SrtC isoforms for these subunits, the molecular mechanisms underlying these interactions remain unclear. Here, we report a series of molecular dynamics simulations of each SrtC enzyme with the sorting signals of RrgA, RrgB, and RrgC to determine the structural and thermodynamic basis of pilin recognition. Results show that, in accordance with previous studies, both SrtC-1 and SrtC-3 are selective for RrgB, while SrtC-2 is selective for RrgA. This specificity is tuned by the sorting signal binding conformation in which the first two residue sidechains complement hydrophobic residues around the active site, while the third residue projects away from the catalytic triad and makes specific interactions based on its charge and reach. Together, these results provided atomic-scale descriptions of the SrtC substrate selectivity mechanisms and extend the emerging model of pilin construction in S. pnuenomae.

The "Lid" in the Streptococcus pneumoniae SrtC1 Sortase Adopts a Rigid Structure that Regulates Substrate Access to the Active Site.

Many species of Gram-positive bacteria use sortase enzymes to assemble long, proteinaceous pili structures that project from the cell surface to mediate microbial adhesion. Sortases construct highly stable structures by catalyzing a transpeptidation reaction that covalently links pilin subunits together via isopeptide bonds. Most Gram-positive pili are assembled by class C sortases that contain a "lid", a structurally unique N-terminal extension that occludes the active site. It has been hypothesized that the "lid" in many sortases is mobile and thus capable of readily being displaced from the enzyme to facilitate substrate binding. Here, we show using NMR dynamics measurements, in vitro assays, and molecular dynamics simulations that the lid in the class C sortase from Streptococcus pneumoniae (SrtC1) adopts a rigid conformation in solution that is devoid of large magnitude conformational excursions that occur on mechanistically relevant time scales. Additionally, we show that point mutations in the lid induce dynamic behavior that correlates with increased hydrolytic activity and sorting signal substrate access to the active site cysteine residue. These results suggest that the lid of the S. pneumoniae SrtC1 enzyme has a negative regulatory function and imply that a significant energetic barrier must be surmounted by currently unidentified factors to dislodge it from the active site to initiate pilus biogenesis.

Solvent interactions stabilize the polyproline II conformation of glycosylated oligoprolines.

In nature, proline residues carry several post-translational modifications (PTMs), including 4R hydroxylation and glycosylation. A recent study synthesized contiguously hydroxylated and glycosylated nonaproline peptides and revealed that both PTMs lead to a significant increase in the thermal stability of PPII relative to the unmodified oligoproline. The increased stability of the hydroxylated peptide can be explained by increased stability of the trans isomer due to stereoelectronic effects. However, the effects of glycosylation cannot be completely explained by stereoelectronics since previous experimental results indicate that 4R-glycosylation does not produce observable changes in the trans preference compared to 4R-hydroxylation. We therefore used sophisticated molecular modeling techniques to determine the reason for the further increase in thermal stability upon glycosylation. Free energy estimates obtained from adaptively biased molecular dynamics calculations in implicit (explicit) solvent are -9 kcal mol(-1) (-20 kcal mol(-1)) for the hydroxylated compound and -9 kcal mol(-1) (-46 kcal mol(-1)) for the glycosylated compound, indicating that direct solvent-peptide interactions are vital for explaining the glycosylation effects on PPII stability. Our data reveals for the first time that interactions between the hydroxyl groups in the glycosylated compound and water act in a complementary fashion with stereoelectronic effects to stabilize the PPII conformation in these substituted oligoproline peptides.

Conformational study of the hydroxyproline-O-glycosidic linkage: sugar-peptide orientation and prolyl amide isomerization in (α/ß)-galactosylated 4(R/S)-hydroxyproline.

Glycosylation is a frequent post-translational modification of proteins that has been shown to influence protein structure and function. Glycosylation of hydroxyproline occurs widely in plants, but is absent in humans and animals. Previous experimental studies on model amides have indicated that α/ß-galactosylation of 4R-hydroxyproline (Hyp) has no measurable effect on prolyl amide isomerization, while a 7% increase in the trans isomer population, as well as a 25-50% increase in the isomerization rate, was observed for the 4S stereoisomer (hyp). In this work, molecular dynamics simulations in explicit water and implicit solvent DFT optimizations are used to examine the structure of the hydroxyproline-O-galactosyl linkage and the effect of glycosylation on the structure and cis/trans isomerization of the peptide backbone. The calculations show two major minima with respect to the glycosidic linkage in all compounds. The C(γ)-exo puckering observed in 4R compounds projects the sugar away from the peptide backbone, while a twisted C(γ)-endo/C(ß)-exo pucker in the 4S compounds brings the peptide and sugar rings together and leads to an intramolecular hydrogen-bonding interaction that is sometimes bridged by a water molecule. This hydrogen bond changes the conformation of the peptide backbone, inducing a favorable n → π* interaction between the oxygen lone pair from the prolyl N-terminal amide and the C═O, which explains the observed increase in trans isomer population in α/ß-galactosylated 4S-hydroxyproline. Our results provide the first molecular level information about this important glycosidic linkage, as well as provide an explanation for the previously observed increase in trans isomer population in 4S-hyp compounds. Moreover, this study provides evidence that sugar-mediated long-range hydrogen bonding between hydroxyl groups and the carbonyl peptide backbone can modify the properties of N-terminal prolyl cis/trans isomerization in peptides.