Structural Analysis of BR3 and SY10 Provides Insight Into the Onset of Bacterial Infective Endocarditis

Bacterial infective endocarditis is an inflammatory disease of the inner lining of the heart, typically caused by opportunistic pathogens. Strains of streptococci and staphylococci bacteria possess adhesin proteins on their surfaces that are associated with binding of the bacterium to the host platelets via sialylated glycans, or ligands. Many adhesin proteins are similar in sequence but have significantly different binding preferences. Better understanding the specificity of ligand-binding preferences may advance preventative measures against the disease. I hypothesized that structural differences underlie the distinct binding patterns between the adhesins of different bacterial species. Therefore, my goal is to structurally characterize two adhesins with known differences in ligands. I focused on two similar streptococcal adhesin proteins; BR3 preferentially binds to the Neu5Ac ligand while SY10 preferentially binds to both Neu5Ac and Neu5Gc ligands. In this study, I generated crystals of BR3 for structural analysis to compare to the known structure of SY10. The DNA encoding for the proteins was cloned into the vector pBG101, including a 6xHis + Glutathione-S-transferase (GST) tag. The protein was expressed in Escherichia coli BL21 (DE3) cells after Isopropyl β-D-1 thiogalactopyranoside (IPTG) induction. Following cell lysis, the proteins were purified using affinity chromatography followed by tag cleavage and size exclusion chromatography. The Mosquito LCP robot was used to generate screening trays to assess different crystallization conditions. Hits from this screen were further optimized using the hanging drop method to yield the biggest single crystals for structural analysis by X-ray crystallography. The structure of BR3 was successfully solved, through a promising diffraction pattern resolution of about 1.8 Å. Further refinement of the BR3 structure is currently underway. Following refinement, comparison of the two protein structures will provide insight into their ligand-binding differences, which could be applicable to other adhesins that cause endocarditis. Further understanding of these interactions could inform new treatments for the disease; it is the interaction between the adhesins and their ligands on platelets that causes bacterial infective endocarditis