Structural studies of the interaction of bacterial lipopolysaccharides with C-reactive protein and lung surfactant protein D

In this thesis, recognition of the complex natural ligand lipopolysaccharide (LPS) by the innate immune proteins C-reactive protein (CRP) from Limulus polyphemus (Atlantic horseshoe crab), and human surfactant protein D, in the form of a recombinant fragment comprising the a-helical coiled-coil neck plus three lectin domains (CRD), has been studied using X-ray crystallography. The intact LPS from three bacteria, E. coli (0111.B4), P. aeruginosa (serotype 10) and H influenzae type b strain RM153 Eagan (wild type and 4A mutant), were subjected to mild acid hydrolysis to separate the lipid A from the extended oligosaccharide fraction (OS) to improve solubility in crystallisation studies. X-ray diffraction data collection using synchrotron radiation on crystals of Limulus CRP grown in the presence of intact E. coli LPS provided diffraction to 2.oA resolution showing a large unit cell with multiple copies of the molecular aggregate of unknown size in the asymmetric unit. Structure solution was not attempted but has since been achieved by other members of the research group although significant difficulties remain with the refinement. Crystallographic analysis at 1.7 A resolution of the recombinant fragment of human SP-D crystals with H influenzae Eagan wild type and Eagan 4A OS shows no binding of the Eagan wild type OS, however, the Eagan 4A OS ligand is found complexed to the protein . CRD in chains Band C in a calcium dependent manner. No ligand binding could be observed in chain A. In both chains the binding is found in one orientation involving the core L,D-heptose C6- and Cr hydroxyl groups of the glycerol side chain, despite the availability of a terminal core glucose bearing vicinal equatorial C3 and C4 hydroxyl groups. Interaction with Kdo, seen in a putative anyhydro closed 5-membered ring form, is also evident suggesting that efficient recognition requires multiple binding interactions