Structural and functional analysis of CD14 and villin headpiece

This dissertation addresses two separate projects. Project I is concerned with the structural and functional analysis of CD14, a protein involved in innate immunity. Project II concerns the structural and functional analysis of villin headpiece, the C-terminal F-actin binding domain of villin.Innate immunity is the front-line defense against invading pathogens. During Gram-negative bacterial infection, the Toll-like receptor 4 and MD-2 complex recognize lipopolysaccharide present in the bacterial cell wall. This recognition can be enhanced 100-1000 fold by CD14. However, the beneficial role provided by CD14 becomes detrimental in the context of sepsis and septic shock. An understanding of how CD14 functions will therefore benefit treatments targeted at both immune suppression and immune enhancement.Mutagenesis is used to determine the role of disulfide bonds, and the sites of N-linked glycosylations and glycosylphophatidylinositol-linkage. The five disulfide bonds are demonstrated to have differential impacts on CD14 folding. The first two disulfide bonds (6-17, 15-32) are essential for protein folding. The third and fourth disulfide bonds (168-198, 222-253) contribute to protein folding. The last disulfide bond (287-333) has no effect on protein folding. Two of the four predicted glycosylation sites, asparagines 132 and 263, are actually involved in N-linked glycosylation of human CD14. Asparagine 326 is confirmed as the glycosylphophatidylinositol-linkage site. Circular dichroism experiments suggest that lypopolysaccharide binds to the inner surface of the CD14 structure. Preliminary crystallization conditions are obtained for human CD14, which will be optimized for crystallization of CD14 with and without lipid A.The headpiece domain of villin is an essential motif for F-actin binding. Mutagenesis analysis shows that two previously untested residues, tryptophan 64 and arginine 37, are important for F-actin binding in an F-actin sedimentation assay. Histidine 41 is shown to contribute to the pH-induced unfolding of the N-terminal subdomain of villin headpiece by characterizing a pH-resistant mutant that replaces this histidine with tyrosine. The first high resolution crystal structures of the wildtype and all of the mutant headpiece domains demonstrate that structural changes do not contribute to these differences.