| Popis: |
Protein-protein and protein-carbohydrate recognition are key to understanding many critical life processes. One such critical life process is host defense, and the determination of the crystal and molecular structures of complexes involved in synthesizing and recognizing clinically-relevant antigens can provide key insight into fields as diverse as immunotherapy or chemo-enzymatic synthesis. In the first part, immunotherapeutic antibody candidates MR1 and BEC2 were studied to gain a structural understanding of their potential in the prevention and treatment of cancer. Antibody MR1 binds specifically and with high-affinity to a mutant of the epidermal growth factor receptor (EGFRvIII) defined by an in-frame deletion that generates a novel glycine residue at the fusion junction. It was our hypothesis that this fusion junction glycine residue would form a key recognition element for antibody MR1. The x-ray crystal structure of MR1 in complex with a peptide antigen of the EGFRvIII shows MR1 to possess a J-shaped groove and the EGFRvIII peptide to adopt a hairpin turn---beta strand conformation. Interestingly, the fusion junction glycine, although a central feature of the EGFRvIII peptide, does not interact specifically with MR1 but allows the hairpin turn, which is both unique to EGFRvIII and crucial for MR1 recognition. Antibody BEC2 is an anti-idiotypic antibody mimic of the tumor-associated GD3 ganglioside. It has been raised against anti-GD3 antibody R24 and used successfully to immunize hosts that natively express the antigen against GD3. It was our hypothesis that key polar groups of BEC2 would structurally mimic the GD3 antigen; however, several endeavors including isolation of R24-like antibodies for crystallization, purification of digested fragments of BEC2, and expression of BEC2 and R24 antibody constructs have all failed to yield sufficient amounts of quality protein for crystallization. In the second part, the structures of mutants of the ABO(H) blood group glycosyltransferase B were solved to gain a greater understanding of substrate specificity and mechanism of catalysis. The x-ray crystal structures of cysteine-to-serine mutants GTB M2 (C80S, C196S) and GTB M3 (C80S, C196S, C209S) show a more ordered catalytic domain with a novel helix being observed as compared to the heavy-atom derivative of wild-type GTB. The addition of the third C-to-S mutation in GTB M3 brings about ordering of the catalytic loop, a loop that is not observed in any of the GTB structures published to date. The C2095 mutation allows two water molecules to position themselves in the active site cleft, with one of these waters bridging S209 to a residue important in donor binding, R188. This bridging interaction locks R188 in one position where R188 can interact with the DXD motif D211 and D302. |
