The mammalian protein transcobalamin II (TCII) is responsible for the transport of vitamin B12 (more accurately termed cobalamin) through the bloodstream. Vitamin B12 is ingested and passed through gut ileal cells to the bloodstream where it is carried by transcobalamin II to cells presenting a specific TCII/cobalamin surface receptor. After receptor mediated endocytosis the vitamin requires further modification before adopting an active cofactor structure required for two enzymes, cytoplasmic methionine synthase (methyl-B12) and mitochondrial methylmalonyl CoA mutase (5'-deoxyadenosyl-B12). Genetic mutants of TCII cause an autosomal recessive disorder that presents itself in infancy with megaloblastic anemia. The 3-dimensional structure of transcobalamin II bound to cobalamin will be solved using X-ray crystallography, and will provide a molecular explanation of a human genetic disorder. The intestinal transport protein, intrinsic factor, shares high homology with TCII and will also be pursued structurally, either by homology modeling or direct structure determination.
Specific Aims : 1) Solve the three-dimensional structure of transcobalamin II by X-ray diffraction techniques, and investigate the structural basis for the physiological disruption of vitamin B12 transport. 2) Determine the critical interactions between TCII and cobalamin, and map the surface of the protein which interacts with the specific cell-surface receptor that recognises the TCII/cobalamin complex. 3) Produce a model of intrinsic factor (IF), haptocorrin and transcobalamin I (TCI) based on the high amino acid sequence identity with TCII.
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