Assembly of the intrinsic factor domains and oligomerization of the protein in the presence of cobalamin

Human intrinsic factor (IF) was purified from the recombinant plant Arabidopsis thaliana by affinity chromatography. Cobalamin (Cbl) saturated protein was separated by gel filtration into peaks I and II, which contained according to SDS electrophoresis the 50 kDa full-length protein IF50 and a mixture of two fragments, respectively. Two components of peak II were identified as the 30 kDa N-terminal peptide IF30 and the 20 kDa C-terminal glycopeptide IF20. Measurements of M, under the nondenaturing conditions were conducted by static light scattering. They revealed 100 kDa IF dimers in peak I, whereas 50 kDa cleaved monomers were found in peak II. The protein devoid of Cbl dissociated to the elementary units incapable of association in the absence of Cbl. The individual proteolytic fragments bound Cbl at high concentration of the ligand; however, neither IF30.Cb1 nor IF20.Cbl oligomerized. A mixture of two fragments IF30 + IF20 and Cbl produced a firm complex, IF30+20.Cbl, which could not associate to dimers. In contrast to IF30+20.Cbl, the saturated full-length monomers IF50.Cbl dimerized with K-d approximate to muM. We suggest a two-domain organization of the full-length protein, where two distant units, IF30 and IF20, can be assembled only by Cbl. They are connected by a protease-sensitive link, whose native structure is likely to be important for dimerization. However, linkage between two domains is not compulsory for Cbl binding. Advantages of the two-domain structure of IF are discussed.