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 IF(50) and a mixture of two fragments, respectively. Two components of peak II were identified as the 30 kDa N-terminal peptide IF(30) and the 20 kDa C-terminal glycopeptide IF(20). Measurements of M(w) 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 IF(30).Cbl nor IF(20).Cbl oligomerized. A mixture of two fragments IF(30) + IF(20) and Cbl produced a firm complex, IF(30+20).Cbl, which could not associate to dimers. In contrast to IF(30+20).Cbl, the saturated full-length monomers IF(50).Cbl dimerized with K(d) approximately 1 microM. We suggest a two-domain organization of the full-length protein, where two distant units, IF(30) and IF(20), 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.
Fedosov
Sergey N
SN
Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Denmark. snf@imsb.au.dk
Fedosova
Natalya U
NU
Berglund
Lars
L
Moestrup
Søren K
SK
Nexø
Ebba
E
Petersen
Torben E
TE
D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007437:Intrinsic Factor / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008024:Ligands; D008027:Light; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D030821:Plants, Genetically Modified; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D020539:Sequence Analysis, Protein; D014805:Vitamin B 12 / Q000378:metabolism*
D029681:Arabidopsis Proteins; D008024:Ligands; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007437:Intrinsic Factor; D014805:Vitamin B 12