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Assembly of the Intrinsic Factor Domains and Oligomerization of the Protein in the Presence of Cobalamin^†

Sergey N. Fedosov,*^‡ Natalya U. Fedosova,^§ Lars Berglund, Søren K. Moestrup, Ebba Nexø,^# and Torben E. Petersen^‡

Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10, Department of Biophysics, University of Aarhus, Ole Worms Alle 185, Cobento Biotech A/S, Science Park, Gustav Wieds Vej 10, Department of Medical Biochemistry, University of Aarhus, Ole Worms Alle 170, and Department of Clinical Biochemistry, AS Aarhus University Hospital, Nrrebrogade 44, 8000 Aarhus C, Denmark

Biochemistry, 2004, 43 (47), pp 15095–15102

DOI: 10.1021/bi048924c

Publication Date (Web): November 5, 2004

†

This work was supported by the Eureka program (CT-T2006) and Cobento Biotech A/S.

To whom correspondence should be addressed. Tel: (+45) 89 42 50 92. Fax: (+45) 86 13 65 97. E-mail: snf@imsb.au.dk.

‡

Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus.

Department of Biophysics, University of Aarhus.

Cobento Biotech A/S.

Department of Medical Biochemistry, University of Aarhus.

Department of Clinical Biochemistry, AS Aarhus University Hospital.

Abstract

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_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 IF₃₀·Cbl 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≈ 1 μM. 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.