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Article

Composite Organization of the Cobalamin Binding and Cubilin Recognition Sites of Intrinsic Factor^†

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

Biochemistry, 2005, 44 (9), pp 3604–3614

DOI: 10.1021/bi047936v

Publication Date (Web): February 10, 2005

†

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

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

‡

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.

AS Aarhus University Hospital.

Abstract

Intrinsic factor (IF₅₀) is a cobalamin (Cbl)-transporting protein of 50 kDa, which can be cleaved into two fragments: the 30 kDa N-terminal peptide IF₃₀ and the 20 kDa C-terminal glycopeptide IF₂₀. Experiments on binding of Cbl to IF₃₀, IF₂₀, and IF₅₀ revealed comparable association rate constants (k₊_Cbl = 4 × 10⁶, 14 × 10⁶, and 26 × 10⁶ M^-¹ s^-¹, respectively), but the equilibrium dissociation constants were essentially different (K_Cbl = 200 μM, 0.2 μM, and ≤1 pM, respectively). The smaller fragment, IF₂₀, had unexpectedly high affinity for Cbl; however, efficient retention of the ligand required the presence of both fragments. Detailed schemes of the interaction of Cbl with IF₅₀ and with IF₃₀ and IF₂₀ are presented, where the sequential attachment of Cbl to the IF₂₀ and IF₃₀ domains plays the key role in recognition and retention of the ligand. Each isolated fragment of IF was tested for the binding to the specific receptor cubilin in the presence or absence of Cbl. Neither apo nor holo forms of IF₂₀ and IF₃₀ were recognized by the receptor. When two fragments were mixed and incubated with Cbl, they associated into a stable complex, IF₃₀₊₂₀·Cbl, which bound to cubilin as well as the noncleaved IF₅₀·Cbl complex. We suggest that formation of the cubilin recognition site on IF is caused by assembly of two distant domains, which allows the saturated protein to be recognized by the receptor. The obtained parameters for ligand and receptor binding indicate that both full-length IF₅₀ and the fragments may be involved in Cbl assimilation.