Composite organization of the cobalamin binding and cubilin recognition sites of intrinsic factor

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 x 10(6), 14 x 10(6), and 26 x 10(6) M-1 s(-1), respectively), but the equilibrium dissociation constants were essentially different (K-Cbl = 200 muM, 0.2 muM, and less than or equal to1 muM, 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.