Mechanisms of discrimination between cobalamins and their natural analogues during their binding to the specific B12-transporting proteins

被引:64
作者
Fedosov, Sergey N.
Fedosova, Natalya U.
Krautler, Bernhard
Nexo, Ebba
Petersen, Torben E.
机构
[1] Aarhus Univ, Prot Chem Lab, Dept Mol Biol, DK-8000 Aarhus C, Denmark
[2] Aarhus Univ, Inst Physiol & Biophys, Dept Biophys, DK-8000 Aarhus C, Denmark
[3] Univ Innsbruck, Inst Organ Chem, A-6020 Innsbruck, Austria
[4] Univ Innsbruck, Ctr Mol Biosci, A-6020 Innsbruck, Austria
[5] AS Aarhus Univ Hosp, Dept Clin Biochem, DK-8000 Aarhus C, Denmark
关键词
D O I
10.1021/bi062063l
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Three proteins, intrinsic factor (IF), transcobalamin (TC), and haptocorrin (HC), all have an extremely high affinity for the cobalamins (Cbls, K-d approximate to 5 fM) but discriminate these physiological ligands from Cbl analogues with different efficiencies decreasing in the following order: IF > TC > HC. We investigated interactions of these proteins with a number of ligands: Cbl, fluorescent conjugate CBC, two base-off analogues [pseudo-coenzyme B-12 (pB) and adenosyl factor A (fA)], and a baseless corrinoid cobinamide. Protein-ligand encounter and the following internal rearrangements in both molecules were registered as a change in the fluorescence of CBC (alone or mixed with other ligands), a transition in absorbance of pB and fA (base-off -> on-base conversion), and alterations in the molecular mass of two split IF domains. The greater complexity of the binding kinetics followed better Cbl specificity (HC < TC < IF). On the basis of the experimental results, we propose a general binding model with three major steps: (1) initial attachment of the ligand to the high-affinity C-domain, (2) primary assembly of N- and C-domains, and (3) slow adjustments and fixation of the ligand at the domain-domain interface. Since step 3 was characteristic of highly specific TC and especially IF, we suggest its particular importance for ligand recognition. The designed models revealed the absolute K-d values for a group of analogues. Calculations show that most of them could potentially bind to the specific transporters IF and TC under physiological conditions. Implications of this finding and the protective role of HC are discussed.
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页码:6446 / 6458
页数:13
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