Comparative Analysis of Cobalamin Binding Kinetics and Ligand Protection for Intrinsic Factor, Transcobalamin, and Haptocorrin

Fedosov, Sergey N.; Berglund, Lars; Fedosova, Natalya U.; Nexø, Ebba; Petersen, Torben E.

doi:10.1074/jbc.m111399200

Cited by 62 publications

(104 citation statements)

References 19 publications

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“…The data of CBC-based measurements (Table 1) showed a good correlation with the results obtained earlier for Cbls by different methods [10,14,18]. Only the rate constant of IF-Cbl dissociation deviated from our previous data and pointed to better retention of the ligand by the current protein preparation ( Table 1).…”

Section: Reliability Of Cbc-fluorescence Methodssupporting

confidence: 84%

“…Bold type indicates the rate constant for CBC differing from the corresponding coefficients for Cbl. *Data for H 2 OCbl and 57 Co-labeled CNCbl from references [9,10,14,18]. RU, response units.…”

Section: Binding Of Cbc To If Fragments If 20 or If 30mentioning

confidence: 99%

“…Affinity of the transporting proteins for Cbl still remains a controversial issue with an extraordinary dispersion of the reported equilibrium dissociation constants K d ¼ 10 )9 )10 )15 m [5,7,[10][11][12][13][14][15]. However, the major reasons of this discrepancy are rather artificial.…”

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confidence: 99%

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Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

et al. 2006

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Fluorescent probe rhodamine was appended to 5′ OH‐ribose of cobalamin (Cbl). The prepared conjugate, CBC, bound to the transporting proteins, intrinsic factor (IF) and transcobalamin (TC), responsible for the uptake of Cbl in an organism. Pronounced increase in fluorescence upon CBC attachment facilitated detailed kinetic analysis of Cbl binding. We found that TC had the same affinity for CBC and Cbl (Kd = 5 × 10−15 m), whereas interaction of CBC with the highly specific protein IF was more complex. For instance, CBC behaved normally in the partial reactions CBC + IF30 and CBC + IF20 when binding to the isolated IF fragments (domains). The ligand could also assemble them into a stable complex IF30–CBC–IF20 with higher fluorescent signal. However, dissociation of IF30–CBC–IF20 and IF–CBC was accelerated by factors of 3 and 20, respectively, when compared to the corresponding Cbl complexes. We suggest that the correct domain–domain interactions are the most important factor during recognition and fixation of the ligands by IF. Dissociation of IF–CBC was biphasic, and existence of multiple protein–analogue complexes with normal and partially corrupted structure may explain this behaviour. The most stable component had Kd = 1.5 × 10−13 m, which guarantees the binding of CBC to IF under physiological conditions. The specific intestinal receptor cubilin bound both IF–CBC and IF–Cbl with equal affinity. In conclusion, the fluorescent analogue CBC can be used as a reporting agent in the kinetic studies, moreover, it seems to be applicable for imaging purposes in vivo.

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Section: Reliability Of Cbc-fluorescence Methodssupporting

confidence: 84%

Section: Binding Of Cbc To If Fragments If 20 or If 30mentioning

confidence: 99%

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Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

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“…Results from our crystal structure analysis combined with previously performed kinetics studies (6,19) using Cbl with different upper axial groups (H 2 O, CN, and N 3 ) allow reconstruction of the main steps during Cbl binding to TC. Cbl is almost completely buried inside the domain interface of holo-TC and thus cannot enter the binding site without an initial gap between the two domains.…”

Section: Discussionmentioning

confidence: 92%

“…The structure of TC may serve as model for the paralogues IF and HC (28). Besides the transporters' similarity in Cbl-binding rate constants of k ϩ Ϸ 10 8 M Ϫ1 ⅐s Ϫ1 (6,19), structural similarity exists in regions corresponding to TC's secondary structure elements (Fig. 9A), especially those forming the inner six-helix bundle.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for mammalian vitamin B ₁₂ transport by transcobalamin

Wuerges

Garau²,

Geremia³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Cobalamin (Cbl, vitamin B12) serves for two essential cofactors in mammals. The pathway for its intestinal absorption, plasma transport, and cellular uptake uses cell surface receptors and three Cbl-transporting proteins, haptocorrin, intrinsic factor, and transcobalamin (TC). We present the structure determination of a member of the mammalian Cbl-transporter family. The crystal structures of recombinant human and bovine holo-TCs reveal a two-domain architecture, with an N-terminal ␣6-␣6 barrel and a smaller C-terminal domain. One Cbl molecule in base-on conformation is buried inside the domain interface. Structural data combined with previous binding assays indicate a domain motion in the first step of Cbl binding. In a second step, the weakly coordinated ligand H 2O at the upper axial side of added H2O-Cbl is displaced by a histidine residue of the ␣6-␣6 barrel. Analysis of amino acid conservation on TC's surface in orthologous proteins suggests the location of the TC-receptor-recognition site in an extended region on the ␣6-␣6 barrel. The TC structure allows for the mapping of sites of amino acid variation due to polymorphisms of the human TC gene. Structural information is used to predict the overall fold of haptocorrin and intrinsic factor and permits a rational approach to the design of new Cbl-based bioconjugates for diagnostic or therapeutic drug delivery.cobalamin ͉ crystal structure ͉ P259R polymorphism ͉ transport protein

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Vitamin B₁₂: eine Methylgruppe ohne Aufgabe?

et al. 2006

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Professor Albert Eschenmoser zum 80. Geburtstag gewidmet Die Faszination, die von den B 12 -Coenzymen ausgeht, den "schönsten" Cofaktoren in der Natur, [1] spiegelt die Einmaligkeit und Komplexität ihrer Struktur und Chemie wider. [2,3] B 12 wurde auch zu einem herausragenden Testsystem für Hypothesen über die Evolution katalytischer Bausteine essenzieller Cofaktoren, [4] und sein Corrin-Ligand könnte gemäß Eschenmoser [5] eine Weiterentwicklung des hypothetischen B 12 -Vorläufers "Protocobyrinsäure" sein. Die Einführung von Methylgruppen und die charakteristische Nucleotidschleife könnten durch (nichtenzymatische) Anpassung und Selbstkonstitution zustande gekommen sein.[4-6] Wir beschreiben hier Norvitamin B 12 (1, Co b -Cyan-5'',6''-dimethylbenzimidazolyl-176-norcobamid) [7] und sein metallorganisches Derivat Methylnorcobalamin (2, Co b -Methyl-5'',6''-dimethylbenzimidazolyl-176-norcobamid), zwei B 12 -Derivate, denen die Methylgruppe des Cobamid-Liganden an C176 fehlt. Veranlasst wurden unsere Studien durch die Entdeckung von Norpseudovitamin B 12 (3, Schema 1), der CyanCo III -Form des Cofaktors von Perchlorethylenreduktase von Sulfurospirillum multivorans, [8] einem natürlichen "vollstän-

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Comparative Analysis of Cobalamin Binding Kinetics and Ligand Protection for Intrinsic Factor, Transcobalamin, and Haptocorrin

Cited by 62 publications

References 19 publications

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

Structural basis for mammalian vitamin B ₁₂ transport by transcobalamin

Vitamin B₁₂: eine Methylgruppe ohne Aufgabe?

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Comparative Analysis of Cobalamin Binding Kinetics and Ligand Protection for Intrinsic Factor, Transcobalamin, and Haptocorrin

Cited by 62 publications

References 19 publications

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics

Structural basis for mammalian vitamin B 12 transport by transcobalamin

Vitamin B12: eine Methylgruppe ohne Aufgabe?

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Structural basis for mammalian vitamin B ₁₂ transport by transcobalamin

Vitamin B₁₂: eine Methylgruppe ohne Aufgabe?