Structural basis for mammalian vitamin B12 transport by transcobalamin

Abstract

Cobalamin (Cbl, vitamin B(12)) 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 alpha(6)-alpha(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(2)O at the upper axial side of added H(2)O-Cbl is displaced by a histidine residue of the alpha(6)-alpha(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 alpha(6)-alpha(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.

Fig. 1.

Overall structure of holo-TC. (A) Secondary structure cartoon (here, bovine TC) showing the N-terminal α-domain (red) and C-terminal β-domain (blue) with the flexible linker (green) and the Cbl (orange) in the central domain interface. (B) Enlarged view to the histidine-coordinated Cbl, which is shown in ball-and-stick representation (magenta, Co ion; lime, phosphorus; red, oxygen; blue, nitrogen; orange, carbon). (C) Topology diagram of the two domains with a separate numbering for helices and strands and the color scheme of A. In the α-domain’s α₆-α₆ barrel, the helix orientations run clockwise in the inner α₆ bundle and counterclockwise in the outer. A 3/10 helix at the bottom center shields the hydrophobic core of the domain. The three disulfide bridges are shown as yellow sticks and Cys residues are labeled (human TC numbering). (D) Superposition of the backbone trace of human holo-TC (blue) with both crystal forms of bovine TC (green and red, chain A in all cases). Arrows indicate the patches of gaps in the sequence alignment of Fig. 3. Stereo versions of A and D are shown in Fig. 6.

Fig. 3.

Sequence alignment and secondary structure elements. The insertion of gaps in the sequence of human TC and the assignment of secondary structure elements are based on the x-ray structures. One-letter residue codes as well as helix and strand symbols are colored in correspondence to Fig. 1 A and C. Cys residues of disulfide bridges are highlighted in yellow, residues involved in direct H-bonds to Cbl in green, and the Co-coordinating His residue in blue. The sequence identity is 73%.

Fig. 2.

Cbl interactions with bovine TC (monoclinic crystal form). (A) Stereoview of the F_o − F_c omit electron density map at 2.0-Å resolution around the coordination of His-175 Nε to the Co ion of Cbl (contour level 3 σ). Some solvent water molecules are shown as red spheres, one of which forms a H-bond to His-175 Nδ. The Co ion (magenta) is axially coordinated by the imidazole Nε at a distance of 2.13 Å (above the corrin plane) and by the dimethylbenzimidazole nitrogen N3B at 2.09 Å. (B) Scheme of polar interactions. H-bonds are shown as dotted lines (red, to residues in the α-domain; blue, to residues in the β-domain; green, solvent-mediated interactions). The main or side chain is indicated for residues in direct contact with Cbl, whereas dots indicate residues linked to Cbl via solvent molecules (11 H₂O and a Cl⁻ ion from NaCl salt). The same scheme of direct contacts is observed in human TC (to translate from bovine to human TC numbering, see Fig. 3).

Fig. 4.

Solvent-accessible surface and electrostatic potential properties of TC. (A) Human (Left) and bovine (Right) TC (color coding: blue surface, positive potential; red surface, negative potential). Inset zooms in on the solvent-accessible portion of Cbl in human TC. (B) A negative electrostatic potential prevails at the domain interface in human TC (Left, α-domain; Right, β-domain). Also illustrated is the hemispherical depression in both domains at their interface, each of which accommodates approximately half of the Cbl molecule.

Fig. 5.

Mapping the amino acid conservation among seven TCs (Fig. 9B) on the molecular surface of human TC. Color coding: red, identity; orange, conserved; yellow, semiconserved; white, not conserved. The view to the α-domain is as in Fig. 1C. The conserved region proposed as TC’s receptor-recognition site is located on the right half and involves the surface of the labeled helices α3–α6 and their loops. A low level of conservation is present on the β-domain surface.