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Structural basis for receptor recognition of vitamin-B12–intrinsic factor complexes

Abstract

Cobalamin (Cbl, vitamin B12) is a bacterial organic compound and an essential coenzyme in mammals, which take it up from the diet. This occurs by the combined action of the gastric intrinsic factor (IF) and the ileal endocytic cubam receptor formed by the 460-kilodalton (kDa) protein cubilin and the 45-kDa transmembrane protein amnionless1,2. Loss of function of any of these proteins ultimately leads to Cbl deficiency in man3,4. Here we present the crystal structure of the complex between IF–Cbl and the cubilin IF–Cbl-binding-region (CUB5–8)5 determined at 3.3 Å resolution. The structure provides insight into how several CUB (for ‘complement C1r/C1s, Uegf, Bmp1’) domains collectively function as modular ligand-binding regions, and how two distant CUB domains embrace the Cbl molecule by binding the two IF domains in a Ca2+-dependent manner. This dual-point model provides a probable explanation of how Cbl indirectly induces ligand–receptor coupling. Finally, the comparison of Ca2+-binding CUB domains and the low-density lipoprotein (LDL) receptor-type A modules suggests that the electrostatic pairing of a basic ligand arginine/lysine residue with Ca2+-coordinating acidic aspartates/glutamates is a common theme of Ca2+-dependent ligand–receptor interactions.

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Figure 1: Crystal structure of CUB 5–8 –IF–Cbl.
Figure 2: Interactions between CUB 5–8 and IF.
Figure 3: Ligand-binding sites of CUB domains and LA modules.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic structure factors and coordinates are deposited at the Protein Data Bank under accession 3KQ4.

References

  1. 1

    Birn, H. et al. Characterization of an epithelial approximately 460-kDa protein that facilitates endocytosis of intrinsic factor-vitamin B12 and binds receptor-associated protein. J. Biol. Chem. 272, 26497–26504 (1997)

    CAS  Article  Google Scholar 

  2. 2

    Fyfe, J. C. et al. The functional cobalamin (vitamin B12)-intrinsic factor receptor is a novel complex of cubilin and amnionless. Blood 103, 1573–1579 (2004)

    CAS  Article  Google Scholar 

  3. 3

    Tanner, S. M. et al. Genetically heterogeneous selective intestinal malabsorption of vitamin B12: founder effects, consanguinity, and high clinical awareness explain aggregations in Scandinavia and the Middle East. Hum. Mutat. 23, 327–333 (2004)

    CAS  Article  Google Scholar 

  4. 4

    Tanner, S. M. et al. Hereditary juvenile cobalamin deficiency caused by mutations in the intrinsic factor gene. Proc. Natl Acad. Sci. USA 102, 4130–4133 (2005)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Kristiansen, M. et al. Molecular dissection of the intrinsic factor-vitamin B12 receptor, cubilin, discloses regions important for membrane association and ligand binding. J. Biol. Chem. 274, 20540–20544 (1999)

    CAS  Article  Google Scholar 

  6. 6

    Christensen, E. I., Verroust, P. J. & Nielsen, R. Receptor-mediated endocytosis in renal proximal tubule. Pflugers Arch. 458, 1039–1048 (2009)

    CAS  Article  Google Scholar 

  7. 7

    Mathews, F. S. et al. Crystal structure of human intrinsic factor: cobalamin complex at 2.6-A resolution. Proc. Natl Acad. Sci. USA 104, 17311–17316 (2007)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Fedosov, S. N. et al. Assembly of the intrinsic factor domains and oligomerization of the protein in the presence of cobalamin. Biochemistry 43, 15095–15102 (2004)

    CAS  Article  Google Scholar 

  9. 9

    Kristiansen, M. et al. Cubilin P1297L mutation associated with hereditary megaloblastic anemia 1 causes impaired recognition of intrinsic factor-vitamin B12 by cubilin. Blood 96, 405–409 (2000)

    CAS  PubMed  Google Scholar 

  10. 10

    Aminoff, M. et al. Mutations in CUBN, encoding the intrinsic factor-vitamin B12 receptor, cubilin, cause hereditary megaloblastic anaemia 1. Nature Genet. 21, 309–313 (1999)

    CAS  Article  Google Scholar 

  11. 11

    Lindblom, A. et al. The intrinsic factor-vitamin B12 receptor, cubilin, is assembled into trimers via a coiled-coil α-helix. J. Biol. Chem. 274, 6374–6380 (1999)

    CAS  Article  Google Scholar 

  12. 12

    Blanc, G. et al. Insights into how CUB domains can exert specific functions while sharing a common fold: conserved and specific features of the CUB1 domain contribute to the molecular basis of procollagen C-proteinase enhancer-1 activity. J. Biol. Chem. 282, 16924–16933 (2007)

    CAS  Article  Google Scholar 

  13. 13

    Gregory, L. A., Thielens, N. M., Arlaud, G. J., Fontecilla-Camps, J. C. & Gaboriaud, C. X-ray structure of the Ca2+-binding interaction domain of C1s. Insights into the assembly of the C1 complex of complement. J. Biol. Chem. 278, 32157–32164 (2003)

    CAS  Article  Google Scholar 

  14. 14

    Teillet, F. et al. Crystal structure of the CUB1-EGF-CUB2 domain of human MASP-1/3 and identification of its interaction sites with mannan-binding lectin and ficolins. J. Biol. Chem. 283, 25715–25724 (2008)

    CAS  Article  Google Scholar 

  15. 15

    Bally, I. et al. Identification of the C1q binding sites of human C1r and C1s. A refined three-dimensional model of the C1 complex of complement. J. Biol. Chem. 284, 19340–19348 (2009)

    CAS  Article  Google Scholar 

  16. 16

    Kristiansen, M. et al. Identification of the haemoglobin scavenger receptor. Nature 409, 198–201 (2001)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Blacklow, S. C. Versatility in ligand recognition by LDL receptor family proteins: advances and frontiers. Curr. Opin. Struct. Biol. 17, 419–426 (2007)

    CAS  Article  Google Scholar 

  18. 18

    Quadros, E. V., Nakayama, Y. & Sequeira, J. M. The protein and the gene encoding the receptor for the cellular uptake of transcobalamin-bound cobalamin. Blood 113, 186–192 (2009)

    CAS  Article  Google Scholar 

  19. 19

    Fisher, C., Beglova, N. & Blacklow, S. C. Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors. Mol. Cell 22, 277–283 (2006)

    CAS  Article  Google Scholar 

  20. 20

    Gordon, M. M., Hu, C., Chokshi, H., Hewitt, J. E. & Alpers, D. H. Glycosylation is not required for ligand or receptor binding by expressed rat intrinsic factor. Am. J. Physiol. 260, G736–G742 (1991)

    CAS  PubMed  Google Scholar 

  21. 21

    Gregory, L. A. et al. The X-ray structure of human mannan-binding lectin-associated protein 19 (MAp19) and its interaction site with mannan-binding lectin and L-ficolin. J. Biol. Chem. 279, 29391–29397 (2004)

    CAS  Article  Google Scholar 

  22. 22

    Appleton, B. A. et al. Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding. EMBO J. 26, 4902–4912 (2007)

    CAS  Article  Google Scholar 

  23. 23

    Bates, P. A., Kelley, L. A., MacCallum, R. M. & Sternberg, M. J. Enhancement of protein modeling by human intervention in applying the automatic programs 3D-JIGSAW and 3D-PSSM. Proteins 45, 39–46 (2001)

    Article  Google Scholar 

  24. 24

    Kozyraki, R. et al. The human intrinsic factor-vitamin B12 receptor, cubilin: molecular characterization and chromosomal mapping of the gene to 10p within the autosomal recessive megaloblastic anemia (MGA1) region. Blood 91, 3593–3600 (1998)

    CAS  PubMed  Google Scholar 

  25. 25

    Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993)

    CAS  Article  Google Scholar 

  26. 26

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    CAS  Article  Google Scholar 

  27. 27

    Kleywegt, G. J. & Jones, T. A. xdlMAPMAN and xdlDATAMAN – programs for reformatting, analysis and manipulation of biomacromolecular electron-density maps and reflection data sets. Acta Crystallogr. D 52, 826–828 (1996)

    CAS  Article  Google Scholar 

  28. 28

    Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  29. 29

    Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  30. 30

    Kleywegt, G. J. & Jones, T. A. Halloween... Masks and Boneshttp://xray.bmc.uu.se/gerard/papers/halloween.html〉.

  31. 31

    Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007)

    CAS  Article  Google Scholar 

  32. 32

    Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993)

    CAS  Article  Google Scholar 

  33. 33

    DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific) 〈http://www.pymol.org/〉 (2002)

    Google Scholar 

  34. 34

    Bond, C. S. & Schuttelkopf, A. W. ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr. D 65, 510–512 (2009)

    CAS  Article  Google Scholar 

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Acknowledgements

We are grateful to G. Biller, G. Ratz and G. Hartvigsen for technical assistance, the staff at MAX-lab and SLS beamlines for help with data collection, and S. Fedosov for advice on expression. The study was essentially supported by a grant to S.K.M. from the Lundbeck foundation. G.R.A. was supported by the Danish Science Research Council and a Hallas-Møller stipend from the Novo-Nordisk foundation. M.M. was supported by the Novo Nordisk Foundation.

Author Contributions C.B.F.A.: cloning, expression, purification, crystallization, data collection, structure determination and analysis, manuscript preparation; M.M.: cloning, expression, purification, size-exclusion chromatographic analyses, manuscript preparation; T.S.: cloning, mutagenesis, expression, size-exclusion chromatographic analyses, cross-linkage experiments; G.R.A. and S.K.M.: study design and manuscript preparation.

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Correspondence to Søren K. Moestrup.

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This file contains Supplementary Figures S1-13 with legends, Supplementary Tables 1-4 and Supplementary References. (PDF 8731 kb)

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Andersen, C., Madsen, M., Storm, T. et al. Structural basis for receptor recognition of vitamin-B12–intrinsic factor complexes. Nature 464, 445–448 (2010). https://doi.org/10.1038/nature08874

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