Abstract
A number of proteins have been shown to modulate canonical Wnt signalling at the cell surface, including members of the Dickkopf (Dkk) family (Baron and Rawadi in J Endocrinol 148:2635–2643, 2007; Cruciat and Niehrs in Cold Spring Harb Perspect Biol 5:a015081, 2013). The Dkk family includes four secreted proteins (Dkk1-4), which are characterised by two highly conserved cysteine-rich regions corresponding to C24–C73 and C128–C201 in human Dkk4 (hDkk4). Here we report essentially complete backbone and comprehensive side chain 15N, 13C and 1H NMR assignments for full length mature hDkk4 (M1–L207) containing a short C-terminal hexa-histidine tag (E208–H222). Analysis of the backbone chemical shift data obtained indicates that there is a very limited amount of regular secondary structure, with only small stretches of β-strand identified in both cysteine-rich regions. The N-terminal region of hDkk4 (M1–G21) and the relatively long linker between the two cysteine-rich regions (E77–Q123) appear to be unstructured and relatively mobile.
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References
Ahn VE, Chu ML, Choi HJ, Tran D, Abo A, Weis WI (2011) Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6. Dev Cell 21:862–873
Baron R, Rawadi G (2007) Targeting the Wnt/beta catenin pathway to regulate bone formation. J Endocrinol 148:2635–2643
Bazan JF, Janda CY, Garcia KC (2012) Structural architecture of functional evolution of Wnts. Dev Cell 23:227–232
Brott BK, Sokol SY (2002) Regulation of Wnt/LRP signaling by distinct domains of Dickkopf proteins. Mol Cell Biol 22:6100–6110
Cavanagh J, Fairbrother WJ, Palmer AG, Skelton NJ, Rance M (2006) Protein NMR spectroscopy: principles and practice, 2nd edn. Elsevier Academic Press, Amsterdam
Chen S, Bubeck D, MacDonald BT, Liang WX, Mao JH, Malinauskas T, Llorca O, Aricescu AR, Siebold C, He X, Jones EY (2011) Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling. Dev Cell 21:848–861
Cheng Z, Biechele T, Wei Z, Morrone S, Moon RT, Wang L, Xu W (2011) Crystal structures of the extracellular domain of LRP6 and its complex with DKK1. Nat Struct Mol Biol 18:1204–1210
Clevers H, Nusse R (2012) Wnt/beta-catenin signaling and disease. Cell 149:1192–1205
Cong F, Schweizer L, Varmus H (2004) Wnt signals across the plasma membrane to activate the beta-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP. Development 131:5103–5115
Cruciat CM, Niehrs C (2013) Secreted and transmembrane wnt inhibitors and activators. Cold Spring Harb Perspect Biol 5:a015081. doi:10.1101/cshperspect.a015081
Daniels DL, Weis WI (2005) Beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation. Nat Struct Mol Biol 12:364–371
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293
Holdsworth G, Slocombe P, Doyle C, Sweeney B, Veverka V, Le Riche K, Franklin RJ, Compson J, Brookings D, Turner J, Kennedy J, Garlish R, Shi J, Newnham L, McMillan D, Muzylak M, Carr MD, Henry AJ, Ceska T, Robinson MK (2012) Characterisation of the interaction of sclerostin with the low density lipoprotein receptor-related protein (LRP) family of Wnt co-receptors. J Biol Chem 287:26464–26477
Karasawa T, Yokokura H, Kitajewski J, Lombroso PJ (2002) Frizzled-9 is activated by Wnt-2 and functions in Wnt/beta-catenin signaling. J Biol Chem 277:37479–37486
Li L, Mao J, Sun L, Liu W, Wu D (2002) Second cysteine-rich domain of Dickkopf-2 activates canonical Wnt signaling pathway via LRP-6 independently of dishevelled. J Biol Chem 277:5977–5981
Logan CY, Nusse R (2004) The Wnt signaling pathway in development and disease. Ann Rev Cell Dev Biol 20:781–810
MacDonald BT, Tamai K, He X (2009) Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17:9–26
Mao B, Wu W, Li Y, Hoppe D, Stannek P, Glinka A, Niehrs C (2001) LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature 411:321–325
Metcalfe C, Bienz M (2011) Inhibition of GSK3 by Wnt signalling—two contrasting models. J Cell Sci 124:3537–3544
Niehrs C (2012) The complex world of WNT receptor signalling. Nat Rev Mol Cell Biol 13:767–779
Piotto M, Saudek V, Sklenar V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 2:661–665
Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44:213–223
Williamson RA, Natalia D, Gee CK, Murphy G, Carr MD, Freedman RB (1996) Chemically and conformationally authentic active domain of human tissue inhibitor of metalloproteinases-2 refolded from bacterial inclusion bodies. Eur J Biochem 241:476–483
Wishart DS, Sykes BD, Richards FM (1991) Relationship between nuclear magnetic resonance chemical shift and protein secondary structure. J Mol Biol 222:311–333
Wishart DS, Sykes BD, Richards FM (1992) The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry 31:1647–1651
Acknowledgments
Dr. Alice Barkell was supported by the award of a Ph.D. studentship from the Biotechnology and Biological Sciences Research Council in partnership with UCB. This work was also supported by a collaborative research grant from UCB to Prof. Mark Carr.
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Barkell, A.M., Holdsworth, G., Waters, L.C. et al. Resonance assignment and secondary structure determination of full length human Dickkopf 4 (hDkk4), a secreted, disulphide-rich Wnt inhibitor protein. Biomol NMR Assign 9, 147–151 (2015). https://doi.org/10.1007/s12104-014-9562-2
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DOI: https://doi.org/10.1007/s12104-014-9562-2