Abstract
Dickkopf (Dkk) family proteins are important regulators of Wnt signaling pathways, which play key roles in many essential biological processes. Here, we report the first detailed structural and dynamics study of a full-length mature Dkk protein (Dkk4, residues 19–224), including determination of the first atomic-resolution structure for the N-terminal cysteine-rich domain (CRD1) conserved among Dkk proteins. We discovered that CRD1 has significant structural homology to the Dkk C-terminal cysteine-rich domain (CRD2), pointing to multiple gene duplication events during Dkk family evolution. We also show that Dkk4 consists of two independent folded domains (CRD1 and CRD2) joined by a highly flexible, nonstructured linker. Similarly, the N-terminal region preceding CRD1 and containing a highly conserved NXI(R/K) sequence motif was shown to be dynamic and highly flexible. We demonstrate that Dkk4 CRD2 mediates high-affinity binding to both the E1E2 region of low-density lipoprotein receptor–related protein 6 (LRP6 E1E2) and the Kremen1 (Krm1) extracellular domain. In contrast, the N-terminal region alone bound with only moderate affinity to LRP6 E1E2, consistent with binding via the conserved NXI(R/K) motif, but did not interact with Krm proteins. We also confirmed that Dkk and Krm family proteins function synergistically to inhibit Wnt signaling. Insights provided by our integrated structural, dynamics, interaction, and functional studies have allowed us to refine the model of synergistic regulation of Wnt signaling by Dkk proteins. Our results indicate the potential for the formation of a diverse range of ternary complexes comprising Dkk, Krm, and LRP5/6 proteins, allowing fine-tuning of Wnt-dependent signaling.
Highlights
Dickkopf (Dkk) family proteins are important regulators of Wnt signaling pathways, which play key roles in many essential biological processes
This study reported considerably higher affinity interactions for both full-length Dkk1 and Dkk2 binding to LRP6 E1 (KD values of 27 and 53 nM, respectively), which identifies LRP6 E1 as the high-affinity Dkk binding site within the E1E2 region of LRP6 and indicates that CRD1 and/or CRD2 is required for a tight interaction [11]
Backbone chemical shift perturbations induced by LRP6 E1E2 binding showed that a small number of signals from residues in both the flexible N terminus and CRD1 were slightly affected by LRP6 E1E2 binding, indicative of a weak transient interaction consistent with the KD determined for the isolated N-terminal region of Dkk4 (Figs. 4D and 5B)
Summary
Dickkopf; CRD, cysteine-rich domain; Krm, Kremen; REDAC, redundant dihedral angle constraints; CANDID, combined automated NOE assignment and structure determination protocol; ECD, extracellular domain; Krg, kringle; 2D and 3D, two- and three-dimensional, respectively; RMSD, root mean square deviation; PDB, Protein Data Bank; IGFBP-4, insulin-like growth factor-binding protein-4; ICK, inhibitor cysteine knot; BLI, biolayer interferometry; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; SEC, size-exclusion chromatography. We report the first detailed structural and dynamics study of a fulllength mature Dkk protein, as typified by Dkk (residues 19 –224), including the first atomic-resolution structure obtained for the CRD1 domain of a Dkk family protein. The detailed structural, dynamics, interaction, and functional data obtained for Dkk, together with reported work on Dkk and Dkk, have allowed the development of a highly informative proposed model for synergistic regulation of Wnt signaling by Dkk and Krm family proteins. This highlights the potential for very fine-tuning of the inhibition of Wnt signaling via a range of ternary LRP–Dkk–Krm complexes
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