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.
Subject(s)
Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Amino Acid Sequence , Humans , Intercellular Signaling Peptides and Proteins/genetics , Low Density Lipoprotein Receptor-Related Protein-6/genetics , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Protein Binding , Protein Domains , Sequence Alignment , Wnt Signaling PathwayABSTRACT
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 (15)N, (13)C and (1)H 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 ß-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.
Subject(s)
Disulfides , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Wnt Proteins/antagonists & inhibitors , Humans , Protein Structure, SecondaryABSTRACT
The asymmetric unit of the title salt, C(28)H(28)O(3)P(+)·Cl(-)·H(2)O, contains a benzyl-triphenyl-phospho-nium cation, a chloride counter-ion, and a water mol-ecule of crystallization. The 3,4,5-trimeth-oxy substituents of the benzylic functionality are arranged with the 3,5-methyl groups lying approximately in the aromatic ring plane while the 4-methyl group is out of the plane.
