Crystal Structure of the Cysteine-Rich Domain of Mouse Frizzled 8 (PDB ID: 1IJY) from Mus musculus

Created by: Michelle Joung

Crystal Structure of the Cysteine-Rich Domain of Mouse Frizzled 8 (PDB ID: 1IJY) from Mus musculusis a receptor for Wnt signaling protein. Cysteine-rich domain of mouse frizzled 8 has a mutation with the elimination of putative N-linked glycosylation sites (5). Wnt-Frizzled signaling pathway provides significant prospects for the treatment of disorders of the nervous system (7). Inappropriate dysregulation of Wnt signaling causes developmental defects and is a key feature of cancer.

 The activity of cysteine rich domain of mouse frizzled 8 is dependent on its structure. Cysteine-Rich domain of Mouse Frizzled 8 has molecular weight of 14928.06 Da and isoelectric point of 4.85 (4).Primary structure of protein is made of 2 identical chains, each consisting of 130 residues.The primary structure of the chain starts with Glycine at the N terminus and ends with aspartic acid at the C terminus (1). Cysteine rich domains of mouse frizzled 8 contains two domains: the N-terminal β-domain composed of cluster of alpha-helices with 10 of the conserved cysteine residues forming five disulfide bridges and C-terminal domain composed of 2 β-strands maintained by six disulfide bridges (5). The Frizzled proteins contain seven hydrophobic domains that are predicted to form transmembrane α-helices (8). The enzyme has 40% alpha helix and 5% beta sheet.Cysteine rich domain of mouse frizzled 8 contains 8 alpha helices made out of 52 residues and 5 beta sheet strands made out of 7 residues.Three of its alpha helices are 3/10 helices (1). Secondary structures of each domain fold inward to form a tertiary structure to provide structural stability. The 2 dimers of Cysteine-rich domain of mouse Frizzled 8 dimerize to form a quaternary structure that allows for the specificity of Wnt signaling (5).          

Cysteine-rich domain of Mouse Frizzled 8 is crystallized using the hanging drop vapor diffusion method under the conditions of using 0.1 M HEPES and 1.9 M ammonium sulfate at pH 7.12% PEG 3350 at 293K (5). In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor diffuses out of the drop until the osmolarity of the drop and the precipitant are equal. The final crystallized structure is missing roughly 30 residues on N-terminal and 500 residues on C-terminal (1).

Cysteine-rich domain of mouse frizzled 8has disulfide bond between cysteine Cys-3 and Cys-62, Cys-11 and Cys-57, Cys-48 and Cys-80, Cys-76 and Cys-115, and Cys-80 and Cys-105 (5). The disulphide bonds between residues help to provide structural stability. The substitution of alanine residues 117 and 118 in the Cysteine-rich domain allows WNT binding. In contrast, deletion of alanine residues 114-120 in mFz8 CRD eliminates WNT binding ability.The residues 114-120 include proline, threonine, asparagine, aspartic acid, leucine and glutamine and act as necessary residue components for efficient Wnt binding (5). Several interdomain hydrogen bonds serve to stabilize the interactions of the two domains (6). Cysteine-rich domain of mouse frizzled 8 does not have any metal ions or ligand molecules (1).  In Wnt binding, Wnt molecule’s Lys-182 forms a salt bridge with the Frizzled 8 Glu-64 and a hydrogen bond with Frizzled Asn-58 (7).

 Cysteine rich-domain of mouse frizzled 8 plays a key role in Wnt signaling pathway. Wnt protein binds to either the Frizzled receptor or a receptor complex consisting of Frizzled and co-receptor of LRP5/6 to activate different signaling cascades that include Wnt signaling pathway or the Wnt/Ca²⁺ pathway (7). Signal transduction by major Wnt signaling pathways is regulated by the interaction of Frizzled domain with the cytoplasmic protein dishevelled (8). In the canonical pathway, β-catenin-dependent signaling is mediated through the cytoplasmic destruction complex (7).  Binding of Wnt activates Frizzled receptor, permitting dishevelled binding and resulting in the stabilization of the destruction complex and the accumulation of non-phosphorylated β-catenin, which then translocate to the nucleus and binds and activates T cell factor/lymphoid enhancer-binding factor transcription factors on the promoter of target genes. Activation and deactivation of Wnt signaling pathway regulates embryonic development, cell proliferation, cell migration, and tissue regeneration. In the absence of Wnt stimulation, the destruction complex is destabilized and β-catenin by CKα and GSK3 is phosphorylated to be degraded, which results in repression of target genes in processes like embryonic development (8).  

The Wnt/Ca²⁺ pathway is activated through Wnt ligands binding to Frizzled receptors, resulting in an increase in an intracellular calcium concentration that activate both calmodulin-dependent protein kinases, which subsequently activate transcription factors NFκB and cAMP-response element-binding protein and cytosolic phosphatase calcineurin (8).  Activated Calcineurin results in dephosphorylation and activation of nuclear factor of activated T cells that lead to the transcription of genes in cardiomyocytes, neuronal cells, and skeletal muscle (7).

The class Frizzled GPCR family, which has an extracellular cysteine-rich domain , consists of smoothened receptor and frizzled receptor (10). Cysteine-rich domain of mouse frizzled 8 is a member of the Frizzled family of seven-pass transmembrane protein with a cyclic symmetry (1)Human smoothened receptor and cysteine-rich domain of mouse frizzled 8 havestructural similarity. Structural similarities between proteins can be analyzed using Blast and Dali server. BLAST is a tool for searching protein and DNA databases for sequence similarities. It finds proteins with similar primary structures to a protein query. The E score from BLAST below 0.05 suggests high primary structure similarity. Smoothened receptor family has 35.94% primary structure similarity with Cysteine-rich domain of mouse frizzled 8, as shown by protein Blast (E= 1e-41) (2).  Dali server compares tertiary structures of proteins and calculates the differences in intramolecular distances. Z score above 2 from Dali server supports that proteins have similar folds. As with all the members of frizzled protein GPCR family, smoothened receptor in complex with TC114 has the same overall tertiary structure as cysteine-rich domain of mouse frizzled 8, as shown by DALI (Z= 10.2) (3). Unlike Cysteine-rich domain of mouse frizzled 8, smoothened receptor has several ligand molecules such Flavin mononucleotide, 2,3-dihydroxypropyl (9Z)-octadec-9-enoate (11) . 

Multi-domain Human smoothened receptor in complex with TC114 from Homo sapiens (pdb ID: 5V57), is a smoothened receptor that also belongs to the class Frizzled of the G protein-coupled receptor superfamily, constituting a key component of the Hedgehog signaling pathway (11).Both Cysteine-rich domain of mouse frizzled 8 and Human smoothened receptor are homo 2-mer and have only 1 unique protein chain (1).  Both  proteins are involved in activation of signaling pathways.Human smoothened receptors have a seven-transmembrane helices domain, a hinge domain and an intact extracellular cysteine-rich domain and is asymmetric (9).  Smoothened receptor's chain consists of 648 amino acid residues. Smoothened receptoris 51% helical with 26 helices made of 335 residues and 10% beta sheet made of 25 strands made of 71 residues (1).

Smoothened receptor is the key receptor in mediating the Hedgehog signaling pathway. The Hedgehog signaling pathway plays a significant role in the normal embryonic development and homeostasis of invertebrates and vertebrates’ pathway of signal transmission from the cell membrane to the nucleus (10). All Hh ligand molecules in pathways are synthesized as precursor proteins that undergo autocatalytic cleavage and cholesterol modification at the carboxy terminus and palmitoylation at the amino terminus (13). Smoothened receptor has a variety of ligand molecules. Three of the ligand molecules are2,3-dihydroxypropyl (9Z)-octadec-9-enoate, flavin mononucleotide andN-methyl-N-[1-[4-(2-methylpyrazol-3-yl)phthalazin-1-yl]piperidin-4-yl]--nitro-2-(trifluoromethyl)benzamide.Binding of a cholesterol to cysteine rich domain of smoothed receptor activates smoothed receptor that leads to translocation of the transcription factor into the nucleus to activate target genes after Hh ligand molecules bind to smoothened receptor (11).  The mechanism of interaction between cholesterol and cysteine rich domain of smoothed receptor activates smoothened receptor is unknown, but interaction shows that activation and deactivation of smoothened receptor help to regulate embryonic development and homeostasis (13).  

Cysteine-rich domain of mouse frizzled 8 is a key component of Wnt pathway that can help prevent errors in activation in Wnt pathway that can lead to development of disorders such as Alzheimer, multiple sclerosis, polycystic kidney disease, type 2 diabetes, lung cancer and breast cancer (12). Similar structures that belong in a frizzled receptor family also help to regulate signaling pathways in different metabolic processes. Although no current evidence implicates dimerization of Wnt or Frizzled proteins, the dimer interface in the crystals suggests that dimerization may be of biological significance (5). Future research on dimerization can be done to test the biochemical significance of dimerization of domains in Wnt signaling pathway.