title: Conformational Freedom of the LRP6 Ectodomain Is Regulated by N-glycosylation and the Binding of the Wnt Antagonist Dkk1.
abstract: LDL-receptor-related protein 6 (LRP6) is a single-pass membrane glycoprotein with a large modular ectodomain and forms a higher order signaling platform upon binding Wnt ligands on the cell surface. Although multiple crystal structures are available for fragments of the LRP6 ectodomain, we lack a consensus view on the overall molecular architecture of the full-length LRP6 and its dynamic aspects. Here, we used negative-stain electron microscopy to probe conformational states of the entire ectodomain of LRP6 in solution and found that the four-module ectodomain undergoes a large bending motion hinged at the junction between the second and the third modules. Importantly, the extent of inter-domain motion is modulated by evolutionarily conserved N-glycan chains proximal to the joint. We also found that the LRP6 ectodomain becomes highly compact upon complexation with the Wnt antagonist Dkk1, suggesting a potential role for the ectodomain conformational change in the regulation of receptor oligomerization and signaling.
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Matoba
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Mihara
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Tamura-Kawakami
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Miyazaki
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Maeda
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Hirai
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
authors.forename: Samuel
authors.initials: S
authors.lastname: Thompson
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Iwasaki
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka 565-0871, Japan. Electronic address: takagi@protein.osaka-u.ac.jp.
authors.forename: Junichi
authors.initials: J
authors.lastname: Takagi
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D020285:Cryoelectron Microscopy; D006031:Glycosylation; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D060488:Low Density Lipoprotein Receptor-Related Protein-6 / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D016624:Negative Staining; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D013329:Structure-Activity Relationship; D051153:Wnt Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: C501126:DKK1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D050505:Mutant Proteins; D011134:Polysaccharides; D051153:Wnt Proteins
keywords: Dkk1; LRP6; N-glycosylation; Wnt signal; conformational flexibility; negative-stain electron microscopy; receptor ectodomain; single particle analysis