Crystal structures of Lgr4 and its complex with R-spondin1

Abstract

The leucine-rich repeat-containing G-protein-coupled receptors (Lgrs) are a large membrane protein family mediating signaling events during development and in the adult organism. Type 2 Lgrs, including Lgr4, Lgr5, and Lgr6, play crucial roles in embryonic development and in several cancers. They also regulate adult stem cell maintenance via direct association with proteins in the Wnt signaling pathways, including Lrp5/6 and frizzled receptors. The R-spondins (Rspo) were recently identified as functional ligands for type 2 Lgrs and were shown to synergize with both canonical and noncanonical Wnt signaling pathways. We determined and report the structure of the Lgr4 ectodomain alone and bound to Rspo1. The structures reveal an extended horseshoe leucine-rich repeat (LRR) receptor architecture that binds, with its concave side, the ligand furin-like repeats via an intimate interface. The molecular details of ligand/receptor recognition provide insight into receptor activation and could serve as template for stem-cell-based regenerative therapeutics development.

Figure 1

Structure of unliganded Lgr4. A. Two orthogonal views of the structure of the unbound Lgr4 ectodomain colored in green. The sugar moieties at the glycosylation sites are drawn as grey spheres. The N and C termini, as well as the individual LRR repeats, are labeled. LRRNT is the N-terminal LRR capping region. B. Two orthogonal views of the Lgr4 dimer (one molecule is colored in green, and the other, in cyan) observed in the crystals. The Lgr4 ectodomain is dimeric in solution as judged by gel filtration and dynamic light scattering.

Figure 2

Structure of the Lgr4/R-spondin complex. A. Two orthogonal views of the ligand/receptor complex. Lgr4 is colored in blue and R-spondin, in magenta. The inserts are superimpositions of ligand-bound (blue) and unliganded (green) Lgr4. Secondary structure elements in R-spondin are labeled. B. The Lgr4/R-spondin complex with either Lgr4 (Left Panel) or R-spondin complex (Right Panel) are drawn as solvent accessible surfaces and color-coded by electrostatic surface potential. This panel illustrates the complementary-charged surfaces that constitute the Lgr4/R-spondin interface.

Figure 3

The Lgr4/R-spondin interface. Two orthogonal views of the binding interface between Lgr4 (colored in blue) and R-spondin (colored in magenta). Interacting residues are drawn as stick figures and labeled. Secondary structure elements in R-spondin are also labeled. The insert in the right panel is a zoom-in of the hydrophobic “clamp” region of the Lgr4/R-spondin interface, with the R-spondin solvent accessible surface drawn in magenta.