Structure of Stem Cell Growth Factor R-spondin 1 in Complex with the Ectodomain of Its Receptor LGR5

Abstract

Leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-LGR6) are receptors for R-spondins, potent Wnt agonists that exert profound trophic effects on Wnt-driven stem cells compartments. We present crystal structures of a signaling-competent fragment of R-spondin 1 (Rspo1) at a resolution of 2.0 Å and its complex with the LGR5 ectodomain at a resolution of 3.2 Å. Ecto-LGR5 binds Rspo1 at its concave leucine-rich-repeat (LRR) surface, forming a dimeric 2:2 complex. Fully conserved residues on LGR4-LGR6 explain promiscuous binding of R-spondins. A phenylalanine clamp formed by Rspo1 Phe106 and Phe110 pinches Ala190 of LGR5 and is critical for binding. Mutations related to congenital anonychia reduce signaling, but not binding of Rspo1 to LGR5. Furthermore, antibody binding to the extended loop of the C-terminal LRR cap of LGR5 activates signaling in a ligand-independent manner. Thus, our data reveal binding of R-spondins to conserved sites on LGR4-LGR6 and, in analogy to FSHR and related receptors, suggest a direct signaling role for LGR4-LGR6 in addition to its formation of Wnt receptor and coreceptor complexes.

Intrinsic disorder in spondins and some of their interacting partners

“…59 However, binding to the LGR5 induced noticeable changes in the Fu1/Fu2 region, especially in its Ru2 part. Fig.…”

“…36,39 Structural information is available for the Fu1/Fu2-containing region of Rspo1 (residues 40-127; e.g., see PDB ID: 4BSP). 59 Although longer regions of Rspo1 are typically used in crystallization studies (residues , no coordinates have been determined for their N-(residues 31-39) and C-terminal regions (residues [128][129][130][131][132][133][134][135][136][137][138][139][140][141][142][143][144][145][146]. Recent structural analysis revealed that in its unbound form, the Fu1/Fu2 region of Rspo1 is characterized by the non-equivalent Fu domains, where the Fu1 domain has 3 cysteineknotted b-hairpins, whereas there are 2 such hairpins in the Fu2 domain, with the C-terminal hairpin being missing from the crystal structure, likely due to the high conformational flexibility.…”

“…Recent structural analysis revealed that in its unbound form, the Fu1/Fu2 region of Rspo1 is characterized by the non-equivalent Fu domains, where the Fu1 domain has 3 cysteineknotted b-hairpins, whereas there are 2 such hairpins in the Fu2 domain, with the C-terminal hairpin being missing from the crystal structure, likely due to the high conformational flexibility. 59 Unbound Fu1/Fu2 region is organized in such a way that the sets of Fu1 and Fu2 b-hairpins are oriented at »90…”

“…Furthermore, the Cterminal part of the Fu1/Fu2 region became visible upon its binding to LGR-5, indicating dramatic binding-induced reduction in the conformational flexibility of this region. 59 Figure 2 represents the results of intrinsic disorder analysis in human Rspo1 (UniProt ID: Q2MKA7) and shows that this protein is expected to have several functional disordered regions. For example, a linker connecting signaling peptide to the rest of Rspo1 is disordered, likely to facilitate the accessibility of site of a proteolytic attack leading to the release of the signaling peptide and generating mature Rspo1 (residues 21-263).…”

“…63 In our analysis, the most stringent criteria were used for selection of interacting proteins by choosing the highest cut-off of 0.9 as the minimal required confidence level. According to these criteria, human Rspo1 interacts with other members of the R-spondin family (Rspo2, Rspo3, and Rspo4), leucinerich repeat containing G protein-coupled receptors 4, 5, and 6 (LGR4, LGR5, and LGR6), low density lipoprotein receptor-related protein 6 (LRP6), zinc and ring finger 3 (ZnRF3), ring finger protein 43 (RNF43), 59 Results of multiple structure alignment are shown in the middle of the plot, whereas individual chains are located around. To show location of each individual chain within the aligned structural ensemble, the remaining structures are made transparent.…”