Extracellular architecture of the SYG-1/SYG-2 adhesion complex instructs synaptogenesis

Abstract

SYG-1 and SYG-2 are multipurpose cell adhesion molecules (CAMs) that have evolved across all major animal taxa to participate in diverse physiological functions, ranging from synapse formation to formation of the kidney filtration barrier. In the crystal structures of several SYG-1 and SYG-2 orthologs and their complexes, we find that SYG-1 orthologs homodimerize through a common, bispecific interface that similarly mediates an unusual orthogonal docking geometry in the heterophilic SYG-1/SYG-2 complex. C. elegans SYG-1's specification of proper synapse formation in vivo closely correlates with the heterophilic complex affinity, which appears to be tuned for optimal function. Furthermore, replacement of the interacting domains of SYG-1 and SYG-2 with those from CAM complexes that assume alternative docking geometries or the introduction of segmental flexibility compromised synaptic function. These results suggest that SYG extracellular complexes do not simply act as "molecular velcro" and that their distinct structural features are important in instructing synaptogenesis. PAPERFLICK:

Figure 1. Structures of SYG-1 and homodimeric SYG-1-like complexes

A. Schematic representation of the domain structures of SYG-1 and SYG-2. All domains are of the Ig type except for the last domain of SYG-2, which is from the related FnIII domain family. Also noted are the Drosophila melanogaster(d) and mammalian (m)orthologs. B. The crystal structure of C. elegans SYG-1 domains 1 and 2 (D1 and D2), in light and dark green, respectively. N-linked glycosylation is depicted in sticks representation. C. The homodimeric structure of Rst D1-D2, demonstrating the near-orthogonal approach of the monomers. D. Overlay of structures solved of Drosophila and mouse SYG-1-like proteins. The close match between the homodimeric structures of Rst (red and orange), Duf/Kirre (yellow), and Neph1 (purple) demonstrate that the crystallographicallyobserved homodimers are conserved and physiological. E. Close-up of the symmetrical Rsthomodimer interface. The 2-fold sign (closed oval) represents the homodimersymmetry axis. The prime sign is added to residue labels for the Rstmonomer displayed in red. F.The Extracellular Interactome Assay (Özkan et al., 2013) for wild-type Rst and mutants against wild-type Rst, Duf, Hbs, and SNS. The assay was performed in both orientations, as wild-type Rst, Duf, Hbs and SNS as bait (above), and as prey (below). The scale, colored as white to blue, represents absorbance values at 650 nm as the assay outcome. See also Figure S1 and Table S1.

Figure 2. Structure of the SYG-1/SYG-2 heterophilic complex

A-B. Two different views of the crystal structure of the complex of SYG-1 (green) and SYG-2 (blue), in which individual Igdomains are labeled in different shades of the respective colors. N-linked glycosylation is represented as sticks. See Table S2 for crystallography statistics. C. Close-up view of the SYG-1/SYG-2 heterophilic interface. Prime signedresidue labels belong to SYG-2 residues. D. Bindingisotherms for the interactions of wild type and mutantSYG-1 with SYG-2 as measured by SPR. See Figure S2 for SPR data for Drosophila SYGs.

Figure 3. Comparison of homophilic and heterophilicSYG-likecomplexes

A.D1s in four complex structures are overlayed to demonstrate the conservation between the homophilic and heterophilic binding modes. B. Surface representation of the interaction footprint (black outline) in the homodimericRst complex. The outline includes residues within 4 Å of the other Rst monomer.Cyan, orange and red represent increasing loss of binding as observed in Figure 1F upon mutagenesis of the labeled residues to alanine. C. Surface representation of the interaction footprint (black outline) of the SYG-1/SYG-2 complex on SYG-1. Within the black outline, blue to red coloring indicates increased loss of binding upon mutagenesis, as measured in Figure 2D, but converted to change in free energy. D. Sequence alignment of first domains (D1) of SYG-1-like and SYG-2-like proteins from the nematodes C. elegans and Brugiamalayi, fruit fly (D. melanogaster), zebrafish(D. rerio), frog (X. laevis), mouse and human. The sequence numbering is for the C. elegans SYG-1. The red, green and blue boxes above the sequences represent residues of Rst, C. elegans SYG-1 and C. elegans SYG-2 that are within 4 Å of their interaction partners. See also Figure S3.

Figure 4. SYG-1 and SYG-2 exist in extended conformations

A. Overlay of five SYG-1 andSYG-1/SYG-2-like complexes solved. The overlay demonstrates that there are only minor movements (“swings”) between the domains. B-D.Electron microscopy of negatively stained SYG-1 and SYG-2. The side length of the individual panels is 25 nm in (B) and 50 nm in (C) and (D). B.Selected class averages of the five-domain ectodomain of Syg-1. All class averages are shown in Figure S4A. C.Selected class averages of the ectodomain of SYG-2. All class averages are shown in Figure S4B. D. Raw particle images of SYG-1/SYG-2 complexes (top), schematic drawings (middle), and the schematic drawings overlaid with the crystal structure of SYG-1-D1-D2/SYG-2-D1-D4 (bottom). See also Figure S4C-G.

Figure 5. Affinity of the SYG-1/SYG-2 complex correlates with synaptic vesicle defects at the HSNL neuron

A-B. Schematic representation of HSNL synapses at the vulva in wild-type (A) and syg-1 worms (B). The dashed box shows wild-type synaptic region. C. Wild-type worms make synapses only at the primary synaptic region at the vulva (within the box). D, E.syg-1 animals show ectopic anterior synaptic vesicles. This is rescued when wild-type syg-1 is expressed in HSN. F. SYG-1 D58A, a mutant with moderate loss of SYG-1 affinity, partially rescues the syg-1 mutant synaptic vesicle phenotype. G, H. SYG-1 F60A and the quadruple mutant, neither of which have appreciable affinity for SYG-2, do not rescue the syg-1 phenotype. I. Correlation between affinities of SYG-1 mutants and the syg-1 phenotype. The syg-1 synaptic vesicle phenotype has been measured as both a fluorescence score, a quantitation of ectopic anterior vesicles over ~10 animals, and as a phenotype penetrance score, an all (1), partial (0.5), or none (0) scoring of the synaptic vesicle phenotype in >100 animals. These are compared against loss of binding energy upon the indicated mutations on SYG-1, and show very high correlations to the fluorescence score (R² = 0.89, blue dashed line) and to the phenotype penetrance (R² = 0.88, red dashed line). See Figure S5 for SYG-1 clustering at the vulva.

Figure 6. SYG-1 and SYG-2 D1s can be replaced with orthologous domains to partially rescue the syg-1;syg-2 double mutant defects

A. Schematic representation of HSNL synapses at the vulva in wild-type worms, in which SYG-2 is expressed in primary vulval epithelial cells. B. Schematic representation of HSNL synapses at the vulva in syg-1;syg-2 double mutant worms co-injected with syg-1 under the control of unc-86 promoter, and syg-2 under the control of egl-17 promoter. Since egl-17 promoter drives syg-2 expression in secondary vulval epithelial cells, a wider region for synaptic vesicle clustering is observed. C.syg-1;syg-2 worms show synaptic vesicles in the ectopic anterior region. The dashed yellow line denotes the extent of the secondary cells. The bracket highlights ectopic clustering of SNB-1 in the anterior axon. D. Co-injection of syg-1;syg-2 animalswith Punc86::syg-1 and Pegl-17::syg-2 results in clustering of synaptic vesicles around the vulva, as explained in (B).Injection of syg-1 alone fails to rescue the synapses in the syg-1;syg-2 mutant (Figure 6A). E-F. Co-injection of syg-1 whereits D1 is replaced with D1 of Rst and syg-2 where its D1 is replaced with D1 of SNS rescues synaptic defects in some animals (F), but not in others (E). Rescue in (F) resembles that in (D). See also Figure S6.

Figure 7. Rescue efficiency of syg-1;syg-2 double mutant defects depends on the approach geometry and rigidity of the interacting ectodomains

A. Comparison of SYG-1/SYG-2 with known structures of Ig-CAM hetero-complexes, all mediated through D1 domains. The structures are ordered from left to right in terms of decreasing similarity to SYG-1/SYG-2 with regards to the approach geometry, where the mouse JAML/CAR complex is most similar,and the CD47/Sirpα complex is most different to the SYG-1/SYG-2 complex. B. A guide to affinities between the studied complexes as dissociation constants (in μM). C. Quantitation of rescue (as phenotype scores) of syg-1;syg-2 worms when D1s are replaced by D1 domains from indicated proteins. ***p< 0.001; **** p< 0.0001;n.s., not significant.CAR/JAML D1s can partially rescue syg-1;syg-2, but the geometrically different CD47/Sirpα cannot. Lack of CD47/Sirpα can be, however, ameliorated when an extremely high-affinity variant of Sirpα (FD6) is used. Also included is rescue with SYG-1 and SYG-2 modified with flexible interdomain linkers (SYG-1-Flex/SYG-2-Flex), which is significantly diminished compared to rigid WT SYG-1/SYG-2. D.Representative images of the localization of SYG-1 chimeras and the flexible SYG-1 variant. For chimeras, SYG-1 D1 domains were replaced with those from other Ig domains involved in Ig-CAM interactions. SYG-1 constructs have been tagged with a C-terminal GFP and expressed in syg-1 syg-2 double mutant background together with the corresponding untagged SYG-2 chimera binding partner in the secondary vulva epithelial cells. (D1) Enrichment of WT SYG-1::GFP to the axonal regions in contact with SYG-2 expressing secondary vulva epithelial cell. The axon segment anterior to the synaptic region is devoid of SYG-1::GFP staining as denoted by yellow arrow. (D2) SYG-1::GFP expression alone without SYG-2 is diffusely localized along the entire axon. (D3) mCAR-SYG-1::GFP and mJAML-SYG-2 which has similar approach geometry as SYG-1 and SYG-2 shows proper localization and enrichment suggestive of binding. (D4) CD47-SYG-1::GFP and Sirpa-SYG-2 with dissimilar approach geometry fails to localize and is found diffused along the entire axon. (D5) Sirpa-FD6-SYG-2 which has very high affinity for CD47::GFP results in the subcellular enrichment of CD47::GFP. (D6) Flexible SYG-1 (SYG-1-Flex::GFP) is found diffused along the entire axon, indicative of proper expression and targeting to the membrane, but is not enriched where SYG-2-Flex is expressed. E. Suggested cellular adhesion model involving SYG-1 (green), SYG-2 (blue).