Structural basis of SALM3 dimerization and synaptic adhesion complex formation with PTPσ

Abstract

Synaptic adhesion molecules play an important role in the formation, maintenance and refinement of neuronal connectivity. Recently, several leucine rich repeat (LRR) domain containing neuronal adhesion molecules have been characterized including netrin G-ligands, SLitRKs and the synaptic adhesion-like molecules (SALMs). Dysregulation of these adhesion molecules have been genetically and functionally linked to various neurological disorders. Here we investigated the molecular structure and mechanism of ligand interactions for the postsynaptic SALM3 adhesion protein with its presynaptic ligand, receptor protein tyrosine phosphatase σ (PTPσ). We solved the crystal structure of the dimerized LRR domain of SALM3, revealing the conserved structural features and mechanism of dimerization. Furthermore, we determined the complex structure of SALM3 with PTPσ using small angle X-ray scattering, revealing a 2:2 complex similar to that observed for SALM5. Solution studies unraveled additional flexibility for the complex structure, but validated the uniform mode of action for SALM3 and SALM5 to promote synapse formation. The relevance of the key interface residues was further confirmed by mutational analysis with cellular binding assays and artificial synapse formation assays. Collectively, our results suggest that SALM3 dimerization is a prerequisite for the SALM3-PTPσ complex to exert synaptogenic activity. Neurons form highly complex networks of specific connection patterns through the cellular junctions at synapses. Synaptic adhesion molecules are emerging synapse organizers, which are localized at the nerve cell membranes at the synaptic cleft, and play a crucial role in the formation, maintenance and refinement of neural circuits 1. Several families of synaptic adhesion molecules have been identified, including e.g. neurexins 2 , neuroligins 3 , synaptic adhesion like molecules (SALMs) 4 , leucine-rich repeat (LRR) transmembrane neuronal proteins (LRRTMs) 5 , leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) 6 and netrin-G ligands (NGLs) 7. The dysfunction of synaptic adhesion molecules have been proposed to be involved in onset and progression of various neurological disorders, which includes autism spectrum disorders, schizophrenia and Alzheimer's disease 8-11. The SALM family of proteins also known as LRR and fibronectin III domain containing proteins (LRFNs), represent a family of LRR-containing synaptic adhesion molecules, with five known members (SALM1-SALM5) 4. All the SALM proteins share a similar domain architecture consisting of an extracellular region with an LRR domain, an immunoglobulin (Ig) domain and a fibronectin III (FnIII) domain followed by a transmembrane domain (TM) and a non-structured cytoplasmic region 12. Notably, SALM1-3 contain type I PDZ binding motifs, which are absent in SALM4 and SALM5 4,12 (Fig. 1a). SALMs are considered to be postsynaptic adhesion proteins, mostly expressed in the neurons and regulate neurite outgrowth and branching 12. In addition, they ...

SALM4 negatively regulates NMDA receptor function and fear memory consolidation

“…Insight into possible mechanisms underlying these changes comes from a previous report that SALM4 associates with and inhibits SALM2-dependent excitatory synapse development 20 . SALM2 forms complexes in vivo with SALM1 and SALM3 in the brain 76 , consistent with a recent crystallographic study showing that SALMs can form homo-and heterodimeric complexes [31][32][33][34] . SALM2 also associates with both NMDARs and AMPARs to promote excitatory synapse development 23 , whereas SALM1 preferentially associates with and promotes dendritic clustering of NMDARs 22 .…”

“…Specifically, SALMs 1-3, but not SALM4 or SALM5, contain a C-terminal PDZ-binding motif that directly interacts with the PDZ domains of PSD-95, an excitatory scaffolding protein that is abundant in the postsynaptic density (PSD) 26,27 . SALM3 and SALM5, but not other SALMs, trans-synaptically interact with presynaptic LAR-family receptor tyrosine phosphatases (LAR-RPTPs) to promote excitatory and inhibitory synapse development [28][29][30] , as recently detailed by X-ray crystallographic studies [31][32][33][34] .…”

Crystallization and low-resolution structure solution of the SALM3–PTPσ synaptic adhesion complex

“…In this study, we report the expression, purification, crystallization and initial low-resolution crystal structure of the mouse SALM3-PTP complex, which verifies the formation of a 2:2 trans-heterotetrameric complex similar to the crystal structure of human SALM5-PTP and confirms the architecture of the previously reported model of the SALM3-PTP complex based on SAXS data (Lin et al, 2018;Karki et al, 2020).…”

“…In a previous study, we determined the structure of the complex of SALM3 with PTP using small-angle X-ray scattering (SAXS), revealing a 2:2 complex similar to the human SALM5-PTP and SALM2-PTP complexes. The relevance of the key interface residues between SALM3 and PTP was further confirmed by mutational analysis with cellular binding assays and artificial synapse-formation assays (Karki et al, 2020). However, the structural details of the SALM3-PTP complex remain to be resolved beyond the model based on the SAXS data.…”

“…LAR-PTPs further contain two tandem tyrosine phosphatase domains in the cytoplasmic region that are presumably involved in regulation of presynaptic signaling (Um & Ko, 2013;Han et al, 2016). The Ig domains of PTP and PTP are involved in interaction with the LRR and Ig domains of SALM3 and SALM5, and the interaction is enhanced in the presence of the LAR-PTP miniexon B (meB) (Lin et al, 2018;Choi et al, 2016;Karki et al, 2020).…”