Biochemical Aspects Of The Interaction Between Neurexin And Neuroligin

The binding between the extracellular domains of neuroligin and P-neurexin is characterized by its dependence upon alternative splicing of both molecules, the glycosylation and dimerization of neuroligin, and the presence of Ca2+ ions. Intracellularly, both transmembrane proteins interact with different binding partners in the pre- and postsynaptic compartments, respectively.

3.1. Isoform-Dependent Binding

Neurexins form a large family of cell-surface proteins7,8. Each of the three vertebrate neurexin genes has two alternative promoters giving rise to a long mRNA transcript encoding a-neurexins, and a small mRNA transcript encoding P-neurexins. Both a- and P-neurexins consist of a conserved, intracellular C-terminus with a type II PDZ-recognition motif, a transmembrane region, a short serine-/threonine-rich sequence, and varying numbers of laminin-like (LNS) domains with interspersed EGF-like sequences. a-Neurexins contain six LNS repeats, whereas P-neurexins only have one9,10. There are at least six principal neurexin isoforms but due to extensive alternative splicing at five (a-neurexins) or two (P-neurexins) conserved splice sites (referred to as #1-5), over 1,000 variants in total may result7,8. a- and P-neurexins are neuron specific, but different neurons may express different combinations of neurexins8. Although they share most of their sequences, a-neurexins play an essential role in neurotransmission that cannot be replaced by P-neurexins11,12, and partly different extracellular binding partners exist for a-neurexins13-15 and P-neurexins13.

Neuroligins, in turn, consist of a short intracellular sequence ending in a type I PDZ-recognition motif, a transmembrane region, and a long extracellular sequence, including an a/P hydrolase domain. This latter domain is homologous to members of the esterase family (e.g., acetylcholine esterase, AChE), although it is catalytically inactive due to the lack of an active site serine1. Like the neurexins, neuroligin mRNA is susceptible to splicing (at two positions referred to as A and B), although it did not appear to affect binding to neurexins until a recent study identified a splice variant (lacking the insert in B) that binds indiscriminately to all P-neurexins and presumably all a-neurexins3. However, the abundance and distribution in brain of the newly investigated neuroligin isoform have yet to be explored before this surprise finding can be fully appreciated.

3.2. Glycosylation and Dimerization

Sequence analysis of both P-neurexin and neuroligin cDNA has highlighted glycosylation sites, including an O-linked carbohydrate-rich domain just N-terminal to the transmembrane regions of both, as well as multiple scattered consensus sequences for N-linked glycosylation1,9. Experimentally, neuroligin obtained from the cell lysate (preglycosylated form) had a size of 98 kDa, whereas protein obtained from the cell surface, had a larger apparent molecular weight of 126 kDa due to glycosylation16. Likewise, glycohydrolase treatment of P-neurexins revealed that they are extensively O-glycosylated, with only some N-glycosylation9. The function of their glycosylation is not fully understood yet, but deglycosylation of neuroligin increased its affinity for P-neurexin as measured by surface plasmon resonance, whereas deglycosylation of P-neurexin had no effect16.

Sedimentative equilibrium studies and analysis of the hydrodynamic properties of neuroligin by size exclusion chromatography have suggested that neuroligin forms dimers in solution16. This was supported by chemical cross-linking of purified recombinant neuroligin, resulting in covalently linked dimers and tetramers17. Further evidence for neuroligin dimerization included the presence of higher molecular weight species of neuroligin 1 following native gel electrophoresis. In addition, dimerization appears to be functionally necessary17.

3.3. Ca2-Binding Sites

From the initial purification of neuroligins by affinity chromatography using the extracellular domain of P-neurexin, it has been noted that Ca2+ is a necessary component for adhesion between P-neurexin and neuroligin1. Ca45 overlay blotting revealed that neuroligin is capable of binding Ca2+, but P-neurexin was not16. In addition, circular dichroism techniques demonstrated that incubation with Ca2+ did not cause any observable structural changes in P-neurexin, nor did it affect P-neurexin stability as assessed by temperature-dependent denaturation16. It appeared from these studies that neuroligin is the Ca2+ binding partner, supported by the identification of a degenerate EF-hand motif in neuroligins18.

We have summarized the available biochemical evidence in a structural model of the neuroligin/P-neurexin complex (Figure 7.1; Colorplate 6). Based on a crystal structure for P-neurexin19, and on homology modeling of neuroligin, this model sufficiently explains the steric hindrance between inserts in splice site #4 of neurexins and splice site B of neuroligins. In addition, the position of dimerization and glycosylation domains of neuroligin is consistent with experimental data. However, the exact whereabouts of the putative Ca2+-binding sites remain obscure because the positions proposed thus far appear very distant from the contact interface of both proteins18, and AChE folds may be too rigid for a calcium-induced conformational change.

3.4. Intracellular Binding Partners

In addition to the extracellular interaction between P-neurexin and neuroligin, their respective intracellular binding partners have also been investigated (see Figure 7.2 and Colorplate 7 for an overview model). All neuroligin isoforms are able to bind to a common postsynaptic density protein, PSD-956. PSD-95 is a postsynaptic scaffolding protein which employs PDZ domains to scaffold proteins together synaptic sites20,21. Using PSD-95 and neuroligin mutants it was established that the C-terminus of neuroligin binds to the third PDZ domain of PSD-956. The first two PDZ domains mediate PSD-95 interactions with the NR2 subunit of the N-methyl D-aspartate (NMDA) glutamate receptor (NMDAR)20,21, thus demonstrating the potential for PSD-95 to couple neuroligin to other postsynaptic components. In addition, PSD-95 has been shown to be responsible for the recruitment of a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor (AMPAR)

subunits22, despite interacting only indirectly via the protein Stargazin23. P-Neurexin, in turn, has been shown to bind to CASK which is enriched in brain at synaptic plasma membranes24. CASK, like PSD-95, is a member of the membrane-associated guanylate kinase (MAGUK) superfamily. The guanylate kinase-like motif lacks catalytic activity, but along with the PDZ and SH3 domains, may mediate protein-protein interactions. The type II PDZ domain of CASK has been shown biochemically to mediate binding to the C-terminus of neurexins24, and pulls down reliably all a- and P-neurexins. Intracellularly, CASK forms a complex with both Mint-1 and Veli26-28 which has been proposed to link neurexin-mediated cell adhesion events to components of the exocytosis machinery and/or to synaptic vesicle trafficking. Along this line, other CASK-binding partners include protein 4.129, calmodulin24, rabphilin 3a,30, and calcium channels31. In addition, CASK has even been proposed as a transcriptional regulator32. Given the promiscuous nature of the PDZ domain interactions and conflicting published results with respect to binding partners and localization27,33, more work will be needed to establish the physiological significance of CASK for the neuroligin/P-neurexin complex.

Figure 7.1. Model Structure of the Extracellular Domains of Neuroligin 1 and Neurexin 1p. An interaction in a head-to-head trans-complex is proposed because it best fits the available biochemical data. The AChE-like domain of neuroligin (green) interacts with the laminin (LNS)-like domain of neurexin (yellow) when the latter contains no insert in splice site #4 (panels A1 and A2, the latter showing the structure at a different angle as indicated). Several loops (red) in neurologin 1 are much longer than in other proteins with an AChE-like fold, including insertions in the two splice sites A and B. This interaction is severely hindered when P-neurexin contains an insert at splice site #4 (panel B), presumably by sterical interfering with the position of the insert B in neuroligin. This model is supported by a recent finding that lack of insert B allows binding to all neurexins irrespective of their splice combination3. While this interaction should be less efficient when neuroligin is glycosylated at the splice site insert B, their Ca2+-dependence is more difficult to explain based on this model structure: the proposed major calcium binding site at a degenerate EF-hand-like motif is located at the C-terminus, and a second potential site is close to splice site B (best seen in panel A2). The structures have been built using coordinates from protein data bank (PDB, www.rcsb.org) entries 1c4r for neurexin, and 1mah & 1fss for neuroligin. The structures of the N-terminal sequences as well as those regions linking the domains have been predicted using software programs SPDBViewer and Threader, and the web service Hmmstr/Rosetta. See Colorplate 6.

Figure 7.2. Model Structure of the Neuroligin/p-Neurexin Trans-Synaptic Complex Together with Their Putative Intracellular Binding Partners PSD-95 and CASK. The size of the complex formed by the laminin (LNS)-like domain (yellow) of P-neurexin with the AChE-like domain (green) of neuroligin is about 10 nm (compare also Figure 7.1) and for itself not sufficient to span the synaptic cleft. Each of these domains is linked to the adjacent membrane by a sequence that may build structurally flexible domains (gray) and would have to expand to the required lengths, building a bridge of at least 16 nm over the synaptic cleft. The model is based on the structures of the LNS domain from P-neurexin, the PDZ and the guanylate kinase domain from CASK, and the three PDZ domains from PSD-95 that have been solved by X-ray crystallography. The structures of the other domains have been obtained by homology modeling using coordinates from the protein data bank (PDB, www.rcsb.org), i.e., entries 1kgd, 1kwa, 1jxm, 1y74, and 1rso for CASK; 1c4r for neurexin; and 1mah & 1fss for neuroligin; 1be9, 1iu0, 1jxm, 1qlc, and 1v1t for PSD-95. Predictions were made using programs SPDBViewer and Threader, and the web service Hmmstr/Rosetta, and represent most compact forms25. The bio-informatical work for this review was supported by grant SFB406-C9 (to MM). See Colorplate 7.

Presynaptic Postsynaptic

Figure 7.2. Model Structure of the Neuroligin/p-Neurexin Trans-Synaptic Complex Together with Their Putative Intracellular Binding Partners PSD-95 and CASK. The size of the complex formed by the laminin (LNS)-like domain (yellow) of P-neurexin with the AChE-like domain (green) of neuroligin is about 10 nm (compare also Figure 7.1) and for itself not sufficient to span the synaptic cleft. Each of these domains is linked to the adjacent membrane by a sequence that may build structurally flexible domains (gray) and would have to expand to the required lengths, building a bridge of at least 16 nm over the synaptic cleft. The model is based on the structures of the LNS domain from P-neurexin, the PDZ and the guanylate kinase domain from CASK, and the three PDZ domains from PSD-95 that have been solved by X-ray crystallography. The structures of the other domains have been obtained by homology modeling using coordinates from the protein data bank (PDB, www.rcsb.org), i.e., entries 1kgd, 1kwa, 1jxm, 1y74, and 1rso for CASK; 1c4r for neurexin; and 1mah & 1fss for neuroligin; 1be9, 1iu0, 1jxm, 1qlc, and 1v1t for PSD-95. Predictions were made using programs SPDBViewer and Threader, and the web service Hmmstr/Rosetta, and represent most compact forms25. The bio-informatical work for this review was supported by grant SFB406-C9 (to MM). See Colorplate 7.

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