Mutational analysis of the neurexin/neuroligin complex reveals essential and regulatory components Carsten Reissner*†, Martin Klose*, Richard Fairless*, and Markus Missler*†‡ *Institute of Anatomy and Molecular Neurobiology, Westfa¨lische Wilhelms University, Vesaliusweg 2, 48149 Mu¨nster, Germany; and †Interdisciplinary Center for Clinical Research, 48149 Mu¨nster, Germany Edited by Thomas C. Su¨dhof, Stanford University School of Medicine, Palo Alto, CA, and approved August 12, 2008 (received for review February 19, 2008) Neurexins are cell-surface molecules that bind neuroligins to form and contacts the Nlgn dimer from opposite sides (19). The -a heterophilic, Ca2؉-dependent complex at central synapses. This crystal structure of the single LNS domain of Nrxn 1␤ demon transsynaptic complex is required for efficient neurotransmission strated that it is related to lectin-like domains (20), and the and is involved in the formation of synaptic contacts. In addition, structure of the second Nrxn 1␣LNS domain revealed the both molecules have been identified as candidate genes for autism. presence of Ca2ϩ binding sites (21). Recently, three cocrystal Here we performed mutagenesis experiments to probe for essen- studies of the Nrxn/Nlgn complex consistently identified the tial components of the neurexin/neuroligin binding interface at the contact areas but differed in the actual residues of the interface single-amino acid level. We found that in neurexins the contact and the identification of Ca2ϩ coordination sites (15–17). For area is sharply delineated and consists of hydrophobic residues of example, the role of R109, N103, and D104 in ␤-Nrxn and of the LNS domain that surround a Ca2؉ binding pocket. Point mu- K306, E397, L399, and R597 in Nlgn1 remained ambiguous in tations that changed electrostatic and shape properties leave Ca2؉ these studies, raising the question of which amino acids are in fact coordination intact but completely inhibit neuroligin binding, essential for binding. Moreover, none of these crystal structures whereas alternative splicing in ␣- and ␤-neurexins and in neuroli- provided a rationale for binding of Nrxn to Nlgn2 (15–17), the gins has a weaker effect on complex formation. In neuroligins, the major binding partner of ␣-Nrxn (22), raising the possibility of contact area appears less distinct because exchange of a more an alternative interface. distant aspartate completely abolished binding to neurexin but Here we reveal through mutagenesis experiments the essential many mutations of predicted interface residues had no strong residues of Nrxn that constitute the hydrophobic contact site ϩ effect on binding. Together with calculations of energy terms for surrounding the Ca2 binding pocket, and we show that these presumed interface hot spots that complement and extend our residues are required for binding to all Nlgn isoforms. Similarly, mutagenesis and recent crystal structure data, this study presents we identified a single essential amino acid in Nlgn but conclude a comprehensive structural basis for the complex formation of from numerous additional mutations and calculation of energy neurexins and neuroligins and their transsynaptic signaling be- terms that the binding site on the surface of the Nlgn CAM tween neurons. domain is less distinct than on the Nrxn LNS domain. calcium ͉ cell adhesion ͉ LNS domain ͉ neurotransmission ͉ synaptogenesis Results .Residues Required for Ca2؉ Coordination in the Nrxn/Nlgn Complex To better define the core structure of the LG/LNS family (9), we he heterophilic complex formed by cell adhesion molecules compared LNS folds of Nrxn to those of agrin, laminin, Gas6, neurexin (Nrxn) and neuroligin (Nlgn) reflects the asym- T SHBG, and pentraxin, employing pairwise structural alignments metric nature of the synapse with presynaptic and postsynaptic [supporting information (SI) Fig. S1A]. The core is composed of specializations (1, 2). Nrxns and Nlgns are essential molecules ␤-strands ␤2-␤11 and is stabilized by hydrophobic residues that because they perform important functions in synaptic transmis- bind a fragment of the ␤10/␤11 loop. The rigid core determines sion (3, 4) and differentiation of synaptic contacts (5, 6), and ϩ that residues of a Ca2 coordination site are placed on top of both molecules have been identified as candidate genes for three loops in agrin, laminin, and Nrxn (i.e., ␤5, turn ␤6/7, and autism (7, 8). loop ␤10/11), characterized by an rmsd of 0.63 Å [from Nrxn 1␤; Nrxns form a family of transmembrane proteins with variable Protein Data Bank (PDB) ID code 1C4R]. To test our definition extracellular sequences. Nrxn genes (Nrxn1–3) give rise to of a core structure, we calculated C␣-C␣ difference distance ␣-neurexins and shorter ␤-neurexins that contain five (␣-Nrxn) ␤ plots and difference Ramachandran values for LNS pairs. or two ( -Nrxn) splice sites (SS1–5) (9). Although they share Changes were mostly located at nonconserved loop regions, most sequences, the essential role of ␣-Nrxn in neurotransmis- 2ϩ ␤ whereas no changes occurred at the Ca sites (Fig. S1B). This sion cannot be replaced by -Nrxn (10), and one ligand exists for rigidity is unique because Ca2ϩ binding is mostly mediated by ␣-Nrxn that does not bind to ␤-Nrxn (11). In contrast, Nlgn was ␤ ␤ flexible loops (23–25). In contrast, the -turns are subject to discovered by its interaction with -Nrxn (12). The cholinester- alternative splicing in Nrxn and agrin and are termed ‘‘hyper- ase-like adhesion molecule (CAM) domain of Nlgn interacts variable regions’’ (20). Because sequence and length of loops ␤ 2ϩ with the extracellular domain of -Nrxn in a Ca -dependent differ between LNS domains, we next explored the possibility manner, and binding is facilitated by the splice variation of ␤-Nrxn that lacks an insert in SS4 (13). Nlgn mRNA is also susceptible to splicing, at two positions referred to as A and B Author contributions: C.R. and M.K. contributed equally to this work; C.R. and M.M. (12), including splice variants with no insert in B that bind to all designed research; C.R., M.K., and R.F. performed research; C.R., M.K., R.F., and M.M. ␤-Nrxns and presumably ␣-Nrxn (14). Therefore, the Nrxn/Nlgn analyzed data; and C.R. and M.M. wrote the paper. complex involves a domain shared by ␣- and ␤-Nrxns, and any The authors declare no conflict of interest. structural characterization needs to account for the Ca2ϩ de- This article is a PNAS Direct Submission. pendence and regulation by alternative splicing (15–17). Freely available online through the PNAS open access option. Extracellularly, both Nrxns contain laminin-like (LG/LNS) ‡To whom correspondence should be addressed. E-mail: [email protected]. domains that in ␣-Nrxn alternate with interspersed EGF-like This article contains supporting information online at www.pnas.org/cgi/content/full/ sequences. ␣-Nrxn contains six (LNS1–6) and ␤-Nrxn only one 0801639105/DCSupplemental. LNS domain (9, 18), which is responsible for Nlgn binding (13) © 2008 by The National Academy of Sciences of the USA 15124–15129 ͉ PNAS ͉ September 30, 2008 ͉ vol. 105 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801639105 Downloaded by guest on September 26, 2021 Fig. 2. Low Ca2ϩ affinity is sufficient for Nrxn/Nlgn complex formation. (A) Electrostatic surface properties of Nrxn 1␣LNS2 domain. (B) Binding of Nlgn to Nrxn 1␣LNS6 (Upper) and binding of neurexophilin to Nrxn 1␣LNS2 (Lower) are mutually exclusive. (C)Ca2ϩ coordination of LNS2 reconstructed in Nrxn 1␤LNS by mutating ␤N238 to glycine. (D) The ␤N238G mutation binds recom- binant Nlgn at a [Ca2ϩ] of 2 mM but not at lower concentrations. The exposure time of lanes 2–4 in D was 10 min, and the exposure time was 1 min for all other lanes. Input controls of recombinant proteins are shown in Fig. S5. respective arginine mutations (Table S1) identifies the Ca2ϩ binding site at the rim of the LNS fold as essential for binding Fig. 1. Ca2ϩ coordination in Nrxn. (A and B) Electrostatic surface analysis to all Nlgns. These data are consistent with a cell culture study surrounding the Ca2ϩ binding site in agrin (A) and Nrxn1␣ LNS6 (B). Negative in which loss of synapse-inducing activity was observed for (red) and positive (blue) charges and long hydrophobic residues (arrowheads) ␤D137A and ␤N238A (27). In that study, the Nrxn mutation are indicated. (C) Immunoblot showing pull-down experiments of Nlgn1 from ␤T235A also showed reduced clustering and preferentially la- brain using IgG fusion proteins of wild-type ␤LNS domain, various alanine beled the surface of cells expressing Nlgn2. However, in our mutations, and control vector. (D) Similar to Nlgn1 (C), Nlgn2 and Nlgn3 are biochemical binding experiments ␤T235A and ␣T1281A pull ␤ precipitated by -Nrxn with and without splice insert SS4 but failed to bind to down all Nlgn isoforms comparable to wild-type levels (Fig. 1 C Nrxn mutations ␤D137A and ␤N238A. Residue T235 in ␤-Nrxn does not inhibit interaction with Nlgn1–3s, in contrast to previous data (27). Original SDS gels and D, lane 3) and fail to discriminate between Nlgn1 (plus insert and input control of fusion proteins for C are shown in Fig. S3. B) (Fig. 1C, lanes 8 and 9) and Nlgn2 (minus B) (Fig. 1D Upper, lane 7). In contrast, the presence of splice insert 4 in Nrxn (ϩSS4) shows reduced binding not only to Nlgn1 (Fig. S3), which that the surface surrounding the Ca2ϩ binding site is responsible confirms earlier results (12), but also to Nlgn2 and 3 (Fig. 1D, for their specific protein–protein binding properties. We mapped lane 4), shown here for the first time. shape and electrostatic potentials, and we observed that, in most LNS domains, the Ca2ϩ site features as a negative pole sur- Ca2؉ Dependence of Nrxn/Nlgn Complex Formation. To explore rounded by a ring of positively charged residues, e.g., in agrin whether Nlgn binding of Nrxn is defined by calcium affinity, we (Fig.