Mutational analysis of the / 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) are cell-surface molecules that bind 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 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 with variable 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. 1A and Figs. S2 and S3). However, the Nrxn1␤ LNS/Nrxn1␣ made use of the LNS2 domain of Nrxn 1␣ (Fig. 2A), which LNS6 domain differs from this ‘‘standard surface’’ because coordinates Ca2ϩ at a position identical to that of LNS6 (21) and hydrophobic residues engulf the Ca2ϩ binding pocket (Fig. 1B), also contains a splice site (SS2). However, the two domains differ consistent with recent crystal structures of the Nrxn/Nlgn com- in that LNS2 is not able to bind Nlgn (Fig. 2B Upper, lane 4), and plex (15–17). the Ca2ϩ site shows a variation: whereas ␣D329, ␣L346, and To test which residues contribute to the Ca2ϩ dependency of ␣M414 are conserved, the residue corresponding to ␣N1284 in the complex, we generated mutations at the Ca2ϩ site of LNS LNS6 (or D3055 in laminin 2␣; Fig. S2D) is replaced by a glycine domains fused to human IgG-Fc. We mutated ␤-Nrxn D137 and (Fig. S2E). The alterability in Ca2ϩ affinity of isolated LNS2 by N238 to alanine (short: ␤D137A and ␤N238A) and arginine splice inserts (21) led to the suggestion that alternative splicing (␤D137R and ␤N238R), thereby disturbing side chains respon- of ␣LNS6/␤LNS may, in analogy, also reduce Ca2ϩ affinity of sible for Ca2ϩ coordination in laminins (26). Because also these domains below a threshold required for Nlgn binding (21, ␣-Nrxn may bind to Nlgn (14), we simultaneously introduced the 28). To directly test the idea if the 10-fold reduction in complex equivalent mutations into the isolated sixth LNS domain of formation caused by insert SS4 in LNS6 (Fig. S6B) depends on ␣-Nrxn (Nrxn 1␣LNS6; Fig. S2) to generate fusion proteins that Ca2ϩ affinity, we mimicked the Ca2ϩ site of LNS2 in Nrxn lack the 38 N-terminal residues specific for Nrxn 1␤ (Fig. S4) (9). 1␣LNS6 and Nrxn 1␤LNS domains by introducing mutations Both wild-type ␤-Nrxn (Fig. 1C, lane 3) and ␣-Nrxn (below, Fig. ␤N238G (Fig. 2C) and ␣N1284G (data not shown). In contrast 2 and Fig. S5) LNS constructs precipitated Nlgns from mouse to the alanine mutations of ␣N1284 and ␤N238 shown above, the brain lysate, suggesting that the ␤-Nrxn-specific residues are glycine mutations surprisingly sustain Nlgn binding (Fig. 2D, dispensable for this interaction (but see refs. 13 and 14 for a lane 4, and data not shown). However, complex formation of different view). In contrast, the alanine mutations of the acidic ␤N238G/Nlgn1 requires a [Ca2ϩ] of 2 mM, which is 200-fold Ca2ϩ binding residues in Nrxn 1␣LNS6 and Nrxn 1␤LNS failed higher compared with wild-type ␤LNS(-SS4)/Nlgn1 (Fig. 6A) to pull down Nlgn1 (Fig. 1C, lanes 4–7), as well as Nlgn2 and and yet 20-fold higher than for ␤LNS(ϩSS4)/Nlgn1 (Fig. 6B).

Nlgn3 (Fig. 1D, lanes 5 and 6). The complete loss of Nlgn binding This is an important result because it reveals that the threshold NEUROSCIENCE for ␣D1183A, ␤D137A, ␣N1284A, and ␤N238A as well as for the for complex formation is far below normal [Ca2ϩ] in the extra-

Reissner et al. PNAS ͉ September 30, 2008 ͉ vol. 105 ͉ no. 39 ͉ 15125 Downloaded by guest on September 26, 2021 Fig. 4. EGF3 domain of ␣NrxnϩSS4 inhibits binding to NlgnϩB. (A) Full- length ␣NrxnϩSS4 binds to IgG-Nlgn1-B but not to Nlgn1ϩB under identical Fig. 3. Nlgn binding requires hydrophobic residues in Nrxn. (A Upper) Nlgn conditions. (B) ␣LNS5-EGF3-LNS6 does not bind Nlgn1ϩB under nonreducing from brain lysate is pulled down by wild-type Nrxn 1␣LNS6 domain but not by conditions (ϪDTT), whereas addition of DTT restores binding. As control, DTT mutations ␣L1280SϩI1282SϩN1284D or ␤L234SϩI236SϩN238D, respectively. does not change Nlgn binding of single ␣LNS6 domains. An extended set of 2ϩ (A Lower) Calcium-45 overlay dot blots demonstrate that Ca binding itself pull-down experiments with more splice combinations is shown in Fig. S6. is maintained in mutations. (B) An artificially extended surface blocks complex formation by mutating ␣G1201VϩT1202A or ␤G155VϩT156A (Upper) but ϩ ϩ leaves Ca2 coordination unscathed (Lower). (C) A laminin Ca2 binding site Nrxn essential for Nlgn1 binding is similarly required for the was mimicked in Nrxn by replacing hydrophobic residues L1280 and I1282 with binding to Nlgn2 and Nlgn3 (Fig. 3D, lanes 4 and 5), suggesting serines and substituting N1284 by aspartate (green). Alternatively, the inter- face was extended by mutating ␣G1201VϩT1202A (red). (D) As for Nlgn1 (A that the same hydrophobic residues of Nrxn are required for all and B), mutations ␤G155VϩT156A and ␤L234SϩI236SϩN238D completely Nrxn/Nlgn complexes. block binding to Nlgn2 and Nlgn3 (lanes 4 and 5). Input control of recombi- To control that the recombinant Nrxn LNS proteins used in nant proteins are shown in Fig. S7. our study were also able to bind to membrane-bound Nlgn, heterologous cells expressing full-length Nlgn with a GFP tag were incubated with fusion proteins. For example, wild-type and 2ϩ cellular space and that factors other than Ca affinity have to ␤T235A domains outlined membrane-bound Nlgn, whereas no ␣ ␤ specify the exclusive binding of LNS6/ LNS (but not LNS2) to surface labeling was observed with ␤G155VϩT156A (Fig. S8). Nlgn. Alternative Splicing in the Nrxn/Nlgn Complex. Splicing at SS4 in Hydrophobic Residues Required for the Nrxn/Nlgn Complex. Our Nrxn and site B in Nlgn was suggested as an important deter- analysis of surface charges indicated that the structure surround- minant in complex formation (12); however, previous studies 2ϩ ␣ ␤ ing the Ca site in LNS6/ LNS (mostly hydrophobic) is have reported conflicting results because different assays and distinct from LNS2 (mostly negatively charged), but these sur- constructs were used. Because we wanted to solve the issue of face properties have not yet been probed experimentally. To how strongly splicing affects complex formation compared with distinguish the effect of candidate residues on shape or electro- the contact site mutations described above, we analyzed all static surface properties at the contact area, we used site- relevant splice combinations under comparable conditions. We directed mutagenesis to probe for Nlgn binding and a calcium-45 ϩ determined the binding of Nlgn containing the 9-aa-long B insert overlay assay to test integrity of Ca2 coordination. First, we (Nlgn1ϩB) to ␤LNS without (␤LNS-SS4) and with the 30-aa- addressed the role of L1280 and I1282 because these residues long splice insert 4 (␤LNSϩSS4). Whereas Nlgn1ϩB binds to differ in LNS domains of laminin or agrin. We inserted muta- ␤ ␮ 2ϩ A ␤ ϩ tions ␣L1280SϩI1282SϩN1284D (Fig. 3A and green in C; and LNS-SS4 at 10 M [Ca ](Fig. S6 ), LNS SS4 requires a Fig. S7) and ␤L234SϩI236SϩN238D into ␣LNS6 and ␤LNS 10-fold-higher concentration (Fig. S6B). In the reverse experi- 2ϩ ment, we observed that ␤LNS-SS4 and ␤LNSϩSS4 precipitated domains, respectively. These triple mutations mimic the Ca ϩ site of laminin 2␣ LNS5 but remove the hydrophobic ring Nlgn from brain lysate, but to a lesser extent if the SS4 insert was present (Fig. 6C). To explore how splicing in Nlgn affects characteristic for Nrxn. We found that complex formation with ␣ ␤ ϩ Nlgn was completely abolished (Fig. 3A Upper, lanes 4 and 5), binding to - and -Nrxns, we expressed Nlgn1 B and Nlgn1-B although Ca2ϩ coordination itself was preserved (Fig. 3A Lower). (lacking the 9-aa insert) and compared binding to different Nrxn ␣ ϩ ␤ ϩ These data suggest that the hydrophobic residues mediate a variants. Nrxn LNS6 SS4 (Fig. S6D) and LNS SS4 (data not ϩ direct interaction with Nlgn. To validate this hypothesis, we shown) were found to bind to both Nlgn1 B and Nlgn1-B, 2ϩ introduced a spacer into the interface by mutating residues indicating that, in contrast to mutations of Ca binding and ␣G1201 and ␤G155 to valine. These changes were introduced contact residues (Figs. 1–3), no splice combination completely together with ␣T1202A and ␤T156A because the bulky valine prevents complex formation. However, when we used Nlgn to may sterically interfere with the methyl group of threonine and pull down full-length ␣-Nrxn, we observed binding of Nlgn1-B to alter the loop conformation (Fig. 3B and red in C). Both double Nrxn 1␣ϩSS4 (Fig. 4A, lane 4) but not of Nlgn1ϩB (Fig. 4A, lane mutations lacked the ability to bind Nlgn but were able to 3), consistent with an earlier report (14). To define the additional coordinate Ca2ϩ (Fig. 3B, lanes 4 and 5), suggesting that contact factor in full-length ␣-Nrxn that prevents complex formation, we between Nrxn and Nlgn is very close in this area and that Ca2ϩ generated IgG fusion constructs of the last extracellular cassette is enclosed within the interface. These mutagenesis data are of Nrxn 1␣ containing the splice insert (␣LNS5࿝E࿝LNS6ϩSS4). consistent with recent cocrystals of the complex (15–17) and Similar to full-length ␣-Nrxn, this construct showed no detect- extend the structural analysis by demonstrating not only that they able binding to Nlgn1ϩB (Fig. 4B, lane 4), suggesting that the are part of the contact site but that their hydrophobicity is complete cassette determines a three-dimensional structure essential. Furthermore, we determined that the contact area on unfavorable of complex formation. Because EGF structures

15126 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801639105 Reissner et al. Downloaded by guest on September 26, 2021 Fig. 5. Residue D271 of Nlgn1 is essentially required for binding Nrxn. (A) Pull-down experiments of mutated recombinant Nlgn1 by ␤LNS-IgGFc fusion protein and IgGFc protein as negative control. (B–D) TsA201 cells expressing wild-type Nlgn1-B (B) or mutant G266D (C) and D271R (D) were surface- labeled with anti-Nlgn antibody 4C12 (45). (Scale bar: 5 ␮m.) Pull-down of additional mutations and input control of recombinant proteins are shown in Fig. S11.

contain cysteine bonds (29), we tested whether reducing condi- tions could change the binding behavior of ␣LNS5࿝E࿝LNS6. In fact, binding to Nlgn1ϩB could be restored by addition of DTT (Fig. 4B, lanes 4 and 7), whereas binding of Nlgn1ϩBtoan isolated ␣LNS6 domain as control was unaffected by DTT (Fig. 4B, lanes 3 and 5). Our results suggest that the preference of full-length Nrxn1␣ϩSS4 for Nlgn1-B (14) may be determined by an EGF-tightened domain arrangement in the ␣LNS5࿝E࿝LNS6 cassette, allowing the first model prediction of their spatial arrangement (Fig. S9).

Nlgn Interface Residues and Complex Formation. Because mostly Fig. 6. Hot spots of the Nrxn/Nlgn complex interface. (A) Summary of Ca2ϩ binding and hydrophobic residues in Nrxn are required for experimentally defined essential residues on Nrxn (Left) and Nlgn (Right) surfaces. Hydrophobic side chains of ␤-Nrxn G155, L234, I236, and R109 Nlgn binding, we aimed at identifying the relevant residues in ϩ (magenta) surround the Ca2 coordination site (yellow) and mediate binding Nlgn. First, we probed the degenerate EF hand (30) for its to Nlgn. Other residues in this area have no effect (cyan). In Nlgn, D271 near contribution to complex formation, but deletion splice inserts A and B and loop L34 (circles) is critical for Nrxn binding (red). A (Nlgn1ϩB⌬EF1) did not alter binding to ␤LNS (Fig. S10), G500A mutation prevents complex formation at least in Nlgn1 (17), whereas thereby validating recent Nlgn crystal structures that did not L399 and N400 only show synergistic effects (16) (in pink), and many other detect Ca2ϩ bound to this motif (15–17, 31). Next, we selected residues do not affect binding (cyan). (B) Summary of calculated hot spots as four presumptive interface sites in Nlgn that match the criteria determined by an alanine scan (see Tables S3 and S4 for details). Three Nrxn analyzed above, i.e., (i) the ϩSS4 and ϩB inserts may interfere hot spots are identified, i.e., I236 (magenta), L234, and L135 (both orange). On to reduce (but not abolish) binding, (ii) the contact site is likely Nlgn, only G500 (pink) and L399 (orange) are calculated as hot spots, whereas residues F499 and N400 (green) have an intermediate effect and G396 and hydrophobic, and (iii) it should contain a residue capable of E397 only marginally reduce complex stability (light green). Calculated ⌬⌬G 2ϩ joining the Ca coordination of Nrxn. We tested candidate sites values are rainbow color-coded from 0 (cyan) to 2 (magenta) kcal/mol. The by mutating key residues: First, A557 and V558 build a hydro- Nrxn/Nlgn complex has been opened by 180° to expose the interface, and phobic site oriented toward the synaptic cleft (19). However, positions of Ca2ϩ ion spheres (1 and 2) are labeled. aspartate and arginine mutations that mimic other cholinester- ases did not change binding to Nrxn (Fig. S10). Second, a proline-rich sequence containing E99 that binds Zn in a deri- resulted in a properly processed protein based on evidence that vatized acetylcholinesterase crystal (PDB ID code 1FSS) it reaches the surface of tsA201 cells (Fig. 5D) and an IgG fusion prompted us to mutate P100 to serine. The change should protein of D271R is produced properly by COS-7 (data not sterically block a close contact if present but failed to alter shown). Importantly, the Nlgn D271R mutation completely complex formation (Fig. S11A, lane 3). Third, close to splice site blocked binding to Nrxn (Fig. 5A, lane 3). When we tested the B, Nlgn contains acidic Q395 and E397 that lie next to a nearby residues with P192A, G266D, L273E, and double muta- hydrophobic residue (L399). We mutated L399 to serine to test tion G266DϩL273D, however, none of these mutations abol- the importance of surface charge in that area and changed H294 ished Nrxn/Nlgn complex formation (Fig. S11 A, lanes 6 and 9, to valine to probe for contribution of a nearby histidine as in B, lanes 3 and 9, and C, lane 3), raising the possibility that the Gas6 (32). None of these mutations, however, altered binding interface on Nlgn may be less distinct as compared with Nrxn. (Fig. 5A, lanes 6 and 9), a surprising finding because a recent Because of available data from cocrystal structures of the cocrystal of the complex suggested that residues L399 and H294 Nrxn/Nlgn complex (15–17, 31), the contribution of suspected may directly contact Nrxn (15), emphasizing the need for residues to complex stability can be calculated and compared mutagenesis studies as performed here (Table S2). Finally, we with mutagenesis analyses (Fig. 6). We estimated binding ener- focused on the area around D271 because it sits near the three gies for wild-type and mutant complexes to assess the effect of

major Nlgn loops and is embedded in a hydrophobic environ- hot spot residues (Tables S3 and S4). In Nrxn, the binding NEUROSCIENCE ment (P192, G266, and L273). Changing D271 to arginine energies for mutants S107R, L135R, G155VϩT156A,

Reissner et al. PNAS ͉ September 30, 2008 ͉ vol. 105 ͉ no. 39 ͉ 15127 Downloaded by guest on September 26, 2021 L234SϩI236SϩN238D, I236R, and N238R have positive ⌬G three from Nlgn (E397 to ␤T235 and ␤I236; N400 to ␤S107). In values, indicating no complex formation, in agreement with addition, ␤R232/D387 and ␤R109/E297 form salt bridges (15). experimental data (Fig. 6A). Interestingly, Nrxn mutations Consistently, mutations of ␤S107A and ␤T235A do not alter its D137A and D137R do not destabilize the complex interface main-chain interaction and sustain complex formation (Fig. 1 according to calculations, supporting its exclusive function in and Table S4) (27). Similarly, no single mutation removing an Ca2ϩ coordination, whereas N238A only slightly destabilized H-bridge or salt bridge abolishes complex formation: Nlgn the complex. Calculated binding energies for all available E297A, D387A, E397A, and N400A only slightly reduce binding alanine mutations (Table S4) are schematically summarized in (Table S3), and Nrxn R232A remained functional (27). Inter- Fig. 6B: only the Nrxn mutations of hydrophobic residues L135 estingly, residue ␤N238 prevents complex formation when (⌬⌬G ϭ 1 kcal/mol), L234 (⌬⌬G ϭ 1 kcal/mol), and I236 (⌬⌬G ϭ changed to A (Fig. 1) but tolerates mutations to G (Fig. 2) or D 2 kcal/mol) resulted in significant destabilization of the complex but (Fig. 3), indicating that it is essential for calcium coordination, no alanine mutation of polar residues such as S107 or R232. In but its side-chain interaction to G396 (15, 16) is dispensable. Nlgn, none of the mutations gave positive ⌬G values, including the Another complex interaction profile is highlighted by ␤R109A, quintuple mutation L399AϩN400AϩD402NϩQ395AϩE397A which showed no synaptogenic activity (27) but sustains complex (16), and only the Nlgn residues L399 and G500 account as stability based on calculated binding energies (Fig. 6B). Consis- moderate hot spots according to energy terms (Fig. 6B and Table tently, single mutations of Nlgn residues E297, H294, and L399 S4). However, our L399S mutation did bind Nrxn experimentally that are in binding distance to ␤R109 did not prevent complex (Figs. 5C and 6A),andNlgn2(carryingaQforthenonconserved formation (Fig. 5) (28), supporting a model of dynamic binding G500) also formed a complex with Nrxn (Fig. 1F). Therefore, of Nrxn R109 with all three residues (15). The hydrophobic ring of Nrxn (Fig. 1B) is expanded by Nlgn mutagenesis and calculated data together indicate that the interface ϩ in Nrxn is sharply delineated, whereas the binding site in Nlgn L399 and F499 to a hydrophobic shell that encloses the Ca2 appears less well defined or even has alternative positions. (15–17). The importance of this shell is highlighted by our triple mutation ␤L234SϩI236SϩN238D, which specifically reduces Discussion the hydrophobicity of the surface but does not introduce a steric hindrance (as imposed by ␤L135R, ␤L234R, ␤I236R, and Using mutagenesis, we identified single residues that deter- N238R; Table S3 and Fig. S12) to prevent complex formation mine the complex between Nrxn and Nlgn. All mutations (Fig. 3) (15). Therefore, we propose that hot-spot residues on the tested thus far are summarized in Tables S1 and S2 and are Nrxn surface do coordinate Ca2ϩ (␤D137 and ␤N238), make displayed in Fig. 6A: essential residues of Nrxn (D137, G155, ␤ ␤ ␤ ␤ hydrophobic contacts ( G155, L234, and I236), or both L234, I236, and N238 for -Nrxn) and Nlgn (D271) showed an (␤I236)—while none of the H-bridges are essential. In addition, all-or-nothing effect in our binding assay (Figs. 1, 3, and 5). 2ϩ our results reveal that the hot-spot residues identified here are Analysis of Ca binding (Fig. 3) and calculation of energy also required for binding to Nlgn2 and Nlgn3 (Figs. 1 and 3) and, terms using available crystal data (Fig. 6B) revealed that 2ϩ presumably, for the structurally identical complex of Nrxn/Nlgn4 essential residues contribute to Ca coordination, to the (17). hydrophobic interface, or to both. In Nlgns, mutagenesis studies are more difficult because several previous changes were retained in the ER (41–43); 2؉ Ca Coordination. Our comparison of LNS domains (21, 26, therefore, we tested each new mutation for its ability to reach the 2ϩ 32–37) revealed a conserved core structure (Fig. S1). Ca cell surface (Fig. 5, Fig. S9, and data not shown). Of all correctly ␤ binding in Nrxn (by D137, V154, I236 and N238 for -Nrxn), delivered mutations, only D271R resulted in complete loss of agrin, and laminins represents a rigid ‘‘socket’’ with no flexible Nrxn binding in our experiments (Figs. 5 and 6). Because D271 states as in EF hands (23), consistent with unchanged CD lies 12 Å apart from Nrxn D104 (15–17), the arginine introduced spectra of Nrxn and agrin (13, 38) and crystal data (15–17, 39). at position 271 may initiate a repelling reaction on R274 and This rigidity allowed us to transfer epitopes from other LNS R311, of which the latter is part of a presumed second Ca2ϩ site ␤ 2ϩ domains to Nrxn: N238G mimics the Ca coordination of (16). 2ϩ Nrxn LNS2 but reduced Ca affinity of the Nrxn/Nlgn According to crystal data, Nlgn possesses up to four more complex Ϸ200-fold (Fig. 2). Because the complex still forms ϩ candidate residues at the interface (G500, L399, N400, and with this mutation at physiological [Ca2 ], it appears unlikely E397) (15–17), but, based on calculated binding energies, only that calcium affinity itself defines splice-code-specific pairings mutations of L399 and G500 could possibly destabilize the (21). Rather, crystal and NMR data revealed that insert SS4 complex (Fig. 6B). Because L399S did not prevent Nrxn binding exists in an equilibrium of two conformations (39, 40). The to Nlgn in our study (Fig. 5), G500 remains as a putatively ϩ inactive form is stabilized by coordinating Ca2 with high important residue. However, Nlgn2 differs from Nlgn1 by a affinity (39), whereas binding follows after unfolding of SS4 G500Q exchange calculated to sterically block Nrxn/Nlgn com- (40). Therefore, the 50% reduction in complex formation plex formation (Table S3), suggesting that G500 may not play a when SS4 is present could sufficiently be explained by a general role. In summary, we conclude that, whereas the binding reduced availability of the active form. In this interpretation, interface on Nrxn is sharply delineated and conserved for the calcium may have a double function for Nrxn/Nlgn: first, it is binding to presumably all Nlgn isoforms, the Nlgn interface required for complex formation, but, second, it slows down the tolerates more mutations and may even contain an alternative process by keeping approximately half of the splice insert hot-spot area in Nlgn2. carrying variants, if present, in a reserve pool. Similarly, the additional presence of the third EGF domain as in full-length Materials and Methods ␣NrxnϩSS4 may lead to a spatial arrangement that makes Original cDNAs used for site-directed mutagenesis have been described (4, 11, binding to splice insert B-positive Nlgns even less likely (Fig. 13, 44). Oligonucleotide primers and procedures for structural modeling, 4 and Figs. S6 and S9). pull-down experiments, live cell labeling, and calcium-45 overlays are based on published protocols (13, 14) and are described in detail in SI Materials and Methods. Binding Residues. Crystal data revealed that the Nrxn/Nlgn1 interface may involve (i) four side-chain to main-chain H- ACKNOWLEDGMENTS. We thank K. Kerkhoff and I. Wolff for excellent tech- bridges, (ii) two salt bridges, and (iii) 19 nonbinding interactions. nical help. This work was supported in part by the Interdisciplinary Center for Hydrogen bridges include one from ␤Nrxn (␤N238 to G396) and Clinical Research, Mu¨nster (Grant Mi3/025/08).

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