Article

Structures of neurexophilin– complexes reveal a regulatory mechanism of

Steven C Wilson1 , K Ian White1 , Qiangjun Zhou1, Richard A Pfuetzner1, Ucheor B Choi1, Thomas C Südhof1,2 & Axel T Brunger1,2,*

Abstract (Nrxns) are a family of presynaptic cell-adhesion mole- cules that are important for specifying the functional properties of Neurexins are presynaptic, cell-adhesion molecules that specify synapses (Su¨dhof, 2017). Through their interactions with various the functional properties of synapses via interactions with trans- binding partners, neurexins constitute part of a molecular code and synaptic ligands. Neurexins are extensively alternatively spliced at act as hub molecules to organize and regulate synaptic function six canonical sites that regulate multifarious ligand interactions, (Su¨dhof, 2017). but the structural mechanisms underlying alternative splicing- Neurexins are encoded by three (Nrxn1–3 in mice), each dependent neurexin regulation are largely unknown. Here, we of which contains multiple promoters to drive expression of determined high-resolution structures of the complex of neurex- Nrxn1a–3a, Nrxn1b–3b, and Nrxn1c. The extracellular sequences of ophilin-1 and the second /neurexin/sex-hormone-binding a-neurexins contain six LNS domains (LNS1–LNS6) interspersed globulin domain (LNS2) of neurexin-1 and examined how alterna- with -like domains (EGFA–EGFC), followed tive splicing at splice site #2 (SS2) regulates the complex. Our data by a cysteine loop and a highly glycosylated stalk region (Fig 1A). reveal a unique, extensive, neurexophilin–neurexin binding inter- The b-neurexins include only the C-terminal region of a-neurexins face that extends the jelly-roll b-sandwich of LNS2 of neurexin-1 from the LNS6 domain onward. Nrxn1c, in turn, lacks all LNS into neurexophilin-1. The SS2A insert of LNS2 augments this inter- domains; its extracellular region contains only the C-terminal stalk face, increasing the binding affinity of LNS2 for neurexophilin-1. region that is conserved in all neurexins (Sterky et al, 2017). All Taken together, our data reveal an unexpected architecture of neurexins are embedded in the membrane by a single transmem- neurexophilin–neurexin complexes that accounts for the modula- brane region followed by a short (~55 residue) cytoplasmic tail. tion of binding by alternative splicing, which in turn regulates the Remarkably, neurexins undergo extensive alternative splicing at six competition of neurexophilin for neurexin binding with other canonical sites (SS1–SS6; Fig 1A). As a result, they are expressed as ligands. thousands of isoforms in the brain (Ullrich et al, 1995; Schreiner et al, 2014; Treutlein et al, 2014). Alternative splicing of neurexins Keywords alternative splicing; neurexin; neurexophilin; synapse; synaptic regulates the interactions of neurexins with a multitude of ligands, cell-adhesion molecules including neurexophilins (Nxphs), neuroligins (Nlgns), LRRTMs, Subject Categories Neuroscience; Structural Biology cerebellins, and dystroglycan (Su¨dhof, 2017). DOI 10.15252/embj.2019101603 | Received 21 January 2019 | Revised 29 August Neurexophilins are a family of secreted, neuropeptide-like glyco- 2019 | Accepted 30 August 2019 originally discovered as a-neurexin ligands and later found The EMBO Journal (2019)e101603 to bind to the LNS2 domain of a-neurexins (Petrenko et al,1996; Missler et al, 1998; in the following we often refer to a-neurexins simply as neurexins or Nrxns). Neurexophilins are encoded by four Introduction genes (Nxph1–4 in mice; Petrenko et al, 1996; Missler & Su¨dhof, 1998; Missler et al, 1998), which are highly conserved in vertebrates Brains process information via complex networks of neural circuits but absent from invertebrates. The neurexophilins are unique in that govern behavior. Neural circuits are, in turn, composed of that they share no detectable homology with other known neurons that communicate with each other at synapses. The func- domains. Given the lack of similarity between neurexophilins and tional properties of these synapses regulate neural circuits and other neurexin ligands or neuropeptides, it is thus not surprising thereby contribute to the performance of all brain functions. that the mechanism of the binding of neurexophilins to neurexins as

1 Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA 2 Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA *Corresponding author. Tel: +1 650 736 1031; E-mail: [email protected]

ª 2019 The Authors. Published under the terms of the CC BY 4.0 license The EMBO Journal e101603 | 2019 1 of 16 The EMBO Journal Steven C Wilson et al

SS1 SS2 SS3 SS6SS4 SS5 A

Nrxn1 N LNS1EGFA LNS2 LNS3 EGFBLNS4 LNS5 EGFC LNS6 C Stalk Intracellular region Pro- mature Nxph1 N domain Nxph1 Presynaptic Membrane

Without Splice Insert With Splice Insert B Nxph1Nxph1-LNS2SS2- Nxph1-LNS2SS2A+

N N 14 14

1 1 C 2 2 13 C 13 12 11 12 11 3 3 4 4

~60 Å 8 8 5 5

6 9 6 9 7 7

8 C 8 SS2A 10 10 C 2 7 2 7 3 4 1 3 4 1 6 N 6 N 5 5

~60 Å

SS2 SS2A+ Figure 1. Domain structure of Nrxn1a and Nxph1 and overall architectures of the Nxph1-LNS2 À and Nxph1-LNS2 complexes. A Schematic showing the domain structure of Nrxn1a and Nxph1 with approximate positions of alternative splice sites indicated by arrows. Colors of LNS2 and Nxph1 correspond to those in the structures in (B). SS2 SS2A+ B Crystal structures of Nxph1 in complex with Nrxn1 LNS2 À and LNS2 . b-strands are numbered, and N- and C-termini are indicated. SS2A insert is shown in magenta. Disordered regions are shown as dashed lines, and disulfide bonds are shown as red sticks.

well as the regulation of this binding by alternative splicing remains Electrophysiological recordings in brain slices from Nxph1 KO mice unknown. revealed altered GABAergic synaptic transmission (Born et al, Different neurexophilin genes are expressed in distinct subpopu- 2014), but the precise scope and mechanisms of Nxph1 function lations of neurons in brain. For example, single-cell RNA-Seq stud- and the role of other neurexophilins remain to be examined. ies showed that neurexophilin-1 (Nxph1) is abundantly produced in Neurexophilins are composed of a signal peptide followed by a hippocampal inhibitory interneurons, Nxph3 in cortical excitatory pro-domain, a polybasic cleavage site, and a mature fragment, thus neurons, and Nxph4 in inhibitory neurons of the hindbrain (Fo¨ldy resembling neuropeptides in that they are synthesized as a precur- et al,2016;Tasicet al, 2016; Saunders et al, 2018; Zeisel et al, sor protein and proteolytically processed to a mature form (Missler 2018). The distinct expression profiles of neurexophilins resemble & Su¨dhof, 1998; Fig 1A). The mature neurexophilin fragment was that of cerebellins throughout the brain, suggesting that they have proposed to contain distinct N-terminal glycosylated and C-terminal cell type-specific roles (Seigneur & Su¨dhof, 2017). Functionally, cysteine-rich domains that are separated in Nxph4 only by a large Nxph1 is the best-characterized neurexophilin, while little work has loop sequence (Missler & Su¨dhof, 1998). However, these putative N- been done on the physiological roles of the other isoforms. and C-terminal domains of mature Nxph1, when individually fused

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to Fc tags, were unable to co-precipitate LNS2, suggesting that the Nxph1 has N-linked glycans at amino acid positions N146, N156, complete mature fragment of Nxph1 represents the minimal LNS2- and N162, while LNS2 is not glycosylated. Crystallization trials binding region of Nxph1 (Reissner et al, 2014). using samples of high mannose-glycosylated Nxph1-LNS2 complex While alternative splicing of neurexins has dramatic effects on did not yield crystals. EndoH or EndoF1 deglycosylated proteins also their interactions with ligands (Su¨dhof, 2017), no structural data could not be crystallized, probably because the glycosidases did not explain how alternative splicing regulates these interactions at the completely deglycosylate Nxph1 as evaluated by SDS–PAGE. There- atomic level. For example, Nrxn1b lacking an insert in SS4 has a fore, in order to crystallize Nxph1-LNS2 complexes, glycosylation of higher binding affinity for neuroligin-1 (Nlgn1) than Nrxn1b Nxph1 was prevented by mutating the aforementioned N-linked containing an insert in SS4 (Boucard et al, 2005; Comoletti et al, glycosylation sites to aspartic acid (Nxph13ND). Crystallization trials 3ND SS2 3ND SS2A+ 2006; Shen et al, 2008; Elegheert et al, 2017), but the structural of Nxph1 -LNS2 À and Nxph1 -LNS2 complexes yielded basis for this regulatory effect is unknown. The structure of the crystals that diffracted to 1.9 A˚ resolution. SS2 Nlgn1-Nrxn1b complex lacking the SS4 splice insert has been deter- Because the structure of the LNS2 À domain of Nrxn1 was SS2 mined (Arac¸ et al, 2007; Tanaka et al, 2011), but no structure is known, the structure of the Nxph1-LNS2 À complex was deter- SS2 available for the Nlgn1-Nrxn1b complex containing the SS4 insert, mined by molecular replacement with the Nrxn1 LNS2 À domain even though most neuroligins bind to Nrxn1b containing an SS4 as the search model, resulting in a well-resolved electron density insert, albeit with a lower affinity. Moreover, no site of alternative map for the unknown structure of Nxph1—roughly half of the splicing in neurexins except for SS4 has been studied in detail. Apart amino acid content of the structure of the complex. This electron from SS4, only SS2 in the LNS2 domain is known to regulate bind- density map was of sufficient quality to build an atomic model of ing of dystroglycan (Sugita et al, 2001). Because LNS2 is also the Nxph1 by automated methods (Materials and Methods; Table 1). In neurexin domain that binds to neurexophilins, dystroglycan turn, the structure of this complex was used as a search model for competes with neurexophilins for binding to LNS2 (Reissner et al, determining the structure of the Nxph1-LNS2SS2A+ complex by 2014), but the significance of dystroglycan binding to neurexins molecular replacement. SS2 SS2A+ remains unclear (Fru¨h et al, 2016). Thus, there is an urgent need for Both the Nxph1-LNS2 À and Nxph1-LNS2 structures insight into how neurexins interact with ligands such as neurex- exhibit a similar overall architecture, with Nxph1 forming a striking ophilins in an alternative splicing-dependent manner via domains extension of the LNS2 jelly-roll b-sandwich described previously other than LNS6. (Sheckler et al,2006;Chenet al, 2011; Miller et al, 2011; Fig 1B). Here, we report splice-dependent interactions between Nxph1 Overall, the LNS2 structure consists of 14 anti-parallel b-strands, and Nrxn1. Specifically, we determined high-resolution structures of one a-helix, and one disulfide bond connecting C444 to C480 Nxph1 in a complex with two Nrxn1 LNS2 SS2 splice variants and (Fig EV2). The structure of the LNS2 domain in both Nxph1-LNS2 performed in vitro binding experiments to relate the structural data complexes is similar to those of LNS2 alone (PDB IDs 2H0B, 3R05, to binding properties. These structures reveal a previously unob- and 3POY) except for the b7 and b10 strands and the loop that served type of protein–protein interface, wherein the b-sandwiches connects b10 to the rest of the molecule (Appendix Fig S1). b7 and of Nxph1 and LNS2 form an extended, contiguous fold. Furthermore, b10 form part of the interface to Nxph1. Thus, complex formation the eight-amino acid splice insert of SS2 augments and stabilizes this with Nxph1 reorders these b-strands and the loop. interface, which results in increased binding affinity. Taken together, The Nxph1 structure comprises eight b-strands and contains three these data reveal a structural mechanism by which alternative splic- disulfide bonds near loop regions. The disulfide bonds in Nxph1 link ing modulates the biochemical properties of a large and previously cysteine pairs C194-C231, C210-C218, and C239-C256 (Figs 1B and unobserved interface in a synaptic cell-adhesion protein complex. EV3). The connectivity pattern of the disulfide bonds in the Nxph1 structure is consistent with the pattern inferred by mass spectrome- try (Reissner et al, 2014). The b-sandwich of Nxph1 consists of two

Results anti-parallel b-sheets formed by b1–b3, b4, b7, and b8, while b5 and b6 run anti-parallel to one another and are solvent-exposed (Fig 1B). Structures of Nxph1 in complex with LNS2 splice variants The single b-sandwich architecture of the Nxph1 structure differs of Nrxn1 markedly from the previously proposed architecture that predicted the presence of distinct N- and C-terminal domains connected by a Attempts at producing Nxph1 using insect cell expression systems linker (Missler & Su¨dhof, 1998). Rather, the b-strands formed by failed due to intracellular retention of the protein, so the BacMam these N- and C-terminal regions are interleaved in the structure, system was used to co-express secreted mature Nxph1 and Nrxn1 mapping to the strands b1–b4 and b5–b8 in the Nxph1 structure,

LNS2 proteins from HEK293S GnTI- cells (Dukkipati et al,2008; respectively (Appendix Fig S2). The loop connecting b4 to b5 Goehring et al, 2014). This led to the efficient secretion of Nxph1- contains a ~50 glycine- and proline-rich amino acid sequence in SS2 Nrxn1 LNS2 complexes. Two splice variants of Nrxn1—LNS2 À Nxph4 but not in the other neurexophilins (Appendix Fig S2). Analy- with no insert in SS2 and LNS2SS2A+ with a conserved eight-residue sis of the Nxph1 structure using DALI (Holm & Laakso, 2016) insert in SS2—were used. Highly homogeneous samples of the revealed that Nxph1 shares only very low-level structural similarity SS2 SS2A+ Nxph1-Nrxn1 LNS2 À and Nxph1-Nrxn1 LNS2 complexes with a few proteins. Among these, the highest scoring structure is a with 1:1 stoichiometry were prepared as assessed by size-exclusion possible leukocidin subunit (z-score = 8.9, sequence iden- chromatography–multi-angle light scattering (SEC-MALS; Fig EV1C tity = 10%, RMSD = 3.2) and the only proteins that have similarity and D). In the following, we refer to these complexes simply as the to the Nxph1 topology are members of the adaptin family (z- SS2 SS2A+ Nxph1-LNS2 À and Nxph1-LNS2 complexes. score = 7.9, sequence identity = 4%, RMSD = 3.3).

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Table 1. Data collection and refinement statistics.

SS2 SS2A+ Structure Nxph1-Nrxn1 LNS2 À Nxph1-Nrxn1 LNS2 Beamline APS NE-CAT 24-ID-C SSRL 12-2 Wavelength 0.97910 Å 0.97946 Å Resolution range 45.60–1.94 (2.01–1.94) 45.92–1.95 (2.02–1.95) Space group C 121 C 121 Unit cell 71.7 61.0 79.6 90 106.7 90 72.0 61.4 79.4 90 106.2 90 Total reflections 317,952 (21,624) 279,800 (12,212) Unique reflections 24,370 (1,662) 23,404 (583) Multiplicity 13.0 (9.1) 12.0 (6.3) Completeness (%) 94.99 (68.58) 83.17 (24.18) Mean I/sigma (I) 14.05 (1.29) 11.40 (0.78) Wilson B-factor 39.50 20.68 R-merge 0.100 (1.214) 0.108 (1.421) CC1/20.999 (0.796) 0.998 (0.559) Reflections used in refinement 23,258 (1,657) 20,360 (583) Reflections used for R-free 1,992 (142) 1,997 (57) R-work 0.198 (0.288) 0.196 (0.314) R-free 0.247 (0.331) 0.240 (0.315) Number of non-hydrogen atoms 2,656 2,791 Macromolecules 2,589 2,611 Solvent 67 180 Protein residues 325 327 RMS (bonds) 0.009 0.005 RMS (angles) 0.88 1.07 Ramachandran favored (%) 97.16 95.58 Ramachandran allowed (%) 2.84 4.42 Ramachandran outliers (%) 00 Rotamer outliers (%) 2.74 0 Clashscore 6.84 5.42 Average B-factor 76.06 35.86 Macromolecules 76.47 35.83 Solvent 59.94 36.28 Number of TLS groups 22 Statistics for the highest resolution shell are shown in parentheses.

The heterodimeric interfaces between the Nxph1 and LNS2 hydrophobic and polar interactions from Nxph1 b8 and LNS2 b10 SS2 SS2A+ b-sandwiches in the Nxph1-LNS2 À and Nxph1-LNS2 spanning the b-sandwich formed by Nxph1 and LNS2 (Appendix Fig structures have buried surface areas of 1,223 A˚ 2 and 1,209 A˚ 2, S3C and D). A PISA search using default parameters identified no respectively, much larger than another interface formed between instances of similar architecture within all deposited structures in Nxph1 molecules in the crystal lattice (Appendix Fig S3A and B). the (RCSB). Thus, this is a striking example of a These interaction interfaces of Nxph1 may be involved in homo- heteromeric anti-parallel b-sandwich extension. meric interactions, as suggested by the presence of dimers and tetra- SS2 mers of Nxph1 alone (based on SEC-MALS and SEC-SAXS Key elements of the Nxph1-LNS2 À interface experiments; Figs EV1E and EV4A; Table EV1). SS2 The Nxph1-LNS2 interface in both complexes is formed via anti- Visual inspection of the Nxph1-LNS2 À structure revealed multiple parallel b-sheet extensions mediated by interactions between b1 and key interface residues (Fig 2A). Among these residues, I401 of SS2 b8 of Nxph1 with b10 and b7 of LNS2, respectively (Fig 1B). Further- LNS2 À interacts with a particularly deep hydrophobic binding more, Nxph1 and LNS2 interlock in a staggered fashion with pocket in Nxph1 (formed primarily by Y249 and L251; Fig 2A and

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A

β7LNS2 A353 F351 V355 L251

Y407 T405 I401 T403

β10 Y249 LNS2

β9LNS2

β8LNS2

B β7 β8 β9 β10 Mouse Nrxn1SS2- 352 362 372 397 407 417 Mouse Nrxn1SS2- GSGAFEA LVEP VNG K FNDN A WH D VK VT RNLRQ V TISVDG *I LT T TGYTQEDYTMLGSDD Mouse Nrxn2SS2- GSGAFEA L VEP VNG K FNDN A WH D VR VT RNLRQ V TISVDG I LT T TGYTQEDYTMLGSDD Mouse Nrxn3SS2- GSGAFEA IVEP VNG K FNDN A WH D VK VT RNLRQ V TISVDG I LT T TGYTQEDYTMLGSDD Nxph1 Nxph1 binding SS2 binding

C D E Contour level = 2

YY249249

I401 L251L251

SS2 Figure 2. Key features of the Nxph1-LNS2 À interface. SS2 A View of the Nxph1-LNS2 À interface showing key residues as sticks. The yellow surface represents the Nxph1 molecule. SS2 B Sequence alignment of mouse Nrxn1–3 LNS2 À showing key regions involved in Nxph1 binding. Sequences are numbered in reference to the canonical UniProt SS2 mouse Nrxn1 sequence Q9CS84-1. Secondary structure elements of LNS2 À are shown at the top and correspond to elements shown in (A). SS2 C Electron density map showing the LNS2 À I401 residue (highlighted in red in B) interacting with a hydrophobic binding pocket formed primarily by Y249 and L251 of Nxph1. SS2 SS2 D Representative BLI traces showing decreased binding of Nxph1 to the Nrxn1 LNS2 À (I401Q) mutant compared to that of wild-type Nrxn1 LNS2 À. Replicate numbers are shown in (E). SS2 SS2 E Comparison of binding affinities for Nxph1 binding to mouse Nrxn1–3 LNS2 À and LNS2 À (I401Q), plotted on a logarithmic scale. Error bars represent SEM, and replicate numbers are indicated in bars. Significance values were calculated using Welch’s t-test, *P < 0.05, **P < 0.01.

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C). These interface residues are highly conserved across both mouse could not assess the effects of glycosylation on Nxph1-neurexin SS2 (Fig 2B) and vertebrate neurexins (Fig EV2) as well as neurex- interactions. The I401Q mutation in Nrxn1 LNS2 À greatly SS2 ophilins (Fig EV3). However, these Nxph-binding sites are not decreased binding of Nxph1 to Nrxn1–3 LNS2 À (Figs 2D and E, conserved in invertebrate neurexins, consistent with the lack of and EV5B; Table 2). The mutant proteins used in the binding experi- neurexophilins in invertebrates (Fig EV2). ments were folded as assessed by circular dichroism (CD) spec- SS2 To validate the interface observed in the Nxph1-LNS2 À crystal troscopy (Appendix Fig S4B), so the reduction in binding affinity is structure, mutagenesis and co-immunoprecipitation experiments not due to a folding defect. Furthermore, the same mutant proteins were performed for selected neurexin residues at the interface exhibited monodisperse size-exclusion chromatography elution pro- (Fig 2A). The residues were selected by visual inspection of the files as evaluated by SEC-MALS experiments with the Nrxn2 SS2 structure, along with energetic analyses of the interacting residues LNS2 À (I401Q) domain (Fig EV1H). Thus, the reduction in bind- SS2 by PISA. Mutagenesis of Nrxn3 LNS2 À residues, corresponding to ing affinity is not due to aggregation. The primary sequence conser- SS2 SS2 I401, T405, and Y407 of Nrxn1 LNS2 À, resulted in decreased co- vation of the interface residues suggests that the Nxph1-LNS2 À SS2 immunoprecipitation of LNS2 À with Nxph1 (Appendix Fig S4A). binding interface is maintained in all vertebrates (Figs EV2 and SS2 These residues are all located in the LNS2 À b10 strand that is part EV3). SS2 of the hydrophobic core of the Nxph1-LNS2 À interface. Individual mutation of these residues to glutamine substantially reduced Nxph1 Structural changes and modulation of binding by co-immunoprecipitation. Of these mutants, I401Q had the greatest alternative splicing SS2 effect on Nxph1 binding to LNS2 À, nearly eliminating the interac- tion (Appendix Fig S4A). Similarly, confocal imaging experiments In the structure of the Nxph1-LNS2SS2A+ complex, the 384-HAM- showed that the I401Q mutation reduced localization of Nxph1 with 386 sequence of SS2A extends the N-terminal part of LNS2 b9 SS2 membrane-bound Nrxn1 LNS2 À when co-expressed in HEK293T (Fig 3A). The other part of the SS2A sequence (379-HSGIG-383) cells (Fig EV5A). Interestingly, this mutation was previously found forms a disordered, solvent-exposed loop as suggested by corre- to have an effect in experiments using scanning mutagenesis and sponding weak electron density. imaging of co-expressed constructs to explore the binding of Nxph1 Structural remodeling at and near the splice insert site as a to LNS2 (Reissner et al, 2014; Neupert et al, 2015). function of SS2 alternative splicing is striking and extensive. Dif- Biolayer Interferometry (BLI) was then used to quantitatively ference density maps calculated by subtracting the observed struc- SS2 assess the strength of the Nxph1-LNS2 interaction (Fig 2D and E; ture factor amplitudes of the Nxph1-LNS2 À structure from those SS2 SS2A+ Table 2). Nxph1 binds Nrxn1 LNS2 À with nanomolar affinity of the Nxph1-LNS2 structure showed strong positive electron (Fig 2E; Table 2). While glycosylation of Nxph1 may stabilize its density throughout the region near SS2A (Fig 3B). Strong positive binding to neurexin (Reissner et al, 2014), we were unable to difference peaks revealed the extension of the b9 384-HAM-386 produce well-behaved Nxph1 without glycans on its own and thus part of the SS2A insert sequence in the Nxph1-LNS2SS2A+

Table 2. Biolayer Interferometry binding data.

Binding pair Mean KD (nM) Kon (1/M*s) Koff (1/s) SS2 Nxph1-Nrxn1 LNS2 À 299.2 58.3 398.6 31.51.2E-04 2.3E-05 Æ Æ Æ SS2 Nxph1-Nrxn1 LNS2 À (I401Q) 16,420 2,299 219.6 22.03.4E-03 1.8E-04 Æ Æ Æ Nxph1-Nrxn1 LNS2SS2A+ 49.0 8.42,043 139.69.7E-05 1.2E-05 Æ Æ Æ Nxph1-Nrxn1 LNS2SS2A+ (I401Q) 472.0 66.4 419.2 27.82.0E-04 3.3E-05 Æ Æ Æ Nxph1-Nrxn1 LNS2SS2AB+ 43.3 4.72,168 2.59.4E-05 1.0E-05 Æ Æ Æ Nxph1-Nrxn1 LNS2SS2AB+ (I401Q) 499.3 17.7 673.8 23.53.4E-04 5.5E-06 Æ Æ Æ SS2 Nxph1-Nrxn2 LNS2 À 124.3 18.7 553.5 13.56.9E-05 1.1E-05 Æ Æ Æ SS2 Nxph1-Nrxn2 LNS2 À (I401Q) 39,340 13,662 380.8 25.32.5E-03 2.1E-04 Æ Æ Æ SS2 Nxph1-Nrxn3 LNS2 À 206.5 12.2 444.5 7.59.2E-05 5.9E-06 Æ Æ Æ SS2 Nxph1-Nrxn3 LNS2 À (I401Q) 1,941,800 555,480 10.5 4.61.2E-02 1.1E-03 Æ Æ Æ Nxph1-Nrxn3 LNS2SS2A+ 31.4 3.52,060 108.26.4E-05 7.2E-06 Æ Æ Æ SS2 Nxph3-Nrxn1 LNS2 À 260.0 83.8 209.3 14.61.0E-04 4.1E-05 Æ Æ Æ SS2 Nxph3-Nrxn2 LNS2 À 212.2 52.0 301.8 14.46.7E-05 1.9E-05 Æ Æ Æ SS2 Nxph3-Nrxn3 LNS2 À 177.3 18.1 178.5 10.03.1E-05 3.3E-06 Æ Æ Æ SS2 Nxph3-Nrxn1 LNS2 À (I401Q) 3,345 276.3 193.8 5.56.4E-04 3.7E-05 Æ Æ Æ SS2 Nxph3-Nrxn2 LNS2 À (I401Q) 1,315 143.2 350.3 37.44.4E-04 2.1E-06 Æ Æ Æ SS2 Nxph3-Nrxn3 LNS2 À (I401Q) 3,501 213.6 153.0 11.15.3E-04 1.6E-05 Æ Æ Æ Error values are SEM.

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8 A β LNS2 RMSD = 0.292 Å

β9 LNS2 M386 SS2A I401 H384 β10LNS2

H125 β1Nxph1

B

L376

L346

Y407 ~90° Q378 I401 M386

C β6β7 β8 β9 β10 Mouse Nrxn1SS2A+ 342 352 362 372 397 407 417 Mouse Nrxn1SS2A+ GAV S LVINLGSGAFEA LVEP VNG K FNDN A WH D V K V T RNLRQ H S GIGHAMVNKLH CS V TISVDG I LT T TGYTQEDYTMLGSDD Mouse Nrxn2SS2A+ GAV W LVINLGSGAFEA LVEP VNG KFNDN A WH D V R V T RNLRQ HA GIGHAM VNKLH YL VTISVDG ILT TTGYTQEDYTMLGSDD Mouse Nrxn3SS2A+ GAV S LVINLGSGAFEA IVEP VNG K FNDN A WH D V K V T RNLRQ H S GIGHAMVNKLH CL VTISVDG ILT TTGYTQEDYTMLGSDD Nxph1 S S 2 AB Nxph1 binding SS2A binding

D E

Figure 3.

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SS2A+ SS2 Figure 3. Comparison of the Nxph1-LNS2 structure to the Nxph1-LNS2 À structure and modulation of Nxph1-Nrxn1 LNS2 binding by alternative splicing. SS2 SS2A+ ◀ A Overall view and close-up of superimposed Nxph1-LNS2 À and Nxph1-LNS2 structures in cartoon representation. SS2 SS2A+ B Electron density maps comparing the Nxph1-LNS2 À and Nxph1-LNS2 structures. These maps cover the same region as shown in the close-up in (A). The SS2A+ 2mFo-DFc map for the Nxph1-LNS2 structure is shown in gray at a contour level of 1 r. The positive and negative Fo–Fo difference density maps (the difference SS2 SS2A+ SS2A+ between Nxph1-LNS2 À and Nxph1-LNS2 structure factor amplitudes using phases from the Nxph1-LNS2 model) are shown in green and red, respectively, at a contour level of 3 r. C Sequence alignment of mouse Nrxn1–3 LNS2SS2A/AB+ showing key regions involved in Nxph1 binding and SS2A and SS2AB inserts. Sequences are numbered in reference to the canonical UniProt mouse Nrxn1 sequence Q9CS84-1. Secondary structure elements are shown at the top and correspond to elements shown in the Nxph1-LNS2SS2A+ structure in (A). SS2A+ SS2AB+ SS2 D Representative BLI traces showing increased binding of Nxph1 to Nrxn1 LNS2 and Nrxn1 LNS2 compared to that of Nrxn1 LNS2 À. Replicate numbers are shown in (E). E Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 LNS2 splice variants and splice insert containing Nrxn1 LNS2 with the I401Q mutation; error bars represent SEM, and replicate numbers are indicated in or above bars. Significance values were calculated using Welch’s t-test, *P < 0.05, **P < 0.01, ****P < 0.0001.

structure. Along with this extension, a variety of secondary rear- that SS2A adds specific inter-molecular interactions to the Nxph1- rangements is evident in the difference density map. For example, LNS2 interface while also inducing regional conformational SS2 the electron density representative of Q378 in the Nxph1-LNS2 À changes to stabilize the complex. structure is replaced by that of M386 in the Nxph1-LNS2SS2A+ Given the extensive effect of the SS2A insert on the Nxph1-LNS2 structure. Furthermore, the M386 and H384 residues of SS2A structure and the proximity of SS2 to the Nxph1-binding site in LNS2

extend across b10 of LNS2 to interact with H125 on b1 of Nxph1. (Figs 1A and 3A–C, and EV2), inserts in SS2 are expected to increase The H384 and M386 residues have buried surface areas upon the affinity of Nxph1 for LNS2. Indeed, Nxph1 binds to Nrxn1 2 2 SS2A+ SS2 complex formation of approximately 13 A˚ and 12 A˚ , respectively, LNS2 with ~6-fold higher affinity than for Nrxn1 LNS2 À

as calculated by PISA. The presence of SS2A also likely stabilizes (Fig 3D and E; Table 2). In these analyses, greater Kon and nearly

the region proximal to the splice insert by the addition of identical Koff values were observed for Nxph1 binding to Nrxn1 hydrophobic interactions. Specifically, in the Nxph1-LNS2SS2A+ LNS2SS2A+ (Table 2), explaining the binding affinity differences SS2 structure, but not the Nxph1-LNS2 À structure, L376 points into (Fig 3D). the core of the molecule toward L346, which shifts along with the In addition to the SS2A insert found in all neurexins, a less

backbone of b7 further into the core of the molecule. In addition, frequent splice variant includes a larger insert extended by seven there is a shift of Y407 into the core of the LNS2 molecule in the additional residues: SS2AB (Ullrich et al,1995)(Figs3CandEV2). Nxph1-LNS2SS2A+ structure. Taken together, these structures show Using BLI, Nrxn1 LNS2 containing this SS2AB insert, LNS2SS2AB+,

AB

Figure 4. The SS2A insert does not affect Ca2+ sensitivity of Nxph1-LNS2 interactions. SS2 SS2A+ 2+ A Representative fluorescence polarization binding curves are shown for LNS2 À and LNS2 interacting with Nxph1 with or without Ca added. Replicate numbers are shown in (B). SS2 SS2A+ 2+ B Comparison of binding affinities of Nxph1 for Nrxn1 LNS2 À and Nrxn1 LNS2 with or without Ca added; replicate numbers are indicated in bars. Error bars represent SEM, and significance values were calculated using Welch’s t-test. ns, not significant.

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ABNxph1 Nxph3

Figure 5. Isoform specificity of neurexin–neurexophilin interactions. A Comparison of binding affinities of Nxph1 binding to different Nrxn1–3 LNS2 isoforms. SS2 B Comparison of binding affinities for Nxph3 binding to Nrxn1–3 LNS2 À. Data information: Error bars show SEM, and replicate numbers are indicated in or above bars. Significance values were calculated using Welch’s t-test, *P < 0.05,****P < 0.0001.

binds Nxph1 with similar kinetics and affinity as LNS2SS2A+ (Fig 3D the effect of Ca2+ on Nxph1-LNS2 interactions. Using a fluorescence and E; Table 2). anisotropy binding assay (Fig 4A), 2 mM Ca2+ had no effect on the Despite their differences, many amino acid interactions are never- binding affinities of Nxph1 for Nrxn1 LNS2SS2A+ or for Nrxn1 SS2A+ SS2 SS2 2+ theless conserved in the Nxph1-LNS2 and Nxph1-LNS2 À LNS2 À (Fig 4B). In addition, inclusion of 100 lM EDTA in Ca - structures. For example, the Nxph1-LNS2SS2A+ interface contains free fluorescence anisotropy binding assays had no effect on the the same key I401 residue found interdigitating with Nxph1 at the binding affinities, ruling out the possibility that trace amounts of SS2 2+ Nxph1-LNS2 À interface (Fig 3A, Appendix Fig S3D). Binding of Ca in the buffer could affect the Nxph1-LNS2 interactions Nxph1 to LNS2SS2A+ and LNS2SS2AB+ is similarly affected by the (Appendix Fig S5). Thus, Ca2+ is not a major regulator of the I401Q mutation in these proteins. In both Nrxn1 LNS2 splice vari- Nxph1-Nrxn1 interaction, consistent with the notion that extracellu- ants, the I401Q mutations also significantly reduced binding of lar Ca2+ is not thought to be a regulatory parameter. Nxph1 (Fig 3E). Again, these mutant proteins are folded as assessed by CD spectroscopy (Appendix Fig S4B) and exhibited monodisperse Isoform specificity of neurexophilin–neurexin interactions size-exclusion chromatography elution profiles, ensuring that the reduction in binding affinity was not due to a folding defect or to In addition to investigating the interactions of Nxph1 with Nrxn1, aggregation. The effects of the I401Q mutation in Nrxn1 LNS2SS2A+ the binding of Nxph1 to mouse Nrxn2 and Nrxn3 was assessed. SS2AB+ SS2 and in Nrxn1 LNS2 on Nxph1 binding are less pronounced Nxph1 binds to the LNS2 À domains of all neurexins (Nrxn1, than those observed for the I401Q mutation in the splice insert-free Nrxn2, and Nrxn3) with similar nanomolar affinities (Fig 5A; SS2 Nrxn1 LNS2 À variant, consistent with the observed stabilization Table 2), consistent with the sequence conservation of neurexin resi- of the Nxph1-Nrxn1 interface via additional interactions contributed dues at the binding interface (Figs 2B and EV2). Nrxn3 LNS2SS2A+ by the SS2A insert. again binds Nxph1 with a ~6-fold higher affinity than Nrxn3 SS2 LNS2 À (Fig 5A; Table 2). As with Nrxn1 binding to Nxph1,

The SS2 insert has no effect on the calcium sensitivity of Nxph1- greater Kon and nearly identical Koff values were observed for Nrxn3 LNS2 interactions LNS2SS2A+ binding to Nxph1 (Table 2). The SS2A-induced increase in Nrxn3 binding affinity for Nxph1 is similar to that of Nrxn1, We did not observe electron density in the Ca2+ binding region of consistent with the high primary sequence conservation of the splice LNS2 (Sheckler et al,2006),presumablybecauseCa2+ was not insert (Fig 3C). We did not test the other splice variants of Nrxn2, included during crystallization. Considering the proximity of the and the Nrxn3 LNS2SS2AB+ variant did not express well, preventing Nxph1-LNS2 interface to the LNS2 Ca2+-binding region, we tested further studies.

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Presynaptic membrane

Glycosylated stalk with cysteine-loop Neuroligin-1 LNS6 +SS4 (Nlgn1) Nlgn1 affinity

~200 Å Neurexin-1 LNS2 +SS2 Neurexophilin-1 Nxph1 affinity (Nxph1)

Postsynaptic membrane

Figure 6. Model of neurexin bound to both neurexophilin and neuroligin in the synapse. A hypothetical composite structure of Nrxn1 bound to both Nxph1 and Nlgn1 is shown. The Nxph1-LNS2SS2A+ complex structure was superimposed on the near full-length crystal structure of Nrxn1a (3R05) containing LNS2-LNS6 domains (Chen et al, 2011). Note that there are no coordinates for the Nrxn1a LNS1 or EGFA domains; structures shown here are models based on the EGFC and LNS6 domains of Nrxn1a (3R05). Nlgn1 from the crystal structure of the Nrxn1b-Nlgn1 complex (3BIW; Aracß et al, 2007) is modeled by superimposition of that complex with the structure of Nrxn1a. Stalk regions, transmembrane domains, and intracellular regions for Nlgn1 and Nrxn1a were modeled based on mouse protein sequences using PyMOL. For Nrxn1, the cysteine loop was modeled based on the reported cysteine-loop sequence (Sterky et al, 2017), and glycosylation of the stalk region was modeled based on predicted O-linked glycosylation sites (Steentoft et al, 2013). Note the opposite effects on binding affinities of inserts at SS2 and SS4 for the Nrxn1 LNS2-Nxph1 and Nrxn1b-Nlgn1 complexes, respectively.

As with the I401Q mutation in Nrxn1, the equivalent mutations may be some isoform specificity to the interaction of Nxph1 with SS2 in Nrxn2 and Nrxn3 LNS2 À domains (referred to as Nrxn2 Nrxn1, Nrxn2, and Nrxn3 that becomes apparent when the interac- SS2 SS2 LNS2 À (I401Q) and Nrxn3 LNS2 À (I401Q) for simplicity) also tion is destabilized by the Nrxn I401Q mutation. SS2 significantly reduced binding affinities for Nxph1 (Fig 2E). Specifi- Binding of Nxph3 to Nrxn1–3 LNS2 À was also tested. Similar SS2 cally, the I401Q mutations decreased the binding affinities of Nxph1 to Nxph1, Nxph3 binds to the Nrxn1, Nrxn2, and Nrxn3 LNS2 À SS2 to the Nrxn2 and Nrxn3 LNS2 À domains from the nanomolar to domains with nanomolar affinities (Fig 5B; Table 2). Furthermore, SS2 the micromolar range and from nanomolar to the millimolar range, using BLI, the I401Q mutation in Nrxn1–3 LNS2 À showed respectively (Fig 2E; Table 2). These binding data suggest that there reduced binding of Nxph3, increasing the KD of Nxph3 to Nrxn1–3

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from the nanomolar to the micromolar range (Fig EV5B; Table 2). explain why LNS2SS2A+ and LNS2SS2AB+ have higher affinities for SS2 Comparison of Nxph1 and Nxph3 binding to wild-type and I401Q Nxph1 than LNS2 À (Fig 3E). Mutation of a key interacting mutant Nrxn1–3 reveals that the reduction in binding by the I401Q isoleucine residue in both Nrxn1 LNS2SS2A+ and Nrxn1 mutation is not as large for Nxph3 as it is for Nxph1 (Fig EV5B; LNS2SS2AB+ also significantly reduced the binding affinity of Table 2). These binding data suggest that isoform specificity may Nxph1 for those domains (Fig 3E), suggesting that SS2 inserts exist for Nxph1 and Nxph3 binding to neurexins. Unfortunately, augment the binding of LNS2 to Nxph1. we were unable to produce recombinant Nxph2 and Nxph4 alone The effects of alternative splicing on the neurexophilin–neurexin SS2 or in a complex with Nrxn1 LNS2 À, precluding quantitative interaction contrast with previous findings for a related synaptic measurements of interactions between neurexins and these protein interface. While LNS2 domains with SS2 inserts show higher proteins. affinity for Nxph1 than insert-free LNS2, the opposite is true for In addition to quantitative binding assays, we performed qualita- neurexin–neuroligin interactions, where Nrxn1b with a 30-amino tive confocal fluorescent imaging assays of HEK293T cells co- acid insert in SS4 (b-Nrxn1SS4+) has a lower affinity for neuroligins SS2 SS2 SS4 expressing membrane-bound Nrxn1 LNS2 À or Nrxn1 LNS2 À than b-Nrxn1 À (Boucard et al,2005;Elegheertet al, 2017; Fig 6). (I401Q) with secreted Nxph1–4(FigEV5A).Theseco-expression In this case, the SS4 insert functions differently than SS2 inserts; the SS2 data show that the I401Q mutation in Nrxn1 LNS2 À reduces the SS4 insert likely sterically interferes with binding of neuroligin to b- SS2 SS4+ localization of Nxph1–3 with Nrxn1 LNS2 À (Fig EV5A). Unfortu- Nrxn1 (Shen et al, 2008), whereas LNS2 with SS2A inserts— nately, Nxph4 did not express well in these cells. and likely SS2AB inserts—form additional interactions with Nxph1 SS2 Together, these BLI and imaging data confirm that mouse not found between Nxph1 and LNS2 À. However, in the case of neurexins bind to Nxph1, Nxph2, and Nxph3, consistent with the LNS2 the role of alternative splicing is similar to that of cerebellin conservation of the Nxph1-binding site in vertebrate neurexins. binding in the context of LNS6 alternative splicing, where cerebellin only binds LNS6 domains augmented with a SS4 insert (Uemura et al, 2010; Matsuda & Yuzaki, 2011; Elegheert et al, 2016). Unfortu- Discussion nately, there is no high-resolution structure available of the cere- bellin-LNS6SS4+ complex to reveal how SS4 affects the interaction. Here, we describe high-resolution structures of a neurexophilin and Nevertheless, low-resolution negative stain electron microscopy analyzed its interaction with neurexins by crystallization of Nxph1 data suggest that LNS6SS4+ interacts with the flexible cysteine-rich SS2 SS2A+ in a complex with the LNS2 À and the LNS2 domains of region in cerebellin-1 (Cheng et al, 2016; Elegheert et al, 2016) and Nrxn1. These analyses reveal new atomic-resolution insights into likely cerebellin-4 (Zhong et al, 2017). how alternative splicing of a neurexin shapes its interactions with a ligand. The structures reveal a unique binding interface wherein the General insights into neurexophilin architecture b-sandwiches of LNS2 and Nxph1 augment one another, essentially and conservation forming a single contiguous domain (Fig 1B). This heteromeric b- sandwich represents a previously unreported mode of a protein– These structural data enable an accurate definition of the domain protein interaction as assessed by PISA searches of all deposited structure of neurexophilins. Previous sequence analyses proposed structures in the Protein Data Bank (RCSB). The Nxph1-LNS2 that the mature fragment of neurexophilins consists of separate N- complexes are formed by anti-parallel b-strand interactions as well and C-terminal domains (Missler & Su¨dhof, 1998; Missler et al, as by additional specific hydrophobic and polar interactions that 1998; Reissner et al, 2014), but the crystal structures presented here span the Nxph1-LNS2 b-sandwich (Figs 1B, 2A, and 3A; reveal that mature Nxph1 does not contain two independently Appendix Fig S3C and D). Together, these interactions contribute to folded domains. Thus, the primary structure of neurexophilins is the strength of Nxph1-LNS2 binding, consistent with its nanomolar redefined here as consisting of a signal peptide, pro-domain, polyba- affinity (Figs 2E, 3E, and 5; Table 2). Furthermore, mutation of a sic cleavage site, and mature fragment. Nxph4 contains an extra key neurexin residue (I401) substantially reduced binding of Nxph1 sequence insert between b4 and b5, which presumably emerges as a SS2 and Nxph3 to the LNS2 À variants of all three neurexins (Figs 2E large loop from the smaller face of the neurexophilin b-sandwich and EV5B). (Appendix Fig S2). Key residues of the Nrxn1 LNS2-binding site in Nxph1 are highly Effects of alternative splicing conserved in all vertebrate neurexophilins (Fig EV3), as are the Nxph1-binding residues in neurexins (Fig EV2). Moreover, binding Intriguingly, alternative splicing of the Nrxn1 LNS2 domains data show that Nxph1 binds to all neurexins with nanomolar affin- modulates the architecture of the Nxph1-LNS2 complexes. Alterna- ity, as does Nxph3 (Fig 5). Importantly, Nxph1 binding is not only tive splicing alters the strength of neurexophilin binding to neur- regulated by SS2 alternative splicing, but may also differ depending exin by remodeling the pattern of interactions at and near the on which of the three neurexins is present (Fig 5); in other words, a interface between the two molecules. Specifically, extension of particular neurexophilin may bind preferentially to a particular

LNS2 b9 by the SS2A insert (Figs 1B and 3A) increases the affinity neurexin with additional fine-tuning achieved through SS2 alterna- of the Nxph1-LNS2 interaction by enhancing the interaction kinet- tive splicing. Although the observed differences in affinities are ics (Fig 3D; Table 2). This is accomplished by the addition of small, they could be amplified in the cellular context with multiple specific contacts at the Nxph1-LNS2 interface in the presence of neurexins and neurexophilins interacting in a restricted space. This SS2A as well as by increasing the number of stabilizing hydropho- exciting possibility would imply that separate isoform-specific, bic interactions nearby (Fig 3B). These structural features likely neurexin–neurexophilin signaling pathways could be organized in

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space. Future research will have to address this possibility by to its N-terminus and an HRV 3C protease-cleavable Twin-Strep tag conducting systematic binding experiments of synaptic assemblies. fused to its C-terminus. Note that Nrxn1 residues numbered here Finally, conservation suggests that this tuning is a relatively recent and referred to throughout the paper are in reference to the canoni- evolutionary innovation. While the Nxph1-binding site is highly cal UniProt mouse Nrxn1 Isoform 1a sequence: Q9CS84-1. In order conserved in vertebrate LNS2, it is not present in invertebrate neur- to prevent artificial inter-molecular disulfides from forming between SS2 SS2 exin sequences (Fig EV2), suggesting that the neurexophilins may LNS2 À, the LNS2 À C293A construct was made using the Quik- have evolved in vertebrates as neurexin ligands. The conservation Change II Site-Directed Mutagenesis Kit (Agilent). We later found of the neurexophilins in vertebrates is striking, and their absence that inclusion of 50 nM TCEP in buffers could prevent inter-mole- from invertebrates may reflect an important role of the neurex- cular disulfides from forming between LNS2, so we used 50 nM ophilins in vertebrate evolution. TCEP during purification of LNS2SS2A+ [mouse Nrxn1 (283–386; 394–480)]. Concluding remarks To obtain sufficient quantities of protein suitable for structural studies, constructs were cloned into the pEG BacMam vector and Nxph1 is likely an important regulator of synaptic transmission were co-expressed using the BacMam expression system in (Born et al,2014),butwhatexactlyNxph1andtheotherneurex- HEK293S GnTI- cells (Dukkipati et al,2008;Goehringet al, 2014). ophilins do and how they perform these functions remains to be Briefly, 293S cells were grown in FreeStyle Expression Media defined. The high binding affinities of Nxph1 and Nxph3 for neurex- (Thermo Fisher) supplemented with 2% FBS at 37°C in 8% CO2 to a ins probably reflect a synaptic role for their interaction. Given that density of 1–2 million cells per milliliter and co-transduced with various neurexophilins are expressed in distinct populations of Nxph1 and LNS2 viruses; approximately 8–24 h after transduction, neurons in the central and enteric nervous systems (Fo¨ldy et al, 10 mM sodium butyrate was added to the expression cultures and 2016; Tasic et al, 2016; Saunders et al, 2018; Zeisel et al, 2018), it is the temperature reduced to 30°C. Proteins were expressed for likely that neurexophilins contribute to the organization of synapses approximately 72 h, and then, cell culture supernatants containing through neurexin binding. However, it is unclear, for example, secreted proteins were harvested. Cell culture supernatants were whether neurexophilins function exclusively at the presynapse or concentrated, and buffer was exchanged into 20 mM Tris pH 8.0 whether they act at all of the output or input synapses of a neuron. with 150 mM NaCl (TBS 20/150 pH 8.0) and 10 mM imidazole at The overall extent to which different neurexophilin and neurexin room temperature. The buffer-exchanged, recombinant protein- isoforms exhibit differential interactions is also unclear. Moreover, containing solution was loaded onto a 5-ml HisTrap (GE) using a SS2 alternative splicing is tightly regulated temporally and spatially A¨ KTA Start (GE) at 4°C. After washing with 20 mM Tris pH 8.0 with (Ullrich et al, 1995), but how that regulation relates to the function 300 mM NaCl (TBS 20/300 pH 8.0) and 50 mM imidazole, a 5-ml of neurexophilins and their possible competition with dystroglycan StrepTrap column (GE) was connected in tandem to the HisTrap. for binding (Reissner et al, 2014) remains to be investigated. The Protein was then eluted off of the HisTrap and directly onto the structural and biochemical data presented here can be used in struc- StrepTrap using TBS 20/300 pH 8.0 and 300 mM imidazole (with or ture-guided functional assays to further reveal the neurological without 50 nM TCEP). Following a brief wash in TBS 20/300 pH 8.0 significance of the neurexophilins. and 300 mM imidazole with or without 50 nM TCEP, protein From the Nxph1-LNS2 structures presented here, we observed a complex bound to the StrepTrap was eluted from the column in TBS new protein interface architecture that is subject to regulation by 20/300 pH 8.0 and 2.5 mM desthiobiotin with or without 50 nM alternative splicing. Our analysis of Nxph1-LNS2 interactions TCEP (Appendix Fig S6). provides insight into how alternative splicing can regulate and fine- Affinity-purified Nxph1-LNS2 complex was then dialyzed into tune protein–protein interactions at the synapse. Furthermore, given 20 mM Tris pH 8.0 with 50 mM NaCl (TBS 20/50 pH 8.0; with or the general role of alternative splicing in many important eukaryotic without 50 nM TCEP) in preparation for anion exchange chromatog- cellular contexts, our structural analysis is broadly relevant to raphy, while affinity purification tags were removed using an HRV understanding how diverse protein interactions can be modulated 3C protease digestion overnight at 4°C. Cleaved Nxph1-LNS2 by alternative splicing. complex was then injected onto a 1-ml HisTrap connected to a 1-ml StrepTrap in TBS 20/50 pH 8.0 and 20 mM imidazole (with or with- out 50 nM TCEP) in order to remove any remaining tagged protein. Materials and Methods The flow-through from that step was then applied to a Mono Q 4.6/ 100 PE column (GE) in TBS 20/50 pH 8.0 with or without 50 nM Protein expression and purification for structural studies TCEP; protein was then eluted with a 40 c.v. gradient of 20 mM Tris pH 8.0 with the concentration of NaCl ranging from 50 to 250 mM For crystallographic studies, the mature form of rat Nxph1 (118– (TBS 20/50 pH 8.0 – TBS 20/250 pH 8.0). Fractions containing pure 271) was expressed with an Igj signal peptide fused to its N- Nxph1-LNS2 were combined and then analyzed by SDS–PAGE and terminus and an HRV 3C protease-cleavable double FLAG tag SEC-MALS. followed by a 6×-polyhistidine tag fused to its C-terminus. In this study, we focus on LNS2 domains, which are only present in the Crystallization and crystallographic data collection long-form Nrxna and not the shorter Nrxnb or Nrxn1c; therefore, when we refer to Nrxn1, Nrxn2, and Nrxn3, we are referring to For crystallization of Nxph1-LNS2 complexes, glycosylation of SS2 Nrxna versions of Nrxn. The LNS2 À domain of mouse Nrxn1 Nxph1 was prevented by mutating its N-linked glycosylation sites to (283–378; 394–480) was expressed with an Igj signal peptide fused aspartic acid (Nxph13ND). Nxph13ND-LNS2 complexes crystallized at

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2–3 mg/ml in the 16% PEG 3350 with 2% Tacsimate pH 5.0 and Protein expression and purification for biochemical studies 0.1 M sodium citrate tribasic condition from the PEG/Ion screen (Hampton Research). Nxph13ND-LNS2 crystals were cryoprotected Neurexin LNS2 mutants were made using the Q5 site-directed muta- in mother liquor containing 10% glycerol and 20% PEG 3350 and genesis kit (NEB). For BLI studies, neurexins were expressed with 3ND SS2 frozen in liquid N2. For the Nxph1 -LNS2 À crystals, data were an Igj signal peptide fused to their N-terminus and an AviTag collected at the Advanced Photon Source (APS) using the microfo- followed by hexahistidine tag on their C-terminus. For co-immuno- cus NE-CAT beamline 24-ID-C (Table 1). For the Nxph13ND- precipitation studies, neurexins were expressed with an Igj signal LNS2SS2A+ crystals, data were collected at the Stanford Synchrotron peptide fused to their N-terminus and a Twin-Strep tag on their Radiation Light Source (SSRL) using the microfocus beamline 12-2 C-terminus. For both co-immunoprecipitation and BLI studies, (Table 1). For simplicity, we refer to Nxph13ND simply as Nxph1 neurexin constructs were transfected into 293F cells in small-scale when discussing the crystal structures. We note, however, that all (25–400 ml) cultures using PEI (Sigma) in a 3:1 PEI-to-DNA ratio biochemical studies were performed with Nxph1 without the 3ND (Aricescu et al, 2006) or using the Expi-293 expression system 3ND mutations, as we were unable to prepare well-behaved Nxph1 (Thermo Fisher); cultures were grown at 37°C in 8% CO2. 10 mM alone. sodium butyrate or enhancers were added to the cultures 8–24 h post-transfection. Approximately 72 h later, secreted neurexin Crystallographic data processing, structure determination, proteins in the media were bound in batch to either Strep-Tactin and refinement resin (IBA Biosciences) or Ni-NTA resin (Qiagen) and purified using affinity chromatography. For co-immunoprecipitation studies, Crystallographic datasets were indexed, integrated, and scaled using neurexins were purified using Strep-Tactin affinity chromatography HLK2000 (Otwinowski & Minor, 1997). We first determined the (Schmidt et al, 2013). For BLI studies, neurexins were purified using SS2 structure of Nxph1 in a complex with LNS2 À using molecular Ni-NTA affinity chromatography, biotinylated enzymatically with replacement with a pre-existing LNS2 structure (2H0B; Sheckler BirA (Avidity) overnight, and further purified using a Superdex et al, 2006) as a search model, with the LNS2 domain amounting to Increase 200 10/300 gel filtration column (GE). SS2 roughly half of the complex. Electron density for the Nxph1 mole- For fluorescence anisotropy binding studies, Nrxn1 LNS2 À- cule emerged in difference maps, and a model of its structure was Twin-Strep and Nrxn1 LNS2SS2A+-Twin-Strep were expressed in built ab initio using phenix.autobuild followed by iterative manual 293F cells using the BacMam expression system. Secreted LNS2 building and refinement using Coot (Emsley et al, 2010) and auto- proteins were purified from media using Strep-Tactin affinity chro- mated refinement using phenix.refine (Adams et al, 2010). We matography, and affinity tags were removed overnight using HRV determined the structure of Nxph1 in a complex with LNS2SS2A+ 3C protease. LNS2 proteins were further purified using anion SS2 using molecular replacement with the Nxph1-LNS2 À structure as exchange chromatography and then labeled on a native thiol on a search model. The Nxph1-LNS2SS2A+ structure was also refined cysteine residue 293 with Alexa FluorTM 488 C5 Maleimide (Thermo with iterative manual building and refinement using Coot and auto- Fisher) overnight in TBS 20/150 pH 8.0 with 50 nM TCEP. SS2 mated refinement using phenix.refine. For the Nxph1-LNS2 À Neurexophilins expressed poorly when transfected into 293F cells SS2 structure, we modeled the full Nrxn1 LNS2 À domain (residues using PEI or ExpiFectamine. Therefore, the BacMam expression 283–378; 394–480) and residues 119–180, 191–212, and 217–263 of system was used to express Nxph1–4 in 293F cultures at 37°C and 8% SS2A+ Nxph1. For the Nxph1-LNS2 structure, we modeled residues CO2. The same Nxph1 construct used for crystallographic studies (but 283–378, 383–386, and 394–480 of LNS2SS2A+ and residues 119– without asparagine residues mutated) was used in all biochemical 180, 189–214, and 217–261 of Nxph1. The regions that were not studies. Rat Nxph2 (108–261), rat Nxph3 (50–252), and rat Nxph4 modeled in the structures, including residues 379–382 of SS2A, had (105–304) were expressed with an Igj signal peptide fused to their N- weak and disordered electron density. terminus and an HRV 3C protease-cleavable double FLAG tag followed by a 6×-polyhistidine tag fused to their C-terminus. All Nxph Analysis of crystallographic interfaces proteins used in biochemical studies contained affinity tags and glycans. We were unable to produce well-behaved Nxph2 and Nxph4. Interface areas were calculated using PISA, and the search of the Protein Data Bank (RCSB) was performed using PISA (Krissinel & SEC-MALS data collection Henrick, 2007). Data were collected using a Superdex 200 10/300 column at a flow rate Structural model figure preparation of 0.5 ml/min in phosphate-buffered saline pH 7.4 (PBS pH 7.4; Sigma). UV absorption, light scattering, and differential refractometry data were Figures showing structural models were made using PyMOL (The recorded for protein elution profiles using Dawn Heleos-II and Optilab PyMOL Molecular Graphics System, version 2.0 Schro¨dinger, LLC). rEX instruments (Wyatt technology). Baselines were corrected with Astra 7.1.2 (Wyatt technology) using a BSA reference and processed Sequence alignments with a differential refractive index value (dn/dc) value of 0.185.

Sequences were aligned using default parameters in CLC Main SEC-SAXS data collection and analysis Workbench 7 and 9 (Qiagen), and secondary structures from the Nxph1-LNS2 structures were assigned to alignments using ESPript 3 SEC-SAXS data were collected at SSRL beamline 4-2. Briefly, protein (Robert & Gouet, 2014). samples were injected onto a Superdex Increase s200 3.2/300

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column (GE) using an UltiMate 3000 HPLC (Thermo Fisher), and Cells were then imaged using a Nikon A1 Eclipse Ti confocal SAXS data were collected with 1-s exposures every 5 s as the microscope using a 20× objective. Images were analyzed using NIS- proteins eluted off of the column. Data were collected using beam Elements AR software (Nikon). energy of 11 keV and a PILATUS3 X 1 M detector (Dectris). Peak SEC-SAXS data points were selected for SAXS data analysis. Crystal- CD spectroscopy lographic models were fit to SAXS data using CRYSOL, and molecu- lar weights were determined using PRIMUS in the ATSAS software Proteins were diluted in TBS 20/150 pH 8.0 and 50 nM TCEP. CD suite (Franke et al,2017). spectra from 190 to 260 nm were collected using a 1.0 mm path length cell, 1.0 nm wavelength step, and 1.0-s averaging time at Co-IP binding assays 25°C with an AVIV Circular Dichroism Spectrometer Model 202-01. For each protein sample, five technical replicates were recorded and For Co-IP binding assays, 10 lg of LNS2 protein was mixed with then averaged. 10 lg of Nxph1 in 20 mM HEPES pH 7.4 with 150 mM NaCl (HBS). These protein mixtures were incubated with M2 anti-FLAG Biolayer interferometry magnetic beads (Sigma) for 15 min at room temperature. The beads were harvested using a magnetic tube rack and washed three Kinetics data were collected with an Octet RED384 (Pall Forte´Bio) times with HBS. Protein complexes were then eluted from the using a double reference subtraction method. Site-specific biotiny- beads using 30 ll of 100 lg/ll3× FLAG peptide (Sigma), and lated neurexin constructs were immobilized onto streptavidin- eluates were filtered using spin columns. Input and elution frac- coated tips and Nxph proteins associated and dissociated from the tions were run on Bio-Rad Criterion TGX Any kD gels in Tris/ neurexin molecules. Kinetics experiments were performed at 25°C Glycine/SDS running buffer (Bio-Rad) and transferred to 0.2 lm in freshly prepared PBS pH 7.4 with 50 nM TCEP and 1% BSA. At nitrocellulose using a Trans-Blot Turbo Transfer System (Bio-Rad). least four independent datasets were collected for each binding pair. Nitrocellulose membranes were blocked overnight in TBST with Dissociation constants were calculated using the global fit method 3% milk and then probed with either (1:2,000) mouse M2 anti- in the Octet Analysis software 9.0 (Pall Forte´Bio). FLAG antibody (Sigma) or (1:1,000) rabbit anti-Strep-tag II anti- body (Abcam) for 2 h at room temp. After washing, nitrocellulose Fluorescence anisotropy binding assays membranes were probed with IRDye 800CW Donkey anti-Mouse IgG (H + L) or IRDye 800CW Donkey anti-Rabbit IgG (H + L) Alexa 488-labeled LNS2 proteins were added at a concentration of secondary antibodies (LI-COR). After final washes, nitrocellulose 50 nM to a 1.5 × dilution series (from 0.07 to 20 lM) of Nxph1 in membranes were imaged using a LI-COR Odyssey CLx imaging TBS 20/150 pH 8.0 and 50 nM TCEP with or without 2 mM CaCl2 system. added. Alexa 488-labeled LNS2 proteins were also added at a concen- tration of 50 nM to wells containing TBS 20/150 pH 8.0 and 50 nM

Co-expression and confocal imaging TCEP with or without 2 mM CaCl2 added to serve as background controls. Ca2+-free binding experiments were repeated as above but in SS2 SS2 2+ Nrxn1 LNS2 À and Nrxn1 LNS2 À (I401Q) were expressed with the presence of 100 lMEDTAtodetermineiftraceamountsofCa an Igj signal peptide fused to their N-terminus and an AviTag in the buffer could affect Nxph1-LNS2 interactions. Plates containing followed by a Myc tag and an HRV 3C protease-cleavable CD8 trans- these mixtures were mixed with shaking for 10 s three times and incu- membrane domain fused to their C-terminus. The same Nxph1 and bated at room temperature for 35 min before measuring fluorescence Nxph3 constructs with C-terminal FLAG tags used in the binding anisotropy using a BioTek Synergy 2 Multi-Mode Microplate Reader. studies were used in these co-expression studies. Rat Nxph2 (108– Fluorescence polarization was calculated using BioTek Gen5 2.09 soft- 261) and rat Nxph4 (105–304) were expressed with an Igj signal ware, and background control values were subtracted from experimen- peptide fused to their N-terminus and an HRV 3C protease-cleavable tal Nxph1-LNS2 samples. Three independent experiments were double FLAG tag followed by a 6×-polyhistidine tag fused to their C- performed for each binding pair and condition. Binding data were plot- terminus. ted in GraphPad Prism 7, and fits to data were made using a non-linear HEK293T cells were transfected using Lipofectamine 3000 one-site total binding model with background constrained to zero. (Thermo Fisher). After approximately 48 h, these cells were washed once with DPBS (Thermo Fisher), fixed with 4% paraformaldehyde, Statistics and then washed three times with DPBS. Fixed cells were then blocked with antibody dilution buffer containing 5% normal goat For comparing binding affinities, two-tailed Welch’s t-tests were serum (Sigma) in DPBS (ADB) for 1 h. These cells were then incu- used. P-values are indicated in the figures as: *P < 0.05, **P < 0.01, bated overnight without agitation at 4°C with 1:1,000 dilutions of ***P < 0.001, and ****P < 0.0001. Crystallographic data and refine- mouse anti-FLAG M2 (Sigma) and polyclonal rabbit anti-cMyc ment statistics are provided in Table 1. (Sigma) in ADB. The cells were then washed three times with DPBS and incubated without agitation in 1:1,000 dilutions of goat anti- mouse Alexa Fluor 546 and goat anti-rabbit Alexa Fluor 647 in ADB Data availability for 1 h at room temp. Finally, stained cells were washed four times with DPBS and mounted on slides using DAPI Fluoromount-G Processed biolayer interferometry traces are available on request. SS2 SS2A+ Mounting Medium (Southern Biotech). The Nxph1-LNS2 À and Nxph1-LNS2 structures and

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diffraction data have been submitted to the Protein Data Bank reveal specific protein-protein and protein-Ca2+ interactions. Neuron 56: (RCSB) [PDB IDs 6PNP (http://www.rcsb.org/pdb/explore/ex 992 – 1003 plore.do?structureId=6pnp) and 6PNQ (http://www.rcsb.org/ Aricescu AR, Lu W, Jones EY (2006) A time- and cost-efficient system for pdb/explore/explore.do?structureId=6pnq)]. high-level protein production in mammalian cells. Acta Crystallogr D Biol Crystallogr 62: 1243 – 1250 Expanded View for this article is available online. Born G, Breuer D, Wang S, Rohlmann A, Coulon P, Vakili P, Reissner C, Kiefer F, Heine M, Pape H-C et al (2014) Modulation of synaptic function Acknowledgements through the a-neurexin-specific ligand neurexophilin-1. Proc Natl Acad Sci We thank the Brunger, Südhof, Garcia, and Weis laboratories for their support USA 111:E1274 – E1283 and input, Kang Shen for discussions, Justin Trotter for Nrxn3 constructs, Anna Boucard AA, Chubykin AA, Comoletti D, Taylor P, Südhof TC (2005) A splice Khalaj for confocal image analysis advice, Lior Almagor for help with the fluo- code for trans-synaptic cell adhesion mediated by binding of neuroligin 1 rescence anisotropy binding experiments, Mark Stolowitz and the Canary to a- and b-neurexins. Neuron 48: 229 – 236 Center at Stanford for use of the Octet RED384, and Tsutomu Matsui for help Chen F, Venugopal V, Murray B, Rudenko G (2011) The structure of neurexin with SEC-SAXS data collection and analysis. The pEG BacMam vector was a gift 1⍺ reveals features promoting a role as synaptic organizer. Structure 19: from Eric Gouaux, and the Nxph13ND construct was a gift from Markus Missler 779 – 789 and Carsten Reissner. S.C.W. is supported by the Stanford Genome Training Cheng S, Seven AB, Wang J, Skiniotis G, Özkan E (2016) Conformational Program. Crystallographic data were collected with support from Raj Raja- plasticity in the transsynaptic neurexin-cerebellin-glutamate receptor shankar and APS NE-CAT beamline 24-ID-E staff and the staff of SSRL beam- adhesion complex. Structure 24: 2163 – 2173 line 12-2. This work is based upon research conducted at the Northeastern Comoletti D, Flynn RE, Boucard AA, Demeler B, Schirf V, Shi J, Jennings LL, Collaborative Access Team beamlines, which are funded by the National Insti- Newlin HR, Südhof TC, Taylor P (2006) selection, alternative splicing, tute of General Medical Sciences from the National Institutes of Health (P30 and post-translational processing regulate neuroligin selectivity for b- GM124165). The PILATUS 6M detector on 24-ID-C beamline is funded by a neurexins. Biochemistry 45: 12816 – 12827 NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Dukkipati A, Park HH, Waghray D, Fischer S, Garcia KC (2008) BacMam system Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science for high-level expression of recombinant soluble and membrane User Facility operated for the DOE Office of Science by Argonne National Labo- glycoproteins for structural studies. Protein Expr Purif 62: 160 – 170 ratory under Contract No. DE-AC02-06CH11357. SEC-SAXS experiments were Elegheert J, Kakegawa W, Clay JE, Shanks NF, Behiels E, Matsuda K, Kohda K, performed at SSRL beamline 4-2. Use of the Stanford Synchrotron Radiation Miura E, Rossmann M, Mitakidis N et al (2016) Structural basis for Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. integration of GluD receptors within synaptic organizer complexes. Science Department of Energy, Office of Science, Office of Basic Energy Sciences, under 353: 295 – 299 Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Elegheert J, Cvetkovska V, Clayton AJ, Heroven C, Vennekens KM, Smukowski Program is supported by the DOE Office of Biological and Environmental SN, Regan MC, Jia W, Smith AC, Furukawa H et al (2017) Structural Research and by the National Institutes of Health, National Institute of mechanism for modulation of synaptic neuroligin-neurexin signaling by General Medical Sciences (including P41GM103393). The contents of this MDGA proteins. Neuron 95: 896 – 913 publication are solely the responsibility of the authors and do not necessarily Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development represent the official views of NIGMS or NIH. of Coot. Acta Crystallogr D Biol Crystallogr 66: 486 – 501 Földy C, Darmanis S, Aoto J, Malenka RC, Quake SR, Südhof TC, Biederer T, Author contributions Freund TF, Tsai L-H (2016) Single-cell RNAseq reveals cell adhesion SCW, TCS, and ATB conceived of the project and designed experiments. SCW, molecule profiles in electrophysiologically defined neurons. Proc Natl Acad KIW, and QZ collected crystallographic data. SCW, KIW, QZ, and ATB deter- Sci USA 113:E5222 – E5231 mined and analyzed crystal structures. SCW generated constructs, purified all Franke D, Petoukhov MV, Konarev PV, Panjkovich A, Tuukkanen A, Mertens proteins, crystallized proteins, collected SEC-SAXS data, performed all binding HDT, Kikhney AG, Hajizadeh NR, Franklin JM, Jeffriesa CM et al (2017) assays, and performed confocal imaging experiments. RAP assisted SCW with ATSAS 2.8: a comprehensive data analysis suite for small-angle SEC-MALS and CD data collection. UBC labeled proteins for fluorescence aniso- scattering from macromolecular solutions. J Appl Crystallogr 50: tropy binding experiments. SCW, KIW, TCS, and ATB wrote the paper with 1212 – 1225 input from all authors. Früh S, Romanos J, Panzanelli P, Bürgisser D, Tyagarajan SK, Campbell KP, Santello M, Fritschy J-M (2016) Neuronal dystroglycan is necessary for Conflict of interest formation and maintenance of functional CCK-positive basket cell The authors declare that they have no conflict of interest. terminals on pyramidal cells. J Neurosci 36: 10296 – 10313 Goehring A, Lee C-H, Wang KH, Michel JC, Claxton DP, Baconguis I, Althoff T, Fischer S, Garcia KC, Gouaux E (2014) Screening and large-scale References expression of membrane proteins in mammalian cells for structural studies. Nat Protoc 9: 2574 – 2585 Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Holm L, Laakso LM (2016) Dali server update. Nucleic Acids Res 44: 351 – 355 Hung LW, Kapral GJ, Grosse-Kunstleve RW et al (2010) PHENIX: a Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from comprehensive Python-based system for macromolecular structure crystalline state. J Mol Biol 372: 774 – 797 solution. Acta Crystallogr D Biol Crystallogr 66: 213 – 221 Matsuda K, Yuzaki M (2011) Cbln family proteins promote synapse formation Araç D, Boucard AA, Ozkan E, Strop P, Newell E, Südhof TC, Brunger AT (2007) by regulating distinct neurexin signaling pathways in various brain Structures of neuroligin-1 and the neuroligin-1/neurexin-1 beta complex regions. Eur J Neurosci 33: 1447 – 1461

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Miller MT, Mileni M, Comoletti D, Stevens RC, Harel M, Taylor P (2011) The Steentoft C, Vakhrushev SY, Joshi HJ, Kong Y, Vester-Christensen MB, crystal structure of the a-Neurexin-1 extracellular region reveals a hinge Schjoldager KT-BG, Lavrsen K, Dabelsteen S, Pedersen NB, Marcos-Silva L point for mediating synaptic adhesion and function. Structure 19: et al (2013) Precision mapping of the human O-GalNAc glycoproteome 767 – 778 through SimpleCell technology. EMBO J 32: 1478 – 1488 Missler M, Hammer RE, Südhof TC (1998) Neurexophilin binding to ⍺- Sterky FH, Trotter JH, Lee S-J, Recktenwald CV, Du X, Zhou B, Zhou P, Neurexins: a single LNS domain functions as an independently folding Schwenk J, Fakler B, Südhof TC (2017) Carbonic anhydrase-related protein ligand-binding unit. J Biol Chem 273: 34716 – 34723 CA10 is an evolutionarily conserved pan-neurexin ligand. Proc Natl Acad Missler M, Südhof TC (1998) Neurexophilins form a conserved family of Sci USA 114:E1253 – E1262 neuropeptide-like glycoproteins. J Neurosci 18: 3630 – 3638 Südhof TC (2017) Synaptic neurexin complexes: a molecular code for the Neupert C, Schneider R, Klatt O, Reissner C, Repetto D, Biermann B, logic of neural circuits. Cell 171: 745 – 769 Niesmann K, Missler M, Heine M (2015) Regulated dynamic trafficking of Sugita S, Saito F, Tang J, Satz J, Campbell K, Südhof TC (2001)A neurexins inside and outside of synaptic terminals. J Neurosci 35: stoichiometric complex of neurexins and dystroglycan in brain. J Cell Biol 13629 – 13647 154: 435 – 445 Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected Tanaka H, Nogi T, Yasui N, Iwasaki K, Takagi J (2011) Structural basis for in oscillation mode. Methods Enzymol 276: 307 – 326 variant-specific neuroligin-binding by a-neurexin. PLoS One 6: Petrenko AG, Ullrich B, Missler M, Krasnoperov V, Rosahl TW, Südhof TC e19411 (1996) Structure and evolution of neurexophilin. J Neurosci 16: 4360 – 4369 Tasic B, Menon V, Nguyen TNT, Kim TTK, Jarsky T, Yao Z, Levi BB, Gray LT, Reissner C, Stahn J, Breuer D, Klose M, Pohlentz G, Mormann M, Missler M Sorensen SA, Dolbeare T et al (2016) Adult mouse cortical cell taxonomy (2014) Dystroglycan binding to a-neurexin competes with neurexophilin-1 revealed by single cell transcriptomics. Nat Neurosci 19: 335 – 346 and neuroligin in the brain. J Biol Chem 289: 27585 – 27603 Treutlein B, Gokce O, Quake SR, Südhof TC (2014) Cartography of neurexin Robert X, Gouet P (2014) Deciphering key features in protein structures with alternative splicing mapped by single-molecule long-read mRNA the new ENDscript server. Nucleic Acids Res 42: 320 – 324 sequencing. Proc Natl Acad Sci USA 111:E1291 – E1299 Saunders A, Macosko EZ, Wysoker A, Goldman M, Krienen FM, de Rivera H, Uemura T, Lee S, Yasumura M, Takeuchi T, Yoshida T, Ra M, Taguchi R, Bien E, Baum M, Bortolin L, Wang S et al (2018) Molecular diversity and Sakimura K, Mishina M (2010) Trans-synaptic interaction of GluRd2 and specializations among the cells of the adult mouse brain. Cell 174: neurexin through Cbln1 mediates synapse formation in the cerebellum. 1015 – 1030 e16 Cell 141: 1068 – 1079 Schmidt TGM, Batz L, Bonet L, Carl U, Holzapfel G, Kiem K, Matulewicz K, Ullrich B, Ushkaryov YA, Südhof TC (1995) Cartography of neurexins: more Niermeier D, Schuchardt I, Stanar K (2013) Development of the Twin- than 1000 isoforms generated by alternative splicing and expressed in Strep-tag® and its application for purification of recombinant proteins distinct subsets of neurons. Neuron 14: 497 – 507 from cell culture supernatants. Protein Expr Purif 92: 54 – 61 Zeisel A, Hochgerner H, Lonnerberg P, Johnsson A, Memic F, van der Schreiner D, Nguyen TM, Russo G, Heber S, Patrignani A, Ahrné E, Scheiffele P Zwan J, Haring M, Braun E, Borm L, La Manno G et al (2018) (2014) Targeted combinatorial alternative splicing generates brain region- Molecular architecture of the mouse nervous system. Cell 174: specific repertoires of neurexins. Neuron 84: 386 – 398 999 – 1014 Seigneur E, Südhof TC (2017) Cerebellins are differentially expressed in Zhong C, Shen J, Zhang H, Li G, Shen S, Wang F, Hu K, Cao L, He Y, Ding J selective subsets of neurons throughout the brain. J Comp Neurol 525: (2017) Cbln1 and Cbln4 are structurally similar but differ in GluD2 3286 – 3311 binding interactions. Cell Rep 20: 2328 – 2340 Sheckler LR, Henry L, Sugita S, Südhof TC, Rudenko G (2006) Crystal structure of the second LNS/LG domain from neurexin 1⍺: Ca2+ binding and the License: This is an open access article under the effects of alternative splicing. J Biol Chem 281: 22896 – 22905 terms of the Creative Commons Attribution 4.0 Shen KC, Kuczynska DA, Wu IJ, Murray BH, Sheckler LR, Rudenko G (2008) License, which permits use, distribution and reproduc- Regulation of neurexin 1b tertiary structure and ligand binding through tion in any medium, provided the original work is alternative splicing. Structure 16: 422 – 431 properly cited.

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Expanded View Figures

Figure EV1. Analysis of Nxph, LNS2, and Nxph1-LNS2 complexes by SEC-MALS. SS2 SS2A+ 3ND SS2 3ND SS2A+ A–H SEC-MALS traces for (A) Nrxn1 LNS2 À, (B) Nrxn1 LNS2 , (C) Nxph1 -Nrxn1 LNS2 À (C293A), (D) Nxph1 -Nrxn1 LNS2 , and (E) Nxph1-2XFLAG-His ▸ SS2 (from HEK293F cells). (F) Overlay of traces shown in panels (A–E), (G) Nxph3-2XFLAG-His (from HEK293F cells), (H) I401Q mutant of Nrxn2 LNS2 À. The mean molecular weight (MW) of each protein is indicated near each peak. The poor fits of the molecular weight curves in (E and G) are due to oligomeric mixtures of Nxph1 and Nxph3 molecules, respectively.

ª 2019 The Authors The EMBO Journal e101603 |2019 EV1 The EMBO Journal Steven C Wilson et al

A Nrxn1 LNS2SS2- B Nrxn1 LNS2SS2A+ 1.0 60000 1.0 60000 dRI (relative) dRI (relative) Molar mass (g/mol) mass Molar Molar Mass (g/mol) (g/mol) mass Molar Molar Mass (g/mol) 0.8 0.8 40000 40000 0.6 0.6

0.4 0.4 MW = 21.3 kDa MW = 20.5 kDa 20000 20000 dRI (relative) dRI (relative) 0.2 0.2

0.0 0 0.0 0 12 14 16 18 12 14 16 18 Elution Volume (mL) Elution Volume (mL)

dRI (relative) dRI (relative) Molar Mass (g/mol) MW = 19.9 kDa Molar Mass (g/mol) MW = 20.7 kDa C Nxph1-Nrxn1 LNS2SS2- (C293A) D Nxph1-Nrxn1 LNS2SS2A+ 1.0 60000 1.0 60000 dRI (relative) dRI (relative) Molar mass (g/mol) mass Molar Molar Mass (g/mol) Molar Mass (g/mol) (g/mol) mass Molar 0.8 0.8 MW = 41.5 kDa MW = 39.6 kDa 40000 40000 0.6 0.6

0.4 0.4 20000

dRI (relative) 20000 dRI (relative) 0.2 0.2

0.0 0 0.0 0 12 14 16 18 12 14 16 18 Elution Volume (mL) Elution Volume (mL)

dRI (relative) dRI (relative) Molar Mass (g/mol) MW = 38.4 kDa Molar Mass (g/mol) MW = 40.2 kDa Nxph1 • dRI • Molar Mass Nxph1 E F Nxph1-Nrxn1 LNS2SS2- (C293A) • dRI • Molar Mass 1.0 60000 Nxph1-Nrxn1 LNS2SS2A+ • dRI • Molar Mass Nrxn1 LNS2SS2- • dRI • Molar Mass Molar mass (g/mol) mass Molar Nrxn1 LNS2SS2A+ • dRI • Molar Mass 0.8 MW = 45.5 kDa

dRI (relative) 40000 0.6 Molar Mass (g/mol)

0.4 20000 dRI (relative) 0.2

0.0 0 12 14 16 18 Elution Volume (mL)

dRI (relative) Molar Mass (g/mol) MW = 46.2 kDa SS2- G Nxph3 H Nrxn2 LNS2 (I401Q)

1.0 1.0 dRI (relative) 60000

Molar Mass (g/mol) (g/mol) mass Molar

MW = 76.1 kDa 80000 (g/mol) mass Molar 0.8 0.8 60000 40000 dRI (relative) 0.6 0.6 Molar Mass (g/mol) 40000 0.4 0.4 MW = 24.1 kDa 20000 dRI (relative) dRI (relative) 0.2 20000 0.2

0.0 0 0.0 0 10 12 14 16 12 14 16 18 Elution Volume (mL) Elution Volume (mL)

Figure EV1.

EV2 The EMBO Journal e101603 |2019 ª 2019 The Authors Steven C Wilson et al The EMBO Journal

Figure EV2. The Nrxn1 LNS2 Nxph1-binding site is highly conserved only in vertebrate neurexins and proximal to the SS2 insert site. Sequence alignment of neurexin LNS2 domains from vertebrate and invertebrate species is shown. Sequences are numbered in reference to the canonical UniProt mouse SS2A+ SS2A+ ▸ Nrxn1 sequence Q9CS84-1. Secondary structure elements from LNS2 in the Nxph1-LNS2 structure are shown at the top. Fully conserved sequences are highlighted in red, semi-conserved sequences are highlighted in blue, and non-conserved sequences are highlighted in white. Inserts in SS2 are highlighted in magenta. Key Nxph1-binding residues are underlined. The disulfide bridge in Nrxn1 LNS2SS2A+ is shown as a red line. UniProt accession numbers for the sequences used are as follows: Mouse Nrxn1 (Q9CS84), Mouse Nrxn2 (E9PUM9), Mouse Nrxn3 (Q6P9K9), Human Nrxn1 (Q9ULB1), Human Nrxn2 (Q9P2S2), Human Nrxn3 (Q9Y4C0), Rat Nrxn1 (Q63372), Rat Nrxn2 (Q63374), Rat Nrxn3 (Q07310), Cow Nrxn1 (Q28146), Cow Nrxn2 (E1BFN9), Zebrafish Nrxn1 (A1XQX0), Zebrafish Nrxn2 (B7ZD56), Zebrafish Nrxn3 (A1XQX8), Frog Nrxn2 (F6YGH1), Frog Nrxn3 (F6WM05), Chicken Nrxn1 (Q9DDD0), Chicken Nrxn3 (D0PRN3), Sea slug Nrxn (G0WLR9), Ant Nrxn3 (F4WZ00), and Fruit fly Nrxn1 (Q9VCZ9). Note: The sequence for Mouse Nrxn3 SS2AB was taken from published coordinates (Schreiner et al, 2014) and manually inserted into the alignment.

ª 2019 The Authors The EMBO Journal e101603 |2019 EV3 The EMBO Journal Steven C Wilson et al

β1 β2 β3 β4 β5 β6 β7 Mouse Nrxn1 283 292 302 312 322 332 342 352 Mouse Nrxn1 IA TF KG S E YF CYD L S Q N ... PIQSS S DEITL SF KT L QR N GL M LHT G KSA DY VNL AL K NG AV S LVIN L GS G AFEA L VE P VN Mouse Nrxn2 VA TF KG N E FF CYD L S H N ... PIQSS T DEITL AF RT L QR N GL M LHT G KSA DY VNL SL K SG AV W LVIN L GS G AFEA L VE P VN Mouse Nrxn3 VA TF RG S E YL CYD L S Q N ... PIQSS S DEITL SF KT W QR N GL I LHT G KSA DY VNL AL K DG AV S LVIN L GS G AFEA I VE P VN Human Nrxn1 IA TF KG S E YF CYD L S Q N ... PIQSS S DEITL SF KT L QR N GL M LHT G KSA DY VNL AL K NG AV S LVIN L GS G AFEA L VE P VN Human Nrxn2 VA TF KG N E FF CYD L S H N ... PIQSS T DEITL AF RT L QR N GL M LHT G KSA DY VNL SL K SG AV W LVIN L GS G AFEA L VE P VN Human Nrxn3 VA TF RG S E YL CYD L S Q N ... PIQSS S DEITL SF KT W QR N GL I LHT G KSA DY VNL AL K DG AV S LVIN L GS G AFEA I VE P VN Rat Nrxn1 IA TF KG S E YF CYD L S Q N ... PIQSS S DEITL SF KT L QR N GL M LHT G KSA DY VNL AL K NG AV S LVIN L GS G AFEA L VE P VN Rat Nrxn3 VA TF RG S E YLS YD L S Q N ... PIQSS SS EITL SF KT W QR N GL I LHT G KSA DY VNL AL K DG AV S LVIN L GS G AFEA I VE P VN Rat Nrxn2 VA TF KG N E FF CYD L S H N ... PIQSS T DEITL AF RT L QR N GL M LHT G KSA DY VNL SL K SG AV W LVIN L GS G AFEA L VE P VN Cow Nrxn1 IA TF KG S E YF CYD L S Q N ... PIQSS S DEITL SF KT L QR N GL M LHT G KSA DY VNL AL K NG AV S LVIN L GS G AFEA L VE P VN Cow Nrxn2 VA TF KG N E FF CYD L S H N ... PIQSS T DEITL AF RT L QR N GL M LHT G KSA DY VNL SL K SG AV W LVIN L GS G AFEA L VE P VN Zebrafish Nrxn1 MA TF KG S E YF CYD L S P N ... PIQSS S DEITL SF KT L QR N GL M LHT G KSA DY VNL AL K NG AV S LVIN L GS G AFEA L VE P VN Zebrafish Nrxn2 VA TF KG N E FFS YD L S QK... PIQSS T DEITL SF RT L QR N GL L LHT G KSA DY VNL SL K SG AV C LVIN L GS G AFEA L VE P SD Zebrafish Nrxn3 VA TF RG S E FF CYD L S Q N ... PIQSS S DEITL SF KT W QR N GL I LHT G KSA DY VNL AL K DG AV S LVIN L GS G AFEA I VE P VN Frog Nrxn2 AA TF RG N E FF CYD L S HS... PIQSS A DEITL SF RT L QR N GL M LHT G KSA DY VNL SL K SG AV W LVIN L GS G AFEA L VE P VN Frog Nrxn3 VA TF RG S E YL CYD L S Q N ... PIQSS S DEITL SF KT R QR N GL I LHT G KSA DY VNL AL K GG AV S LVIN L GS G AFEA I VE P TN Chicken Nrxn1 IA TF KG S E YF CYD L S Q N ... PIQSS S DEITL SF KT L QR N GL M LHT G KSA DY VNL AL K NG AV S LVIN L GS G AFEA L VE P VN Chicken Nrxn3 MA TF RG S E YL CYD L S Q N ... PIQSS S DEITL SF KT W QR N GL I LHT G KSA DY VNL AL K DG AV S LVIN L GS G AFEA I VE P VN Nxph1- binding Sea slug Nrxn DA TF MG TQYFS Y NL S SRG..DVMVDQ D RVR L DF RT K Q AN SL LFY TG N S KDY MTVG L MD G AV F L T IN L GS G MYQ A EIR P SG Ant Nrxn3 EA FF RG T E YLTI DL S K..GE P VL S TQES I S L QF KT RAA N GL LFYS G EGD DY LTIS L RD G GAAVSMS L AK G RLDLHIK P V K Fruit fly Nrxn1 EA TF RG TQFLS YD L GQTGAE PI V S AQ D A I SFY F RT R Q PN GL LFY TG HGT DY L NL AL RD G G V S L TMG L AN G KQ E MHIK P SK

β8 β9 β10 β11 Mouse Nrxn1 362 372 397 407 417 427 437 Mouse Nrxn1 GK F ND NA WH DV K VT R NLRQ HS GIG HAM V NKLHCS VTISV D GILTTTGYTQEDYTM L GS DDFFYVGGSP S TAD L PG SPVSN Mouse Nrxn2 GK F ND NA WH DV R VT R NLRQ HA GIG HAM V NKLHYL. TISV D GILTTTGYTQEDYTM L GS DDFFY I GGSP N TAD L PG SPVSN Mouse Nrxn3 GK F ND NA WH DV K VT R NLRQ HS GIG HAM V NKLHCL VTISV D GILTTTGYTQEDYTM L GS DDFFYVGGSP S TAD L PG SPVSN Human Nrxn1 GK F ND NA WH DV K VT R NLRQ HS GIG HAM V ...... TISV D GILTTTGYTQEDYTM L GS DDFFYVGGSP S TAD L PG SPVSN Human Nrxn2 GK F ND NA WH DV R VT R NLRQ HA GIG HAM V NKLHYL VTISV D GILTTTGYTQEDYTM L GS DDFFY I GGSP N TAD L PG SPVSN Human Nrxn3 GK F ND NA WH DV K VT R NLRQ ...... VTISV D GILTTTGYTQEDYTM L GS DDFFYVGGSP S TAD L PG SPVSN Rat Nrxn1 GK F ND NA WH DV K VT R NLRQ HS GIG HAM V NKLHCS VTISV D GILTTTGYTQEDYTM L GS DDFFYVGGSP S TAD L PG SPVSN Rat Nrxn3 GK F ND NA WH DV K VT R NLRQ ...... VTISV D GILTTTGYTQEDYTM L GS DD SS YVG P SP S TAD L PG SPVSN Rat Nrxn2 GK F ND NA WH DV R VT R NLRQ HA GIG HAM V NKLHYL VTISV D GILTTTGYTQEDYTM L GS DDFFY I GGSP N TAD L PG SPVSN Cow Nrxn1 GK F ND NA WH DV K VT R NLRQ TS GIG HAM V NKLHCS VTISV D GILTTTGYTQEDYTM L GS DDFFYVGGSP S TAD L PG SPVSN Cow Nrxn2 GK F ND NA WH DV R VT R NLRQ HA GIG HAM V NKLHYL VTISV D GILTTTGYTQEDYTM L GS DDFFY I GGSP N TAD L PG SPVSN Zebrafish Nrxn1 GK F ND NA WH DV K VT R NLRQ HS GIG HAM V NKLHCS VTISV D GILTTTGYTQEDYTM L GS DDFFYVGGSP S TAD L PG SPVSN Zebrafish Nrxn2 GK F ND NA WH A V R V SR NLRQ RA G V G LPT V NKLHYM VTISV D G L LTTTGYTQEDYTM L GS DDFFYVGGSP N TAD L PG SPVSN Zebrafish Nrxn3 GK F ND N SWH DV K VT R NLRQ HS GIG HAM V NKLHCL VTISV D GILTTTGYTQEDYTM L GS DDFFYVGGSP S TAD L PG SPVSN Frog Nrxn2 GK F ND N SWH DV R VT R NLRQ HA GIG HAL V ...... TISV D GILT S TGYTQEDYTM L GS DDFFY I GGSP N TAD L PG SPVSN Frog Nrxn3 D KF ND N EWH E V K VT R NLRQ HS GIG HAM V NKLHCL VTISV D GILTTTGYTQEDYTM L GS DDFFYVGGSP S TAD L PG SPVSN Chicken Nrxn1 GK F ND NA WH DV K VT R NLRQ HS GIG HAM V NKLHCS VTISV D GILTTTGYTQEDYTM L GS DDFFYVGGSP S TAD L PG SPVSN Chicken Nrxn3 GK F ND NA WH DV K VT R NLRQ HS GIG HAM V NKLHCL VTISV D GILTTTGYTQEDYTM L GS DDFFYVGGSP S TAD L PG SPVSN SS2AB Nxph1- SS2A binding Sea slug Nrxn TR F DD N RWH QLLIQ R EA R ELPRDAGV...... CF VT MEL D G MYRKQ G SITGKFIR L SS N.LL YV A GSP N T ET L PG S RV RT Ant Nrxn3 LR F DD RQ WH KIV V HR KVQEISS I TSF...... CRLSAI VD G VYAEH G H T AGSF T HL AS D .RLL VGG GADARS L QG AKGII Fruit fly Nrxn1 VR F DD HQ WH K V T V HR RIQEISS I TSF...... CRLVTV VD DVY T DHSHIAGKF TM L SS S.RV YVGG AVNPRA L LG AR V HT

β12 β13 α1β14 Mouse Nrxn1 TT 447 457 467 477 Mouse Nrxn1 NF M GCL K E VVYKNND VR LE LSRL A KQ GD PKMK IH G VVA F .K C Mouse Nrxn2 NF M GCL K D VVYKNND FK LE LSRL A KE GD PKMK LQ G DLS F .R C Mouse Nrxn3 NF M GCL K E VVYKNND IR LE LSRL A RI GD TKMK IY G EVV F .K C Human Nrxn1 NF M GCL K E VVYKNND VR LE LSRL A KQ GD PKMK IH G VVA F .K C Human Nrxn2 NF M GCL K D VVYKNND FK LE LSRL A KE GD PKMK LQ G DLS F .R C Human Nrxn3 NF M GCL K E VVYKNND IR LE LSRL A RIA D TKMK IY G EVV F .K C Rat Nrxn1 NF M GCL K E VVYKNND VR LE LSRL A KQ GD PKMK IH G VVA F .K C Rat Nrxn3 NF M GCL K E VVYKNND IR LE LSRL A RI G AT KMK IY G EVV F .K C Rat Nrxn2 NF M GCL K D VVYKNND FK LE LSRL A KE GD PKMK LQ G DLS F .R C Cow Nrxn1 NF M GCL K E VVYKNND VR LE LSRL A KQ GD PKMK IH G VVA F .K C Cow Nrxn2 NF M GCL K D VVYKNND FK LE LSRL A KE GD PKMK LQ G DLS F .R C Zebrafish Nrxn1 NF M GCL K E VVYKNND VR LE LSRL A KL GD PKMK VS G VVA F .K C Zebrafish Nrxn2 NF M GCL K D VVY TNN EFR LE LS HL A ELR D PK VTLH G DLT F .R C Zebrafish Nrxn3 NF M GCL K E VVYKNND IR LE LSRL A RIV D PKMK IQ G DVV F .K C Frog Nrxn2 NF M GCL K D VVYKNND FK LE LSRL A SE GD PKM RLS G DLV F .R C Frog Nrxn3 NF M GCL K E VVYKNND IR LE LSRL A RI GD TKMK IY G DVV F .K C Chicken Nrxn1 NF M GCL K E VVYKNND VR LE LSRL A KQ GD PKMK IH G VVA F .K C Chicken Nrxn3 NF M GCL K E VVYKNND IR LE LSRL A RI GD TKMK IY G EVK F .V C

Sea slug Nrxn NF K GCL RK V RY RADGVL L D L TD L A RREHSL M QVT G DVI F DK C Ant Nrxn3 NF V GCL RK V EFAAERIRM E L IEA A KS G AAGVAAW G KMD F .F C Fruit fly Nrxn1 NF V GCL RK V EFSADTLN L N L ID L A KS G SKLIQVA GNLEY.Q C

C444-C480

Figure EV2.

EV4 The EMBO Journal e101603 |2019 ª 2019 The Authors Steven C Wilson et al The EMBO Journal

Figure EV3. The Nxph1 LNS2-binding site is highly conserved in vertebrate neurexophilins. SS2 Sequence alignment of neurexophilins from vertebrates is shown. Secondary structure elements from Nxph1 in the Nxph1-LNS2 À structure are shown at the top. This ▸ alignment only shows regions of neurexophilins aligned to mature Nxph1. Fully conserved sequences are highlighted in red; semi-conserved sequences are highlighted in yellow, and non-conserved sequences are highlighted in white. Key Nrxn1 LNS2-binding residues are underlined. Disulfide bridges are shown as orange lines. GenBank or RefSeq accession numbers for the sequences used are as follows: Rat Nxph1 (AAI05771.1), Rat Nxph2 (XP_008759839.2), Rat Nxph3 (AAI66603.1), Rat Nxph4 (AAH81805.1), Mouse Nxph1 (NP_032777.3), Mouse Nxph2 (EDL08154.1), Mouse Nxph3 (EDL15984.1), Mouse Nxph4 (EDL24512.1), Cow Nxph1 (AAX08911.1), Cow Nxph2 (NP_776831.1), Cow Nxph3 (NP_001179753.1), Cow Nxph4 (DAA29666.1), Human Nxph1 (BAE45730.1), Human Nxph2 (BAD11132.1), Human Nxph3 (AAQ88890.1), Human Nxph4 (AAQ88977.1), Frog Nxph1 (NP_001120374.1), Frog Nxph3 (NP_001096477.1), Zebrafish Nxph1 (AAH76424.1), Zebrafish Nxph2 (XP_698522.2), Zebrafish Nxph3 (XP_691473.2).

ª 2019 The Authors The EMBO Journal e101603 |2019 EV5 The EMBO Journal Steven C Wilson et al

β1 β2 β3 β4 Rat Nxph1 118 127 137 147 157 167 176 Rat Nxph1 MFGWGDF H SNIK T VKLN LL ITGK IVDH GNG TFSV YF RHN STG QGN VS VS LVPP TK I VE F ...... DL.AQ Q TVID..... Rat Nxph2 MFGWGDF H SNIK T VKLN LL ITGK IVDH GNG TFSV YF RHN STG LGN VS VS LVPP SK I VE F ...... EV.ST Q SSLE..... Rat Nxph3 IFGWGDF Y SNIK T V A LN LL VTGK IVDH GNG TFSV HF RHN A TG QGN IS IS LVPP SK A VE F ...... H.QEQ Q IFIE..... Rat Nxph4 IFGWGDF YFRVH T L K FS LL VTGK IVDH VNG TFSV YF RHN S SSL GN LS VS IVPP SK R VE F GGVWLPGPAPHPLQSTLALEGVLPG Mouse Nxph1 MFGWGDF H SNIK T VKLN LL ITGK IVDH GNG TFSV YF RHN STG QGN VS VS LVPP TK I VE F ...... DL.AQ Q TVID..... Mouse Nxph2 MFGWGDF H SNIK T VKLN LL ITGK IVDH GNG TFSV YF RHN STG LGN VS VS LVPP SK V VE F ...... EI.SP Q STLE..... Mouse Nxph3 IFGWGDF Y SNIK T V A LN LL VTGK IVDH GNG TFSV HF RHN A TG QGN IS IS LVPP SK A VE F ...... H.QEQ Q IFIE..... Mouse Nxph4 IFGWGDF YFRVH T L K FS LL VTGK IVDH VNG TFSV YF RHN S SSL GN LS VS IVPP SK R VE F GGVWLPGPASHPLQSTLALEGVLPG Cow Nxph1 MFGWGDF H SNIK T VKLN LL ITGK IVDH GNG TFSV YF RHN STG QGN VS VS LVPP TK I VE F ...... DL.AQ Q TVID..... Cow Nxph2 MFGWGDF H SNIK T VKLN LL ITGK IVDH GNG TFSV YF RHN STG LGN VS VS LVPP SK V VE F ...... EV.SP Q STLE..... Cow Nxph3 IFGWGDF Y SNIK T V A LN LL VTGK IVDH GNG TFSV HF RHN A TG QGN IS IS LVPP SK A VE F ...... H.QEQ Q IFIE..... Cow Nxph4 IFGWGDF YFRVH T L K FS LL VTGK IVDH VNG TFSV YF RHN S SSL GN LS VS IVPP SK R VE F GGVWLPGPAPHPLQSTLALEGVLPG Human Nxph1 MFGWGDF H SNIK T VKLN LL ITGK IVDH GNG TFSV YF RHN STG QGN VS VS LVPP TK I VE F ...... DL.AQ Q TVID..... Human Nxph2 MFGWGDF H SNIK T VKLN LL ITGK IVDH GNG TFSV YF RHN STG LGN VS VS LVPP SK V VE F ...... EV.SP Q STLE..... Human Nxph3 IFGWGDF Y SNIK T V A LN LL VTGK IVDH GNG TFSV HF QHN A TG QGN IS IS LVPP SK A VE F ...... H.QEQ Q IFIE..... Human Nxph4 IFGWGDF YFRVH T L K FS LL VTGK IVDH VNG TFSV YF RHN S SSL GN LS VS IVPP SK R VE F GGVWLPGPVPHPLQSTLALEGVLPG Frog Nxph1 MFGWGDF H SNIK T VKLN LL ITGK IVDH GNG TFSV YF RHN STG QGN VS VS LVPP TK I VE F ...... DL.AQ Q SVID..... Frog Nxph3 VFGWGDF Y SNIK T VKLN LL ITGK VVDH GNG SFSV YF RHN STG HGN VS VS LVPP SK ILD F ...... DL.EQ Q MYVE..... Zebrafish Nxph1 MFGWGDF H SNIK T VKLN LL ITGK IVDH GNG TFSV YF RHN STG QGN VS VS LVPP TK I VE F ...... DLTAQ Q SVID..... Zebrafish Nxph2 MFGWGDF N SNIK T VKLN LL ITGK IVDH GNG TFSV YF RHN STG LGN VS VS LVPP SK V VE F ...... EI.AQ Q STLE..... Zebrafish Nxph3 MFGWGDF Y SNIK T V R LN LL ITGK IVDH GNG TFSV YF RHN STG QGN IS VS LVPP VK A VE F ...... DL.ER Q SVVY..... Nrxn1 LNS2-binding β5β6 Rat Nxph1 187 197 207 217 227 Rat Nxph1 ...... AK D SK SFNC R I EYE K VDKAT K NT LC NY DP SK TC YQ E Q TQ S HVS W LC SK Rat Nxph2 ...... T K E SK SFNC HI EYE K TD R AK K TA LC NF DP SK IC YQ E Q TQ S HVS W LC SK Rat Nxph3 ...... AK A SK IFNC R M E W EK VE R GRRTS LC TH DP A K IC SRDHA QS SAT W SC S Q Rat Nxph4 LGTPLGMA....GQGLGGNLGGALAGPLGGALGVPG AK E S RA FNC HV EYE K TN R AR K HRP C LY DP S QV C FT E H TQ S QAA W LC AK Mouse Nxph1 ...... AK D SK SFNC R I EYE K VDKAT K NT LC NY DP SK TC YQ E Q TQ S HVS W LC SK Mouse Nxph2 ...... T K E SK SFNC HI EYE K TD R AK K TA LC NF DP SK IC YQ E Q TQ S HVS W LC SK Mouse Nxph3 ...... AK A SK IFNC R M E W EK VE R GRRTS LC TH DP A K IC SRDHA QS SAT W SC S Q Mouse Nxph4 LGPPLGMA....GQGLGSNLGGALAGPLGSALGVPG AK E S RA FNC HV EYE K TN R AR K HRP C LY DP S QV C FT E H TQ S QAA W LC AK Cow Nxph1 ...... AK D SK SFNC R I EYE K VDKAT K NT LC NY DP SK TC YQ E Q TQ S HVS W LC SK Cow Nxph2 ...... T K E SK SFNC R I EYE K TD R AK K TA LC NF DP SK IC YQ E Q TQ S HVS W LC SK Cow Nxph3 ...... AK A SK IFNC R M E W EK VE R GRRIS LC TH DP A K TC SRDHA QS SAT W SC S Q Cow Nxph4 LGAPLGIAAA..GPGLGGTLGGALAGPLGGALGVPG AK E S RA FNC HV EYE K TN R AR K HRP C LY DP S QV C FT E H TQ S QAA W LC AK Human Nxph1 ...... AK D SK SFNC R I EYE K VDKAT K NT LC NY DP SK TC YQ E Q TQ S HVS W LC SK Human Nxph2 ...... T K E SK SFNC R I EYE K TD R AK K TA LC NF DP SK IC YQ E Q TQ S HVS W LC SK Human Nxph3 ...... AK A SK IFNC R M E W EK VE R GRRTS LC TH DP A K IC SRDHA QS SAT W SC S Q Human Nxph4 LGPPLGMAAAAAGPGLGGSLGGALAGPLGGALGVPG AK E S RA FNC HV EYE K TN R AR K HRP C LY DP S QV C FT E H TQ S QAA W LC AK Frog Nxph1 ...... AK D SK SFNC R I EYE K VDKAT K NT LC NY DP SK TC YQ E Q TQ S HVS W LC SK Frog Nxph3 ...... AK E SK IFNC R V E F EK VDKAI K TG LC PH DP SK TC HQQQ T HS RVS W TC S H Zebrafish Nxph1 ...... AK D SK SFNC R I EYE K VE R GS K NT LC NF DP SK TC FQ E Q TQ S HVS W LC SK Zebrafish Nxph2 ...... T K DT K SFNC R I EY QK TD R SK K TSH C SY DP S NV C YQ E S TQ S HVS W LC SK Zebrafish Nxph3 ...... P K D SK IFNC R ID YE K VD R SKRTS LC NY DP SK TC FQ E Q TQ S HVS W IC SK

C210-C218

C194-C231 β7β8 Rat Nxph1 237 247 257 267 Rat Nxph1 PFK VI C IYI SF YS TDY KLVQK VCPDYN YHS D TP Y FP SG Rat Nxph2 PFK VI C I H I AF YS IDY KLVQK VCPDYN YHS E TP Y LT SG Rat Nxph3 PFK V VC V YI AF YS TDY RLVQK VCPDYN YHS D TP Y YP SG Rat Nxph4 PFK VI C I FVS F L S FDY KLVQK VCPDYN FQ S EH PY F.. G Mouse Nxph1 PFK VI C IYI SF YS TDY KLVQK VCPDYN YHS D TP Y FP SG Mouse Nxph2 PFK VI C I H I IF YS VDY KLVQK VCPDYN YHS E TP Y LSF G Mouse Nxph3 PFK V VC V YI AF YS TDY RLVQK VCPDYN YHS D TP Y YP SG Mouse Nxph4 PFK VI C I FVS F L S FDY KLVQK VCPDYN FQ S EH PY F.. G Cow Nxph1 PFK VI C IYI SF YS TDY KLVQK VCPDYN YHS D TP Y FP SG Cow Nxph2 PFK VI C IYI AF YS VDY KLVQK VCPDYN YHS E TP Y LS SG Cow Nxph3 PFK V VC V YI AF YS TDY RLVQK VCPDYN YHS D TP Y YP SG Cow Nxph4 PFK VI C I FVS F L S FDY KLVQK VCPDYN FQ S EH PY F.. G Human Nxph1 PFK VI C IYI SF YS TDY KLVQK VCPDYN YHS D TP Y FP SG Human Nxph2 PFK VI C IYI AF YS VDY KLVQK VCPDYN YHS E TP Y LS SG Human Nxph3 PFK V VC V YI AF YS TDY RLVQK VCPDYN YHS D TP Y YP SG Human Nxph4 PFK VI C I FVS F L S FDY KLVQK VCPDYN FQ S EH PY F.. G Frog Nxph1 PFK VI C IYI SF YS TDY KLVQK VCPDYN YHS D TP Y FP SG Frog Nxph3 PFK IV C V Y VS F Y TT DY RLVQK VCPDYN YH NSS PY SP SG Zebrafish Nxph1 PFK VI C IYI SF YS TDY KLVQK VCPDYN YHS D T SY FP SG Zebrafish Nxph2 PFK VI C IYI AF YS TDY KLVQK ICPDYN YHS D TP Y AST G Zebrafish Nxph3 PFK VI C IYI SF YS TDY RLVQK VCPDYN YH NEM PY LP SG

C239-C256 Nrxn1 LNS2-binding

Figure EV3.

EV6 The EMBO Journal e101603 |2019 ª 2019 The Authors Steven C Wilson et al The EMBO Journal

A Nxph1 SEC-SAXS Nxph1 SAXS Nxph1

100 50 2 0.06 I(s) 80 40 1 model fit 0.04 60 30 R 2 = 9.479 g I(s)

[Å] qmin I 10 0 P(r) , 0

I 40 20

Log 0.02 -1 20 10

0 0 -2 0.00 300 350 400 0.0 0.2 0.4 20 40 60 80 100 Image number s r (Å)

B Nxph13ND-LNS2SS2A+ SEC-SAXS Nxph13ND-LNS2SS2A+ SAXS Nxph13ND-LNS2SS2A+

30 30 2 0.05 I (s) 1 model fit 0.04 20 20 2 R 0 = 0.250 0.03 g

[Å] qmin I P(r) , 0

I -1 0.02 10 10 Log I(s) -2 0.01

0 0 -3 0.00 300 400 500 0.0 0.2 0.4 20 40 60 80 100 Image number s r (Å)

C LNS2SS2A+ SEC-SAXS LNS2SS2A+ SAXS LNS2SS2A+

30 25 2 0.08 I (s) 20 1 model fit 0.06 20 2 15 R 0 = 0.282 g

[Å] qmin

I 0.04 P(r) , 0

I 10 -1 10 Log I(s) 0.02 5 -2

0 0 -3 0.00 350 400 450 500 550 0.0 0.2 0.4 0 20 40 60 80 100 Image number s r (Å)

Figure EV4. SEC-SAXS analysis of Nxph1 and Nrxn1 LNS2 alone and in complex. A–C SEC-SAXS data for (A) Nxph1, (B) Nxph13ND-Nrxn1 LNS2SS2A+, and (C) LNS2SS2A+. For each sample, SEC-SAXS traces are shown along with peak SAXS data with crystallographic model fit and pair-wise distance distribution functions. Scattering data represent the average of five technical replicates, and error bars in the middle panels represent SD.

SS2 Figure EV5. The I401Q mutation reduces the affinity of Nxph-Nrxn1 LNS2 À interactions. SS2 A Confocal images of co-expressed wild-type and I401Q mutant Nrxn1 LNS2 À with Nxph1–4. Scale bar: 50 lm. ▸ SS2 B BLI data showing reduced binding affinity of Nxph1 and Nxph3 to Nrxn1–3 LNS2 À (I401Q) mutants. Error bars represent SEM, and replicate numbers are indicated in bars. Significance values were calculated using Welch’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

ª 2019 The Authors The EMBO Journal e101603 |2019 EV7 The EMBO Journal Steven C Wilson et al

SS2- SS2- A wtNrxn1 LNS2 Nrxn1(I401Q) LNS2 merge/ DAPI merge/ DAPI

Nxph1

Nxph2

Nxph3

Nxph4

B

Figure EV5.

EV8 The EMBO Journal e101603 |2019 ª 2019 The Authors Table EV1 – Molecular weights calculated from SEC-SAXS data SEC-SAXS sample Calculated molecular weight (predicted monomer MW) kDa Major Nxph1 peak 58.15 (29) High molecular weight Nxph1 shoulder 124.45 (29) Nxph13ND-LNS2SS2A+ peak 41.08 (41) LNS2SS2A+ peak 18.68 The calculated molecular weights in the table were determined using the Bayesian Inference method in ATSAS. Appendix

Structures of neurexophilin-neurexin complexes reveal a regulatory mechanism of alternative splicing

Steven C. Wilson1, K. Ian White1, Qiangjun Zhou1, Richard A. Pfuetzner1, Ucheor B. Choi1, Thomas C. Südhof1,2, and Axel T. Brunger1,2

1Department of Molecular and Cellular Physiology, Stanford University Medical School, Stanford, CA 94305, USA

2Howard Hughes Medical Institute

Table of Contents

Appendix Figure S1 Superposition of structures of uncomplexed LNS2SS2- and Nxph1-complexed LNS2 SS2- ...... 2

Appendix Figure S2 Glycosylated and cysteine-rich regions in neurexophilin ...... 3

Appendix Figure S3 Packing of complexes in Nxph1-LNS2SS2A+ crystals and details of Nxph1-LNS2SS2A+ interface architecture ...... 4

Appendix Figure S4 Co-immunoprecipitation of Nrxn3 LNS2SS2- mutants with Nxph1 and Circular Dichroism spectra of LNS2 and Nxph proteins ...... 5

Appendix Figure S5 Trace amounts of Ca2+ in buffer do not affect the binding affinity of Nxph1 for Nrxn1 LNS2SS2- and Nrxn1 LNS2SS2A+ ...... 6

Appendix Figure S6 Tandem affinity purification of Nxph1-Nrxn1 LNS2 complexes ...... 7

Appendix References ...... 8

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Appendix Figure S1 Superposition of structures of uncomplexed LNS2SS2- and Nxph1- complexed LNS2 SS2-. Aligned LNS2SS2- domains from Nxph1-LNS2SS2-, 2H0B, 3POY, and 3R05 structures are shown. Arrows indicate differences between the conformations of β7 and β10, and the loop after β10 in the Nxph1-LNS2SS2- and uncomplexed LNS2SS2- structures.

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Appendix Figure S2 Glycosylated and cysteine-rich regions in neurexophilin. (A) Views of the Nxph1-LNS2SS2A+ structure. The previously proposed neurexophilin N- and C- terminal domains (Missler & Südhof, 1998) are colored green and purple, and the loop region connecting strands β4 and β5 is colored orange. Disulfides are colored red, and N-linked glycosylation sites are colored blue. The β-strands of LNS2SS2A+ and Nxph1 are numbered. The SS2A insert is colored magenta in teal-colored LNS2SS2A+. (B) Sequence alignment of rat Nxph1-4 with the color-coded regions in A are indicated with text and lines. The disulfide bonds are indicated with red connecting lines, and the highly conserved N-linked glycosylation sites in the sequence alignment are outlined with blue boxes. Sequences were aligned using Clustal Omega (Sievers et al, 2011), and the secondary structure from the Nxph1 structure is shown at the top of the sequence alignment.

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Appendix Figure S3 Packing of complexes in Nxph1-LNS2SS2A+ crystals and details of Nxph1-LNS2SS2A+ interface architecture. (A) Selected ac plane of molecules in crystals of the Nxph1-LNS2SS2A+ complex. The face of the ac plane is outlined in black. (B) Close-up view and 180° rotation of the section of the ac plane outlined in red in panel A. Of note are the large heterodimeric interfaces between Nxph1 and LNS2SS2A+ and the smaller homodimeric interfaces between Nxph1 molecules. Interfaces are outlined with dotted ovals and buried surface areas of interfaces indicated. (C) Stick representation of β-strands in the complex interface. β-strands are labeled and highlighted. Polar interactions traversing the Nxph1-LNS2 β sandwich are shown as dotted lines. (D) Rotated view of the complex in C, with all detected polar interactions shown as dotted lines.

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Appendix Figure S4 Co-immunoprecipitation of Nrxn3 LNS2SS2- mutants with Nxph1 and Circular Dichroism spectra of LNS2 and Nxph proteins. (A) Nrxn3 LNS2-TwinStrep proteins were co-immunoprecipitated with Nxph1-2×FLAG-His using M2 anti-FLAG magnetic beads. (Mutated Nrxn3 residues are numbered at the top according to the corresponding residue numbers in the UniProt Nrxn1 sequence Q9CS84-1). EGFP- TwinStrep was used as a negative control. Inputs and elutions were probed with mouse anti-FLAG or rabbit anti-Streptag II and appropriate LI-COR secondary antibodies. (B) CD spectra of LNS2 proteins. (C) CD spectra of Nxph1 and Nxph3.

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Appendix Figure S5 Trace amounts of Ca2+ in buffer do not affect the binding affinity of Nxph1 for Nrxn1 LNS2SS2- and Nrxn1 LNS2SS2A+. (A) Representative binding curves are shown for LNS2SS2- and LNS2SS2A+ interacting with Nxph1 in the presence of 2 mM Ca2+ or 100 µM EDTA. (B) Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2- and Nrxn1 LNS2SS2A+ in the presence of 2 mM Ca2+ or 100 µM EDTA. Replicate numbers are indicated in bars. Replicate numbers for experiments represented in A are shown in B. Error bars represent SEM and significance values were calculated using Welch’s t- test.

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Appendix Figure S6 Tandem affinity purification of Nxph1-Nrxn1 LNS2 complexes. (A) Nxph1-LNS2 complexes were co-expressed and secreted from HEK293S GnTI- cells using the BacMam expression system. His-tagged Nxph1 was co-purified from concentrated media in complex with TwinStrep-tagged LNS2 using tandem Ni-NTA- StrepTactin affinity chromatography. Affinity tags were removed with overnight HRV 3C protease digestion. Purified complexes were re-run over tandem Ni-NTA-StrepTactin columns to remove any complexes with un-cleaved affinity tags and further purified using anion exchange chromatography. (B) SDS-PAGE of peak anion exchange fractions of the Nxph13ND-LNS2SS2- (C293A) complex.

7 Appendix References

Missler M & Südhof TC (1998) Neurexophilins form a conserved family of neuropeptide- like glycoproteins. J. Neurosci. 18: 3630–3638 Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD & Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7: 539

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