Structural insights into the exquisite selectivity of neurexin/neuroligin synaptic interactions
2010; Springer Nature; Volume: 29; Issue: 14 Linguagem: Inglês
10.1038/emboj.2010.123
ISSN1460-2075
AutoresPhilippe Leone, Davide Comoletti, Géraldine Ferracci, Sandrine Conrod, Simon U Garcia, Palmer Taylor, Yves Bourne, P. Marchot,
Tópico(s)Receptor Mechanisms and Signaling
ResumoArticle11 June 2010free access Structural insights into the exquisite selectivity of neurexin/neuroligin synaptic interactions Philippe Leone Philippe Leone Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS/Université d'Aix-Marseille, Campus Luminy, Marseille, FrancePresent address: Centre d'Immunologie Marseille-Luminy (CIML, UMR-6102), Case 906—Campus de Luminy—163 Av de Luminy, F-13288 Marseille cedex 09, France Search for more papers by this author Davide Comoletti Davide Comoletti Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Géraldine Ferracci Géraldine Ferracci Centre d'Analyse Protéomique de Marseille (CAPM), Institut Fédératif de Recherche Jean Roche, Faculté de Médecine—Secteur Nord, Marseille, France Search for more papers by this author Sandrine Conrod Sandrine Conrod Centre de Recherche en Neurobiologie-Neurophysiologie de Marseille (CRN2M), CNRS/Université d'Aix-Marseille, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine—Secteur Nord, Marseille, France Search for more papers by this author Simon U Garcia Simon U Garcia Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Palmer Taylor Palmer Taylor Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Yves Bourne Corresponding Author Yves Bourne Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS/Université d'Aix-Marseille, Campus Luminy, Marseille, France Search for more papers by this author Pascale Marchot Corresponding Author Pascale Marchot Centre de Recherche en Neurobiologie-Neurophysiologie de Marseille (CRN2M), CNRS/Université d'Aix-Marseille, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine—Secteur Nord, Marseille, France Search for more papers by this author Philippe Leone Philippe Leone Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS/Université d'Aix-Marseille, Campus Luminy, Marseille, FrancePresent address: Centre d'Immunologie Marseille-Luminy (CIML, UMR-6102), Case 906—Campus de Luminy—163 Av de Luminy, F-13288 Marseille cedex 09, France Search for more papers by this author Davide Comoletti Davide Comoletti Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Géraldine Ferracci Géraldine Ferracci Centre d'Analyse Protéomique de Marseille (CAPM), Institut Fédératif de Recherche Jean Roche, Faculté de Médecine—Secteur Nord, Marseille, France Search for more papers by this author Sandrine Conrod Sandrine Conrod Centre de Recherche en Neurobiologie-Neurophysiologie de Marseille (CRN2M), CNRS/Université d'Aix-Marseille, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine—Secteur Nord, Marseille, France Search for more papers by this author Simon U Garcia Simon U Garcia Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Palmer Taylor Palmer Taylor Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Yves Bourne Corresponding Author Yves Bourne Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS/Université d'Aix-Marseille, Campus Luminy, Marseille, France Search for more papers by this author Pascale Marchot Corresponding Author Pascale Marchot Centre de Recherche en Neurobiologie-Neurophysiologie de Marseille (CRN2M), CNRS/Université d'Aix-Marseille, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine—Secteur Nord, Marseille, France Search for more papers by this author Author Information Philippe Leone1, Davide Comoletti2, Géraldine Ferracci3, Sandrine Conrod4, Simon U Garcia2, Palmer Taylor2, Yves Bourne 1 and Pascale Marchot 4 1Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS/Université d'Aix-Marseille, Campus Luminy, Marseille, France 2Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA, USA 3Centre d'Analyse Protéomique de Marseille (CAPM), Institut Fédératif de Recherche Jean Roche, Faculté de Médecine—Secteur Nord, Marseille, France 4Centre de Recherche en Neurobiologie-Neurophysiologie de Marseille (CRN2M), CNRS/Université d'Aix-Marseille, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine—Secteur Nord, Marseille, France *Corresponding authors: ToxCiM, Dépt Signalisation Neuronale, Centre de Recherche en Neurobiologie-Neurophysiologie de Marseille (CRN2M, UMR-6231), Faculté de Médecine—Secteur Nord, CS80011, Bd Pierre Dramard, F-13344 Marseille cedex 15, France. Tel.: +33 491 698 908; Fax: +33 491 698 839; E-mail: [email protected] et Fonction des Macromolécules Biologiques (AFMB, UMR-6098), Case 932—Campus de Luminy—163 Av de Luminy, F-13288 Marseille cedex 09, France. Tel.: +33 491 825 566; Fax: +33 491 266 720; E-mail: [email protected] The EMBO Journal (2010)29:2461-2471https://doi.org/10.1038/emboj.2010.123 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The extracellular domains of neuroligins and neurexins interact through Ca2+ to form flexible trans-synaptic associations characterized by selectivity for neuroligin or neurexin subtypes. This heterophilic interaction, essential for synaptic maturation and differentiation, is regulated by gene selection, alternative mRNA splicing and post-translational modifications. A new, 2.6 Å-resolution crystal structure of a soluble neurexin-1β–neuroligin-4 (Nrx1β–NL4) complex permits a detailed description of the Ca2+-coordinated interface and unveils concerted positional rearrangements of several residues of NL4, not observed in neuroligin-1, associated with Nrx1β binding. Surface plasmon resonance analysis of the binding of structure-guided Nrx1β mutants towards NL4 and neuroligin-1 shows that flexibility of the Nrx1β-binding site in NL4 is reflected in a greater dissociation constant of the complex and higher sensitivity to ionic strength and pH variations. Analysis of neuroligin mutants points to critical functions for two respective residues in neuroligin-1 and neuroligin-2 in governing the affinity of the complexes. Although neuroligin-1 and neuroligin-2 have pre-determined conformations that respectively promote and prevent Nrx1β association, unique conformational reshaping of the NL4 surface is required to permit Nrx1β association. Introduction The post-synaptic neuroligins and presynaptic neurexins are transmembrane adhesion proteins whose extracellular domains interact to form trans-synaptic associations (Ichtchenko et al, 1996; Nguyen and Südhof, 1997; Gerrow and El-Husseini, 2006; Dalva et al, 2007). Neuroligins and neurexins are required for the maturation of glutamatergic excitatory and GABAergic inhibitory synapses, rather than for establishing the initial contact between neurons. Indeed, their over-expression in non-neuronal cells is sufficient to induce differentiation of co-cultured neurons (Scheiffele et al, 2000; Fu et al, 2003; Graf et al, 2004; Nam and Chen, 2005), whereas knockout mice for the neuroligin or neurexin genes display no major defect in synapse formation (Missler et al, 2003; Varoqueaux et al, 2006). The essential function of the neuroligins is further highlighted by the identification of mutations related to autism spectrum disorders and mental retardation in the neuroligin-3 (NL3) and neuroligin-4 (NL4) isoforms (Jamain et al, 2003; Laumonnier et al, 2004; Yan et al, 2005; Talebizadeh et al, 2006; Zhang et al, 2009). Proper central nervous system function depends in part on the ratio between excitatory and inhibitory synapses, and mutations in neuroligin structure may alter this parameter (Cline, 2005; Levinson and El-Husseini, 2005; Tabuchi et al, 2007; Südhof, 2008). Five neuroligin genes have been identified in humans (NL1–5) and four in rodents (Ichtchenko et al, 1996; Philibert et al, 2000; Bolliger et al, 2001, 2008). The crystal structures of the extracellular cell adhesion domains of rodent NL1 and NL2 and human NL4 were solved recently (Araç et al, 2007; Fabrichny et al, 2007; Chen et al, 2008; Koehnke et al, 2008). The overall architecture of this domain falls within the α/β-hydrolase fold exemplified in the acetylcholinesterase (AChE) structure. When compared with the AChE subunit, distinct features such as a more hydrophobic dimerization interface, different conformations of surface loops surrounding the vestigial active centre and distinct electrostatic surface potentials are also evident. Furthermore, alternative RNA splicing for all neuroligins except NL4 yields isoforms containing or lacking inserts at splice sites A and B (SSA and SSB) (Ichtchenko et al, 1996; Bolliger et al, 2001). The different isoforms of neuroligins exhibit different localization patterns in brain. Whereas NL1 and NL2 are predominantly present in glutamatergic and GABAergic synapses, respectively, NL3 is present in both types of synapses (Song et al, 1999; Varoqueaux et al, 2004; Budreck and Scheiffele, 2007). The low expression levels of NL4 (<5% of the total neuroligins in adult mouse brain) precluded ascertaining its precise synaptic localization (Varoqueaux et al, 2006). The majority of point mutations in neuroligins that relate to autism spectrum disorders reside within the AChE-like domain of NL3 and NL4. The molecular basis underlying their involvement in congenital brain disorders remains to be investigated, but the remote locations of these mutations from the neurexin-binding site preclude direct alteration at the neurexin-binding surface. Rather, these mutations likely affect neuroligin function through defective folding and trafficking (Comoletti et al, 2004; Zhang et al, 2009) or interactions with yet unidentified partner(s). In humans, three neurexin genes are under the control of two promoters, which lead to long neurexins-α (Nrxα) and short neurexins-β (Nrxβ) (Ushkaryov et al, 1992). Alternatively spliced inserts in the extracellular domain of neurexins could generate over 2000 possible isoforms (Rowen et al, 2002; Tabuchi and Südhof, 2002). The Nrxα extracellular sequences contain six laminin-neurexin-sex (LNS) hormone-binding globulin domains separated by three epidermal growth factor (EGF) domains, whereas Nrxβs are composed of a short specific N-terminal sequence followed by a single LNS domain, corresponding to the sixth LNS domain of Nrxα. This LNS domain common to two forms of neurexins is responsible for the interaction with neuroligins (Boucard et al, 2005) and may contain an insert at the alternative splicing site 4 (SS4). The crystal structures of the LNS domains of Nrx1β and Nrx2β and of the LNS-2 and LNS-4 domains of Nrx1α reveal a similar lectin-like-fold made of a β jelly roll with a Ca2+-binding site located on the hypervariable-loop edge, near the SS4 position (Rudenko et al, 1999; Sheckler et al, 2006; Koehnke et al, 2008; Shen et al, 2008). Emerging evidence indicates that variations in copy number and rare variants within the NRXN1 and NRXN3 genes contribute to autism spectrum disorder susceptibility and mental retardation (Feng et al, 2006; The Autism Genome Project Consortium, 2007; Kim et al, 2008; Yan et al, 2008; Glessner et al, 2009). As neuroligin and neurexin interactions have a crucial function in synapse validation and regulation (Chubykin et al, 2007), molecular-based studies are essential to understanding the determinants that govern complex formation and function. The large number of different isoforms of both partners makes a systematic in vitro analysis and thorough in vivo transgene studies laborious. Nevertheless, in vitro studies revealed that an intricate recognition code controls the Ca2+-dependent interaction between different neuroligin and neurexin isoforms. In broad outline, the NL1 interaction with Nrxβ promotes formation of glutamatergic excitatory synapses, whereas NL2 in contact with Nrxα induces assembly of GABAergic inhibitory synapses (Boucard et al, 2005; Chih et al, 2006; Graf et al, 2006; Kang et al, 2008). Either interaction is further regulated by alternative splicing of both partners, ionic strength or N-linked glycosylation of neuroligins (Comoletti et al, 2003, 2006; Chen et al, 2008). After the initial study of small angle X-ray and neutron scattering by Comoletti et al (2007), the molecular bases for recognition of neuroligins by neurexins have been approached at atomic resolution with crystal structures of Nrx1β–NL1 and Nrx1β–NL4 complexes (Araç et al, 2007; Fabrichny et al, 2007; Chen et al, 2008). These studies have revealed the position and orientation of the bound Nrx1β at the neuroligin surface and confirmed the requirement for Ca2+ at the complex interface. They also provide initial templates to understand the dynamic interaction network between different neuroligin and neurexin isoforms. For example, the spatial locations of SSB in NL1 and SS4 in Nrx1β, both proximal to the binding interface, may explain partial inhibition of complex formation in splice variants. Despite the availability of structural templates, the determinants of selective recognition between neuroligins and neurexins remain partially unsolved. For example, we earlier proposed that the very low affinity of Nrx1β for NL2 (KD∼8.8 μM) (Comoletti et al, 2006) might be related to the presence of a bulky Gln side chain at position 475 in the neurexin-binding site of NL2, in place of Gly500 in NL1 and Gly464 in NL4 (Fabrichny et al, 2007), but experimental data to confirm this are lacking. As well, the Nrx1β affinity for NL1 is about four-fold higher than for NL4 (KD=29 and 132 nM, respectively) (Comoletti et al, 2006), but the structural basis for this difference remains unclear. Finally, the limited resolution of our initial structure of the Nrx1β–NL4 complex (Fabrichny et al, 2007) precluded a detailed comparison with structures of the related Nrx1β–NL1 complex or of free NL1 (Araç et al, 2007; Chen et al, 2008). We have solved a new, 2.6 Å-resolution structure of the Nrx1β–NL4 complex that permits precise positioning of all side chains at the complex interface, including those involved in Ca2+ coordination. Comparison with the structure of unbound NL4, solved at a similar resolution (Fabrichny et al, 2007), unveils conformational and positional rearrangements of loops and side chains at the NL4 surface upon Nrx1β binding. Moreover, we used site-directed mutagenesis and surface plasmon resonance (SPR) to (i) explore in real time the dependency of Nrx1β binding to NL1, NL4 and NL2 as a function of ionic strength and pH; (ii) analyse the relative contributions of structure-guided Nrx1β mutants to complex formation and stability and (iii) determine the energetic contributions of two different interfacial residues selected from the structures, His294 in NL1 and Gln475 in NL2, in governing Nrx1β binding. Our comprehensive analysis shows that whereas NL1 and NL2 are respectively preset conformationally to bind and not bind Nrx1β, conformational reshaping at the NL4 surface is required for accommodating Nrx1β. Results and discussion Improved accuracy of the Nrx1β–NL4 complex structure The overall 2.6 Å-resolution structure of the Nrx1β–NL4 complex is similar to earlier structures of the same complex (3.9 Å; Fabrichny et al, 2007) and of Nrx1β–NL1 complexes (2.4 and 3.5 Å; Araç et al, 2007; Chen et al, 2008) (Figure 1A). The extracellular domain of NL4 is composed of a twisted central β sheet surrounded by multiple α-helices, typical of the α/β-hydrolase fold. Two NL4 subunits related by a two-fold symmetry axis assemble as a non-covalent anti-parallel dimer, through a tightly packed four-helix bundle made of helices α7,83 and the C-terminal helix α10 from each subunit. The Nrx1β-binding site on NL4, which consists of three surface loops encompassing residues His267–Gly271, Gln359–Asp366 and Gln462–Pro466, is located on the face opposite to the Cys-loop (Figure 1A), an equivalent to the long Ω loop at the active site gorge entrance of AChE (cf. Fabrichny et al, 2007). The extracellular domain of Nrx1β is composed of two anti-parallel β sheets of six and seven β strands, arranged in a jelly roll β-sandwich typical of lectin and lectin-like domains (Rudenko et al, 1999). Nrx1β is bound with its β-sandwich oriented perpendicular to the long axis of the NL4 dimer and its concave side facing the NL4 four-helix bundle. The NL4-binding site on Nrx1β corresponds to the hypervariable-loop edge, opposite to the N- and C-termini (Figure 1A). Compared with the structures of unbound and NL1-bound Nrx1β (Araç et al, 2007; Chen et al, 2008; Shen et al, 2008), no conformational change is detected in the NL4-bound Nrx1β molecule. Figure 1.The Nrx1β–NL4 complex and binding interface at 2.6 Å resolution. (A) Overall structure of the Nrx1β–NL4 complex, with a yellow NL4 dimer, magenta four-helix bundle, cyan Cys-loops, green Nrx1β molecules and grey Ca2+; the N- and C-termini are labelled. (B) The amphiphilic character of the binding interface, most evident at the Nrx1β surface with the contiguous blue electropositive and white hydrophobic/apolar patches, insures attraction and proper orientation of the partnering molecules. Electrostatic surface potentials of the Nrx1β (top) and NL4 (bottom) molecules are contoured at −0.3/+0.3 V, in which red describes negative and blue positive potentials. Both molecules are rotated 90° from (A) in opposite directions along the horizontal axis, and the molecular surface involved in the interaction is outlined in black. (C) The Ca2+ position and coordination in the Nrx1β–NL4 (left) and Nrx1β–NL1 (middle) complexes and the Nrx1β LNS domain (3BOD; Koehnke et al, 2008) (right) are similar. The omit difference electron density map (blue) for the Ca2+ is contoured at 5σ in the Nrx1β–NL4 complex. Water molecules were not visible in the structure of the Nrx1β–NL1 complex because of its lower resolution (3.5 Å). Download figure Download PowerPoint This new structure of the Nrx1β–NL4 complex permits a detailed description of the interface, notably in the vicinity of the Ca2+ coordination sphere. Ca2+ lies central to the interface where it is coordinated by six ligands and adopts a typical octahedral geometry (Figure 1C). Four coordinating oxygen atoms are provided by Nrx1β residues (Asn238, Asp137, Val154, Ile236) and two others by water molecules. NL4 interactions with Ca2+ only involve residues Glu361 and Gln359 through water-mediated contacts. The position and coordination of Ca2+ in the Nrx1β–NL4 complex are similar to those in unbound Nrx1β and the Nrx1β–NL1 complex (Araç et al, 2007; Chen et al, 2008; Shen et al, 2008), arguing for conservation of the Ca2+-binding site geometry in the neurexin–neuroligin interaction. The binding interface is flat and covers a surface area of ∼620 Å2 (buried to a 1.4 Å probe radius) on each molecule, a value consistent with the moderate affinity of the complex partners (Supplementary Movie S1). Two contiguous subsites that mainly encompass electrostatic interactions and hydrophobic/apolar contacts, respectively, are clearly identified (Figure 1B; Table I). In the first subsite, an electropositive patch formed by the Nrx1β Arg109 and Arg232 side chains and the Ca2+ nestled at the Nrx1β surface is complementary, in overall shape, dimensions and charge distribution, to the electronegative patch formed by the Glu270, Asp351 and Glu361 side chains at the NL4 surface. This subsite holds the Ca2+-mediated interactions and consists mainly of hydrogen-bonding interactions involving four water molecules. In turn, only two Nrx1β apolar side chains lie within this positively charged subsite, Leu234 and Ile236, the latter having an important function for Nrx1β binding (see below). Hence, charge complementarity between the two partners highlights the crucial function of the Ca2+, whose coordination to negatively charged residues at the Nrx1β surface reinforces the electropositive motif and hence, not only prevents charge repulsion of the partners, but also favours their electrostatic attraction. In the second subsite, the binding interface mainly encompasses long-range hydrophobic/apolar contacts along with a few hydrogen bonds. Most of these contacts involve the peptidic backbone of each partner and small side chain residues, except for a hydrophobic stacking interaction between Nrx1β Leu135 and the phenol ring of NL4 Tyr463. Hence, the amphiphilic character of the complex interface likely accounts for the proper orientation of the two partnering proteins despite the limited contact area. Finally, three protruding Nrx1β side chains, Arg109 and Arg232 in the electropositive subsite and Asn103 at the edge of the hydrophobic/apolar subsite, are suitably positioned to serve as boundary clamp for stabilizing the complex, consistent with our earlier interpretation (Fabrichny et al, 2007). Table 1. Nrx1β and NL4 residues in contact, and NL1-specific contacts Nrx1β NL4a NL1b Hydrogen bonds Asn103 Arg561 OD1c NH2c Od,e, d,e Asp366 OD1/OD2d,e, d,e Ser107 Asn364 NHd OD1d O Lys306 NZf Arg109 Glu270b NHe OE2e NH1 OE1 NH1 Tyr365 OHe Arg232 Asp351 NH1c OD1c NH2 Gln359 OE1 Thr235 Glu361 NH OE1 (d)/OE2(c) OG1 OE2 Ile 236 Glu361 NHc OE1c Asn238 Gly360 ND2 O Hydrophobic contactsg Asn103 Pro466 Arg105d Asn364d Pro106 Asn364 Lys306h Ser107 Asn364Leu363 Thr108 Leu363 Arg109 His267Glu270b Leu135 Gln462Tyr463 Leu234 Gln359Glu361 Thr235 Glu361 Ile236 Glu361Phe362Leu363 Asn238 Tyr463 Ser239 Tyr463Gly464 a NL4 residues conserved among all neuroligins are labelled in bold. b Contacts in the Nrx1β–NL4 complex are conserved in the Nrx1β–NL1 complex, except for those involving Glu270 (corresponding to Glu297 in NL1) because of the insertion of Lys306 in NL1. c Contact observed only in subunit B of the dimer present in the asymmetric unit. d Contact observed only in subunit A of the dimer present in the asymmetric unit. e Interaction through a water molecule. f Contact observed only in subunits A and C of the two dimers present in the asymmetric unit. g Within a 4.5 Å distance between carbon atoms from each partner in the complex. h Contact observed only in subunits A and B of the two dimers present in the asymmetric unit. Conformational rearrangements in NL4 associated with Nrx1β binding Structural analysis of Nrx1β-bound versus unbound NL4 reveals conformational differences in two distinct regions at the NL4 surface. The first region corresponds to the Nrx1β-binding site, the topology of which is entirely reshaped through concerted positional rearrangement of several side chains to accommodate Nrx1β binding (Figure 2A; Supplementary Movie S2). Most notably, the side chains of Glu361 and Leu363 at the centre of the interface along with those of Glu270 and His267 on the one edge and Tyr463 on the other edge of the interface undergo marked swapping and/or rotation motions upon Nrx1β association (Supplementary Table S1). These conformational displacements disrupt the intricate tripartite hydrogen-bonding network formed by the side chains of Glu270 and Glu361, that form a glutamic acid bridge, and of His267, observed in unbound NL4. As a consequence, the Glu361 side chain is stabilized in a suitable position for Ca2+ coordination, whereas a cavity formed by Leu363, His267 and Glu270 is created to accommodate the guanidinium group in Arg109 of Nrx1β. Formation of a glutamic acid bridge between Glu270 and Glu361 in NL4 requires protonation of at least one of their two carboxyl groups. In fact, NL4 was crystallized at pH4.3, a value close to the glutamate pKa value, but the Glu270–Glu361 interaction is likely to occur under physiological conditions as well. In unbound NL4, the side chain of His267 interacts with Glu270 and could locally modify its pKa value, thus participating to stabilization of the Glu270–Glu361 bridge. In NL1, the corresponding His294 contributes to pH dependency of the Nrx1β–NL1 interaction (see below). Figure 2.Conformational rearrangements of the Nrx1β-binding site occur in NL4, but not in NL1 upon binding to Nrx1β. (A) The Nrx1β-binding site has a distinct conformation in Nrx1β-bound NL4 (NL4 in yellow and Nrx1β in green) compared with unbound NL4 (orange). Black arrows depict displacement of NL4 residues upon Nrx1β binding. In unbound NL4, the tripartite interaction between His267, Glu270 and Glu361 prevents Nrx1β binding and indirect Ca2+ coordination. In one subunit of the unbound NL4 dimer, His267 adopts an alternate conformation corresponding to that found in Nrx1β-bound NL4 (not shown). (B) The Nrx1β-binding site in unbound NL1 (blue) is similar to that in NL4 bound to Nrx1β (both displayed as in A). Numbering in parentheses corresponds to NL1 residues. Owing to the insertion of Lys306 in NL1, the Nrx1β-binding site in unbound NL1 is complementary for Nrx1β binding and does not undergo conformational rearrangements upon complex formation. Download figure Download PowerPoint The second region where the topologies of Nrx1β-bound and unbound NL4 diverge significantly is located on the subunit face that is opposite to the Nrx1β-binding site, and where the active site gorge opens in AChE (Supplementary Figure S1). Here, the Cys-loop (Cys110–Cys146) adopts distinct conformations in the two unbound NL4 subunits, both differing from that observed in Nrx1β-bound NL4, whereas the neighbouring loops L3 (Gln477–Ser487) and L4 (Ile502–Ser513) display different conformations in the two structures. This observation further illustrates flexibility of the NL4 Cys-loop (Fabrichny et al, 2007) and highlights sequence variations associated with conformational mobility of the corresponding surface loops among members of the α/β-hydrolase-fold family. The function of these loops in neuroligins and the relevance of conformational rearrangements occurring on this face of the NL4 subunit upon Nrx1β binding on the opposite face are unclear. Presence of a vestigial active centre cavity filled with water molecules (Fabrichny et al, 2007) may impart overall flexibility and a more distensible NL4, reflected in conformational changes of surface loops on the opposite side. These conformational rearrangements may also be associated with recognition and binding of a still non-identified second neuroligin partner. Structural comparison of the Nrx1β–NL4 and Nrx1β–NL1 complexes The extracellular domains of NL4 and NL1 (without splice inserts A and B) share ∼80% of sequence identity and display very similar structures (see RMSD values in Materials and methods). All the residues forming the Nrx1β-binding surface of NL4 are conserved in NL1, except for two conservative substitutions (Asn498 and Phe499 in NL1 for Gln462 and Tyr463 in NL4) that are most unlikely to alter the Nrx1β interaction network. Indeed, neither the side chain of NL4 Gln462 nor that of NL1 Asn498 do interact with bound Nrx1β, whereas NL4 Tyr463 interacts through its phenol ring, as does NL1 Phe499 (Araç et al, 2007), with no contribution of its hydroxyl group (Figure 2B). However, for NL1, the insertion of Lys306 between Glu297 and Gly307 in the absence of splice insert B (Supplementary Figure S2), found at the Nrx1β-binding site, is a unique feature of this neuroligin subtype. In the Nrx1β–NL1 complex, Lys306 interacts with Nrx1β Pro106 and Ser107 (Table I), and pushes the adjacent Glu297 side chain towards NL1 Arg259 at the edge of the interface. In the Nrx1β–NL4 complex, the corresponding negatively charged Glu270 residue faces Nrx1β and interacts with its Arg109. There is no conformational rearrangement of the Nrx1β-binding site of NL1 upon Nrx1β binding, as shown by perfect superimposition of this region in the unbound and Nrx1β-bound NL1 structures (Araç et al, 2007). In fact, as a result of the Lys306 insertion, the Glu297 position and stabilization through a salt bridge with Arg259 prevent any interaction with Glu397, contrary to that observed between the corresponding Glu270 and Glu361 residues in unbound NL4 (Figure 2A). Consequently, the binding site topology in unbound NL1 is preset for Nrx1β binding. Indeed, the Glu397 side chain orientation is compatible with Ca2+ coordination and the cavity formed by His294 and Leu399 (but not Glu297, because of its interaction with Arg259) can accommodate Nrx1β Arg109 (Figure 2B). Characterization of Nrx1β binding to neuroligins The energetics and the dynamic properties of the interaction between Nrx1β
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