Deciphering the structural framework of glycine receptor anchoring by gephyrin
2006; Springer Nature; Volume: 25; Issue: 6 Linguagem: Inglês
10.1038/sj.emboj.7601029
ISSN1460-2075
AutoresEun Young Kim, Nils Schrader, Birthe Smolinsky, Cécile Bedet, Christian Vannier, Günter Schwarz, Hermann Schindelin,
Tópico(s)Plant Molecular Biology Research
ResumoArticle2 March 2006free access Deciphering the structural framework of glycine receptor anchoring by gephyrin Eun Young Kim Eun Young Kim Department of Biochemistry, Center for Structural Biology, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Nils Schrader Nils Schrader Department of Plant Biology, Technical University Braunschweig, Braunschweig, GermanyPresent address: Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany Search for more papers by this author Birthe Smolinsky Birthe Smolinsky Department of Plant Biology, Technical University Braunschweig, Braunschweig, Germany Institute of Biochemistry, University of Cologne, Köln, Germany Search for more papers by this author Cécile Bedet Cécile Bedet Laboratoire de Biologie Cellulaire de la Synapse Normal et Pathologique, INSERM, École Normale Supérieure, Paris, France Search for more papers by this author Christian Vannier Christian Vannier Laboratoire de Biologie Cellulaire de la Synapse Normal et Pathologique, INSERM, École Normale Supérieure, Paris, France Search for more papers by this author Günter Schwarz Corresponding Author Günter Schwarz Department of Plant Biology, Technical University Braunschweig, Braunschweig, Germany Institute of Biochemistry, University of Cologne, Köln, Germany Search for more papers by this author Hermann Schindelin Corresponding Author Hermann Schindelin Department of Biochemistry, Center for Structural Biology, State University of New York at Stony Brook, Stony Brook, NY, USA Rudolf Virchow Center for Experimental Biomedicine and Institute of Structural Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Eun Young Kim Eun Young Kim Department of Biochemistry, Center for Structural Biology, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Nils Schrader Nils Schrader Department of Plant Biology, Technical University Braunschweig, Braunschweig, GermanyPresent address: Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany Search for more papers by this author Birthe Smolinsky Birthe Smolinsky Department of Plant Biology, Technical University Braunschweig, Braunschweig, Germany Institute of Biochemistry, University of Cologne, Köln, Germany Search for more papers by this author Cécile Bedet Cécile Bedet Laboratoire de Biologie Cellulaire de la Synapse Normal et Pathologique, INSERM, École Normale Supérieure, Paris, France Search for more papers by this author Christian Vannier Christian Vannier Laboratoire de Biologie Cellulaire de la Synapse Normal et Pathologique, INSERM, École Normale Supérieure, Paris, France Search for more papers by this author Günter Schwarz Corresponding Author Günter Schwarz Department of Plant Biology, Technical University Braunschweig, Braunschweig, Germany Institute of Biochemistry, University of Cologne, Köln, Germany Search for more papers by this author Hermann Schindelin Corresponding Author Hermann Schindelin Department of Biochemistry, Center for Structural Biology, State University of New York at Stony Brook, Stony Brook, NY, USA Rudolf Virchow Center for Experimental Biomedicine and Institute of Structural Biology, University of Würzburg, Würzburg, Germany Search for more papers by this author Author Information Eun Young Kim1, Nils Schrader2, Birthe Smolinsky2,3, Cécile Bedet4, Christian Vannier4, Günter Schwarz 2,3 and Hermann Schindelin 1,5 1Department of Biochemistry, Center for Structural Biology, State University of New York at Stony Brook, Stony Brook, NY, USA 2Department of Plant Biology, Technical University Braunschweig, Braunschweig, Germany 3Institute of Biochemistry, University of Cologne, Köln, Germany 4Laboratoire de Biologie Cellulaire de la Synapse Normal et Pathologique, INSERM, École Normale Supérieure, Paris, France 5Rudolf Virchow Center for Experimental Biomedicine and Institute of Structural Biology, University of Würzburg, Würzburg, Germany *Corresponding authors: Department of Biochemistry, Center for Structural Biology, State University of New York at Stony Brook, Stony Brook, NY 11794-5115, USA. Tel.: +1 631 632 1022; Fax: +1 631 632 1555; E-mail: [email protected] of Biochemistry, University of Cologne, Otto-Fischer-Strasse, 12-14, 50674 Köln, Germany. Tel.: +49 221 470 6432; Fax: +49 221 470 6731; E-mail: [email protected] The EMBO Journal (2006)25:1385-1395https://doi.org/10.1038/sj.emboj.7601029 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Glycine is the major inhibitory neurotransmitter in the spinal cord and brain stem. Gephyrin is required to achieve a high concentration of glycine receptors (GlyRs) in the postsynaptic membrane, which is crucial for efficient glycinergic signal transduction. The interaction between gephyrin and the GlyR involves the E-domain of gephyrin and a cytoplasmic loop located between transmembrane segments three and four of the GlyR β subunit. Here, we present crystal structures of the gephyrin E-domain with and without the GlyR β-loop at 2.4 and 2.7 Å resolutions, respectively. The GlyR β-loop is bound in a symmetric 'key and lock' fashion to each E-domain monomer in a pocket adjacent to the dimer interface. Structure-guided mutagenesis followed by in vitro binding and in vivo colocalization assays demonstrate that a hydrophobic interaction formed by Phe 330 of gephyrin and Phe 398 and Ile 400 of the GlyR β-loop is crucial for binding. Introduction The glycine receptor (GlyR) is the main inhibitory neurotransmitter receptor in the spinal cord and brain stem. Binding of glycine results in channel opening and allows Cl− flux across the synaptic membrane. As a member of the ligand-gated ion channel superfamily, functional GlyRs have been generally believed to display a pentameric subunit assembly with an α3β2 stoichiometry in the adult organism; however, recently an alternative α2β3 stoichiometry was reported (Grudzinska et al, 2005). In contrast, prenatal GlyRs are predominantly α5-homopentamers. Up to date, four α-subunits (α1–α4) and one β-subunit have been identified, but it is not well understood how subunit diversity is related to the function and distribution of GlyRs (Lynch, 2004). For fast and precise signal transmission at inhibitory synapses, GlyRs as well as γ-aminobutyric acid type A receptors (GABAAR) are required to be highly concentrated at the postsynaptic membrane, a process which is dependent on the receptor-associated scaffolding protein gephyrin (Kneussel and Betz, 2000; Sheng and Pak, 2000; Legendre, 2001; Moss and Smart, 2001). Gephyrin is a 93 kDa cytoplasmic protein, which was initially discovered through copurification with GlyRs from rat spinal cord (Pfeiffer et al, 1982). It consists of an N-terminal G-domain and a C-terminal E-domain, which are connected by a central region composed of ∼160 amino-acid residues. The two domains display significant similarities to the bacterial MogA and MoeA proteins, respectively, which are essential for molybdenum cofactor (Moco) biosynthesis. Strong experimental evidence has accumulated, which demonstrates that gephyrin is critical for GlyR clustering. Gephyrin colocalizes with GlyRs through the hydrophobic interaction between its E-domain and the large cytoplasmic loop connecting transmembrane (TM) segments 3 and 4 of the GlyR β-subunit (Kirsch et al, 1995; Kneussel et al, 1999; Schrader et al, 2004), which will be referred to as GlyR β-loop. Furthermore, a loss of gephyrin synthesis, either by antisense oligonucleotides in spinal cord neurons or by gene knockout in mice, prevents GlyR clustering (Kirsch et al, 1993; Feng et al, 1998). Although no direct interaction between gephyrin and GABAAR has been demonstrated, gephyrin also plays a crucial role in the stabilization of GABAAR clusters. Furthermore, gephyrin interacts with the microfilament components profilin, Mena/VASP and G-actin (Giesemann et al, 2003) as well as tubulin (Kirsch et al, 1991), and the simultaneous binding to GlyR and microtubules or the actin-based cytoskeleton forms the basis of the neuroreceptor-anchoring function of gephyrin. In addition, a variety of other cellular components such as GABARAP, collybistin, RAFT1, and dynein light chains (Sabatini et al, 1999; Kins et al, 2000; Kneussel et al, 2000; Fuhrmann et al, 2002) also interact with gephyrin, although the physiological significance of these interactions is not well characterized at present. As expected from the primary sequence homology, gephyrin is also involved in Moco biosynthesis, specifically the last step of molybdenum insertion into the apo-cofactor. It was shown that gephyrin is able to reconstitute Moco biosynthesis in Moco-deficient bacteria, plants and mammalian cells (Stallmeyer et al, 1999), and a loss of gephyrin causes Moco deficiency in mice (Feng et al, 1998) and humans (Reiss et al, 2001). It remains unclear whether the described two functions of gephyrin are related to each other. Nevertheless, together they define gephyrin as a 'moonlighting' protein. The gene encoding gephyrin is highly mosaic, and, as a result, its heterogeneity by alternative splicing could potentially contribute to its functional diversity (Meier et al, 2000; Rees et al, 2003), yet a correlation between the different functions and splice variants remains to be established. The crystal structures of Escherichia coli MogA, the G-domains of gephyrin and the plant homologs Cnx1 and E. coli MoeA have been determined recently (Liu et al, 2000; Schwarz et al, 2001; Xiang et al, 2001). On the basis of these structural data, gephyrin was proposed to trimerize and dimerize via its G-domain and E-domain, respectively, and eventually assemble into a hexagonal lattice, which is proposed to connect inhibitory neuroreceptors to components of the cytoskeleton (Kneussel and Betz, 2000; Xiang et al, 2001). More recently, the crystal structure of gephyrin's E-domain in complex with a putative hexapeptide derived from GlyR β-loop suggested that the binding site of GlyR resides in subdomain IV of the E-domain (Sola et al, 2004), whereas our in vitro interaction assays suggested subdomain III instead (Schrader et al, 2004). This complex structure, however, was refined at a relatively low resolution of 3.25 Å, and neither the identity of the residues involved nor the details of the interaction could be identified. Here, we present the 2.4 Å crystal structure of gephyrin's E-domain in complex with the GlyR β-loop and the apo-structure of the E-domain at 2.7 Å. The cocrystal structure defines the interaction between GlyR and gephyrin in atomic detail. The contributions of selected residues to the binding have been probed by site-directed mutagenesis coupled with in vitro and in vivo binding studies providing insights into the molecular details of gephyrin-mediated clustering of glycine receptors. Results and discussion Structure determination of Geph-E alone and in complex with the GlyR β-loop The E-domain of rat gephyrin (Geph-E, residues 318–736) was crystallized in space group P21212 containing one dimer per asymmetric unit. An initial model of Geph-E was built using a lower-resolution data set (3.3 Å) by a combination of a two-wavelength MAD experiment and molecular replacement using E. coli MoeA as the search model (Table I). Subsequently, a higher-resolution data set was obtained, and a final structure of Geph-E was determined at 2.7 Å resolution (Table II), resulting in an R-factor of 20.5% (Rfree=27.7%). The overall structure of Geph-E (Figure 1A) shows a high structural similarity to E. coli MoeA (Xiang et al, 2001), as indicated by an overall root mean square (r.m.s.) deviation of 2.02 Å, and is even more closely related to the recently published E-domain structure (Sola et al, 2004), with an overall r.m.s. deviation of 0.95 Å. Each Geph-E monomer consists of four subdomains (Figure 1A and B) as first described for MoeA, and two monomers interact to form an elongated dimer with subdomains II on opposite ends. A comparison of the two monomers reveals that the core structure (subdomains I, III and IV) is rather rigid, as reflected by an r.m.s. deviation of 0.7 Å for the Cα atoms, whereas subdomain II is not only oriented differently with respect to the core but also displays substantially higher r.m.s. deviations of 2.5 Å. This is probably due to its flexibility, which results in poorly defined electron density of subdomain II in one of the monomers. Figure 1.Overall structure of Geph-E and complex with the GlyR β-loop. (A) Ribbon diagram of the Geph-E apo-structure. The four subdomains of one monomer are color-coded as shown in the schematic representation (upper left), with the other monomer in gray. The N- and C-termini of Geph-E are indicated. The amino-acid numbering is based on the P1 splice variant (Prior et al, 1992). (B) Ribbon diagram of Geph-E in complex with the GlyR β-loop-derived peptide. The bound peptide is 13 residues long and colored in salmon. The orientation of the left view is identical to the apo structure, whereas the right view is rotated 90° relative to the left view around the horizontal axis. (C) Electron density of the GlyR β-loop. Stereo view of a simulated annealed omit map (contoured at 0.8 times the r.m.s. deviation) superimposed with the final model of the GlyR β-loop. The peptide is shown in atom-based color code (C: salmon, O: red, and N: blue) using a stick representation. The N- and C-termini of the peptide are indicated. Download figure Download PowerPoint Table 1. Data collection and MAD statistics SeMet Geph-E Geph-E Peak Remote Geph-E/GlyR β-loop Data collection Wavelength (Å) 1.1 0.98021 0.96496 1.1 Resolution limits (Å) 50–2.7 50–3.5 50–3.5 50–2.4 Number of reflections 24 624 21 485 21 781 18 251 Completeness 0.99 (0.999) 1.0 (0.999) 1.0 (1.0) 0.935 (0.77) Mean redundancy 4.8 5.8 3.8 5.0 Rsyma 0.10 (0.54) 0.16 (0.43) 0.17 (0.51) 0.09 (0.56) 〈I/σI〉b 13.7 (2.6) 21.5 (8.3) 14.5 (4.9) 21.5 (2.7) Space group P21212 P21212 P21212 C2221 Unit cell dimensions a, b, c (Å) 108.8, 156.2, 51.4 109.0, 157.2, 51.5 109.0, 157.2, 51.5 51.3, 123.5, 155.0 MAD Number of sites 18 Rcullisc 0.86 NA FOMd 0.2395 aRsym=∑hkl∑i∣Ii−〈I〉∣/∑hkl∑iIi, where Ii is the ith measurement and 〈I〉 is the weighted mean of all measurements of I. b〈I/σI〉 indicates the average of the intensity divided by its standard deviation. Numbers in parentheses refer to the respective highest resolution data shell in each data set. cRcullis is the lack of closure divided by the absolute of the difference between FPH and FP for the anomalous differences. dFOM is the mean figure of merit at 4 Å resolution. Table 2. Refinement statistics Geph-E Geph-E/GlyR β-loop Resolution limits (Å) 20–2.7 20–2.4 Number of reflections used in refinement 23 473 17 248 Number of protein/ solvent atoms 6191/66 3298/150 Rcrysta (Rfreeb) 0.205 (0.277) 0.192 (0.274) Deviations from ideal values in Bond distances (Å) 0.022 0.019 Bond angles (deg) 1.60 1.59 Torsion angles (deg) 4.4, 35.6, 16.3 4.9, 37.9, 14.9 Planar groups (Å) 0.005 0.005 DPI (Å)c 0.39 0.31 Ramachandran statistics (%)d 84.8/12.2/2.6/0.4 91.0/7.8/0.8/0.3 Overall average B-factor (Å2) 62.1 40.5 aRcryst=∑hkl∣∣Fo∣–∣Fc∣∣/∑hkl∣Fo∣, where Fo and Fc are the observed and calculated structure factor amplitudes. bRfree is the same as Rcryst for 5% of the data randomly omitted from refinement. The number of reflections excludes the Rfree subset. cDPI is the data precision index based on the free R factor as calculated by REFMAC. dRamachandran statistics indicate the fraction of residues in the most favored, additionally allowed, generously allowed, and disallowed regions of the Ramachandran diagram as defined by PROCHECK. To characterize the interaction between Geph-E and GlyR in atomic detail, individually purified Geph-E and residues 378–426 of the GlyR β-loop were cocrystallized. Two different crystal forms belonging either to space group P212121 containing one Geph-E dimer and two peptides, or C2221 with one monomer and a single peptide were obtained under the same crystallization conditions. In the C2221 crystals, the functional unit again is a dimer, which is formed via a crystallographic twofold symmetry axis. Both structures were solved by molecular replacement using the apo-structure. An initial 3.0 Å data set of the P212121 crystal confirmed the presence of the peptide and revealed that ∼10 residues were well-ordered and their sequence could be unambiguously assigned already at this resolution. The complex structure was ultimately refined at 2.4 Å resolution to an R-factor of 19.2% (Rfree=27.4%) against a data set collected from a C2221 crystal (Tables I and II). In this structure 13 residues, corresponding to the segment from 398 to 410 of the GlyR β-loop, were found to be ordered (Figure 1C). Due to the higher resolution, the following discussion will focus on the crystallographic dimer from the C2221 crystal, which is displayed in Figure 1B. The Geph-E/GlyR β-loop complex in the P212121 and C2221 crystals showed very similar structures (r.m.s. deviation of 0.88 Å), except for small changes in a few poorly defined loops. In addition, there are also no deviations between the two monomers in the P212121 form, indicating that the monomers in the dimer behave independently. This observation is in contrast to a published proposal (Sola et al, 2004), which suggests that the two Geph-E monomers display different affinities for the GlyR β-loop. Our previous biochemical studies have shown that each Geph-E monomer has two binding sites for the GlyR β-loop, one with high and the other with low affinity (Schrader et al, 2004). In our complex crystals prepared with a twofold excess of ligand, the lower-affinity binding site of Geph-E appears not to be occupied, as we cannot observe additional electron density corresponding to another bound peptide. The peptide derived from the GlyR β-loop does not display any secondary structure, with the exception of a short 310 helix formed by residues 406–410. Although a minor shift in subdomain II was observed upon binding of the GlyR β-loop (r.m.s. deviation of 1.67 Å), no significant conformational changes in the core structure of the E-domain (r.m.s. deviations of 0.65 Å) were seen. Therefore, association of the GlyR β-loop into gephyrin can be primarily described as following a lock-and-key, rather than an induced-fit mechanism. Details of the interface between Geph-E and the GlyR β-loop The 13 residues of the GlyR β-loop adopt the shape of the letter C and fit tightly into a groove created by subdomains III and IV from one monomer and subdomain IV′ from the other monomer (Figure 2). The Geph-E/GlyR β-loop interface buries ∼950 Å2 of the total surface area, which constitutes ∼53% of the total peptide surface area. Complex formation is stabilized mainly by hydrophobic interactions, which contribute more than 60% to the buried interface, and also by seven direct hydrogen bonds. The primary contact region on Geph-E can be divided into four smaller segments (Figure 2C): a loop from subdomain IV, including residues 713–719 (L713–719), the central four-stranded β-sheet of subdomain IV, the first α-helix and several neighboring residues in subdomain III, and a loop in subdomain IV′ comprised of residues 682–685 (L682–685). Figure 2.Detailed representation of Geph-E in complex with the GlyR β-loop. (A) Overall architecture of the complex. Subdomains of Geph-E are colored as in Figure 1, with the exception of subdomain IV′ from the second subunit, which is colored in dark blue. Both GlyR β-loops are shown in stick representation, utilizing the same color code as in Figure 1C. (B) Close-up view into the binding pocket. The GlyR β-loop is tightly packed in the cleft formed by subdomains III and IV from one monomer, as well as subdomain IV′ from the other monomer. (C) Detailed stereo view of the interactions between Geph-E and the GlyR β-loop. Left, right and lower walls of the peptide-binding pocket on Geph-E are displayed in orange, blue and yellow, respectively, and the bottom is colored in cyan. Geph-E is shown in ribbon representation and GlyR β-loop in stick representation. All residues in close proximity to the interface are highlighted and numbered (in black for Geph-E and magenta for the GlyR β-loop). Hydrogen bonds are shown as dotted red lines. Download figure Download PowerPoint L713–719, which is not conserved in the MoeA homologs, creates the 'left' wall of the binding pocket (as shown in Figure 2C), where Pro 713 and Tyr 719 make close contacts with Ser β403, Leu β404, Pro β405 and Arg β406 (the 'β' indicates residues in the GlyR β-loop). To a smaller extent Pro 714 and Thr 716 also contribute to this interaction, while Gln 718 forms a hydrogen bond with Arg β406, which, however, should be treated with caution as the arginine side chain is only poorly defined in the electron density maps (Figure 1C). A recent study has demonstrated the importance of this loop for receptor binding via in vivo colocalization and in vitro interaction assays using the E-domain mutant in which L713–719 was replaced with that of MoeA (Sola et al, 2004). Based on a superposition of the loops in the MoeA and Geph-E/GlyR β-loop structures, the charged residues Glu 387 and Arg 390 of MoeA (corresponding to Pro 713 and Lys 715 of gephyrin, respectively) protrude into the peptide-binding site, which presumably interferes with peptide binding. In contrast, Lys 715 points into the opposite direction away from the peptide and is not involved in interactions. Lys 658, Tyr 673, Met 711 and Asp 729 are aligned across the β-sheet of subdomain IV and primarily form the bottom of the binding pocket, which also involves four hydrogen bonds: one between Ile β400 and Tyr 673, two between Ser β403 (main chain amide and side chain OH) and Asp 729 and one between the side chains of Ser β403 and Lys 658. Moreover, Ile 656 and Val 727 participate in hydrophobic interactions with Phe β398 and Phe β408, respectively, at either end of the bound peptide. The 'lower' wall of the binding pocket is comprised of the first helix of subdomain III, as well as residues Leu 637 and Arg 653. Asp 327 forms two hydrogen bonds, one with the hydroxyl of Ser β399 and the other with the amide of Ile β400, which also makes hydrophobic contacts with Phe 330, Met 326 and Leu 637. In addition, the side chain of Arg 653 is parallel to the aromatic ring of Phe β398. Finally, L682–685 from the second monomer builds the 'right' wall of the binding pocket, fitting into the inside of the C-shaped peptide. In particular, Gln 683 is the residue closest to the peptide, which is embraced by Ser β403 and Phe β408. In addition, Pro 685 and His 682 are engaged in weak nonpolar interactions with Phe β408 and Phe β398. The fact that the peptide binds into a groove formed by both monomers suggests that GlyR binding might affect Geph-E dimerization. In vitro pulldown assays with mutants of gephyrin and the GlyR β-loop In order to identify which of the residues in the binding interface are critical for the interaction between gephyrin and GlyR, nine Geph-E variants (D327A, F330A, R653A, I656A, Y673F, Q683A, P713E, P713A/P714A and D729A) and seven GlyR β-loop variants (βF398A, βS399A, βI400A, βS403A, βF408A, βF398A/I400A and βF398A/I400A/F408A) were generated by site-directed mutagenesis. With the exception of the Geph-E Y673F variant, all Geph-E and GlyR β-loop mutants were overexpressed and purified in the same manner as the wild-type (WT) proteins. Initially, the contribution of these residues was studied in pulldown assays using either WT GlyR β-loop and Geph-E mutants or GlyR β-loop variants and WT Geph-E. The experiments with WT GlyR β-loop revealed that the F330A variant significantly reduces GlyR β-loop binding, while the P713E variant abolishes binding (Figure 3A). Interaction with P713A/P714A (PPAA) is also significantly abated, whereas the D327A, R653A, I656A, Q683A and D729A variants show similar levels of binding as the WT E-domain. Phe 330 is located in the first helix of subdomain III and is in the center of the lower wall of the binding pocket in close proximity to Phe β398. Loss of binding in the P713E variant was not unexpected, since not only a proline with its unique structural features is removed, but at the same time the negatively charged glutamate is introduced. Based on the MoeA structure, it is predicted to point towards the peptide-binding region and thus interferes with binding. In addition, similar results obtained with the double proline mutant PPAA may be explained by structural perturbations resulting from the replacement of the two proline residues, thus suggesting that Pro 713 and Pro 714 play an important role in maintaining L713–719 in a conformation that allows productive interactions with the GlyR β-loop. In contrast, the four hydrogen bonds formed by Asp 327 and Asp 729 are dispensable, and the other examined residues in the interface (Arg 653, Ile 656 and Gln 683) do not show a critical impact on binding either. Figure 3.Pulldown assays with Geph-E and GlyR β-loop mutants. (A) Various Geph-E mutants were purified and co-precipitated with WT GlyR β-loop beads. WT Geph-E was also tested with WT GlyR β-loop beads as the control. Identical amounts of protein were applied to equal volumes of the beads. Bound protein in the pellet (upper panel) and unbound protein in the supernatant (lower panel) were subjected to SDS–PAGE analysis. The lane labeled M contains a molecular weight standard. (B) Cosedimentation of WT Geph-E with various GlyR β-loop mutants immobilized to the beads. Download figure Download PowerPoint A second set of pulldown assays was performed to evaluate the GlyR β-loop variants. Previous colocalization assays revealed that only multiple, but not single, substitutions of hydrophobic side chains of GFP-49 (GlyR β-loop fused to a green fluorescent protein (GFP)) eliminated the interaction with gephyrin (Kneussel et al, 1999). In our current binding assays, surprisingly, the single substitution βF398A significantly impaired the interaction with Geph-E (Figure 3B). The βI400A substitution also showed a reduction in binding, albeit to a smaller extent, whereas βS403A and βF408A behaved like WT. The double (βF398A/I400A, βFI) and triple (βF398A/I400A/F408A, βFIF) variants containing βF398A completely abolished binding, which is thought to result from synergistic effects of βF398A and βI400A. Phe β398 and Ile β400 form a hydrophobic core in the lower binding wall together with Geph-E Phe 330, which, as outlined above, is also a major contributor to the strength of Geph-E/GlyR β-loop interaction. Analysis of the interactions involving either Asp 327 and Ser β399 or Asp 729 and Ser β403 through the respective single alanine substitutions revealed that the hydrogen bonds are not critical for this interaction. Subsequently, the same mutations in Geph-E as well as the additional Y719A variant were introduced into full-length gephyrin, splice variant P2 (Prior et al, 1992), followed by in vitro binding assays to verify whether residues critical for Geph-E/GlyR β-loop interactions are indeed required for P2/GlyR β-loop complex formation. All P2 mutants were overexpressed and purified following the protocol for the WT protein, and pulldown assays using WT and variants of P2 and GlyR β-loop were performed as described above. The overall results are consistent with the binding studies involving only the E-domain (as seen in Figure 3 and Supplementary Figure S1), showing that F330A, P713E, βF398A, βFI and βFIF significantly reduce or abolish binding, while the PPAA, βS399A and βI400A variants considerably weaken the interaction. As described above, Tyr 673 forms a hydrogen bond with the main-chain carbonyl of Ile β400 via its phenolic OH-group. The importance of this hydrogen bond could not be tested in Geph-E, since the Geph-E Y673F variant could not be overexpressed and purified. Unexpectedly, deletion of this hydrogen bond by conversion of tyrosine into phenylalanine drastically reduced the interaction of full-length gephyrin and the GlyR β-loop, whereas disruption of the hydrogen bonds in the D327A and D729A variants did not significantly alter P2/GlyR peptide binding. In contrast, an additional point mutation in L714–719, Y719A, behaved just like WT. This demonstrates that among the observed hydrogen bonds only the one involving the side chain of Tyr 673 is critical to the binding interaction. Overall, there is good agreement of the results from the pulldown assays between the isolated E-domain and full-length gephyrin, thus suggesting that the cocrystal structure of the Geph-E/GlyR β-loop complex faithfully reproduces the interactions in the gephyrin/GlyR complex. ITC studies of the Geph-E/GlyR interaction For further confirmation of the pulldown assays and quantitative analyses, those Geph-E and GlyR β-loop variants, which showed significant effects in the pulldown assays, were subjected to isothermal titration calorimetry (ITC). To test gephyrin mutants, purified Geph-E WT and the F330A, P713E and PPAA variants as well as P2 WT and the Y673F variant were individually titrated with GlyR β-loop WT (Figure 4A). The resulting binding enthalpy was corrected for the h
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