Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide Ingemar von Ossowski 1 , Esko Oksanen 2 , Lotta von Ossowski 1 , Chunlin Cai 1 , Maria Sundberg 1 , Adrian Goldman 2,3 and Kari Keina ¨ nen 1 1 Department of Biological and Environmental Sciences (Division of Biochemistry), University of Helsinki, Finland 2 Institute of Biotechnology, University of Helsinki, Finland 3 Neuroscience Center, University of Helsinki, Finland Selective insertion ⁄ removal of GluR-A (GluR1) sub- unit, containing a-amino-5-methyl-3-hydroxy-4-isoxa- zole propionic acid (AMPA) receptors to⁄ from the postsynaptic membrane, is a key early event in some experimental models of activity-dependent regulation of synaptic strength [1–5]. Rapid insertion of GluR-A to synaptic membrane is dependent on its cytoplasmic C-terminal domain of 80 amino acid residues [6]. Keywords crystal structure; GluR1; GluR-A; PDZ domain; SAP97 Correspondence K. Keina ¨ nen, Department of Biological and Environmental Sciences (Division of Biochemistry), Viikinkaari 5, P.O.Box56, FI-00014 University of Helsinki, Helsinki, Finland Fax: +358 9191 59068 Tel: +358 9191 59606 E-mail: kari.keinanen@helsinki.fi (Received 25 August 2006, revised 28 September 2006, accepted 2 October 2006) doi:10.1111/j.1742-4658.2006.05521.x Synaptic targeting of GluR-A subunit-containing glutamate receptors involves an interaction with synapse-associated protein 97 (SAP97). The C-terminus of GluR-A, which contains a class I PDZ ligand motif (-x-Ser ⁄ Thr-x-/-COOH where / is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97 PDZ2 ). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97 PDZ2 variant in complex with an 18-mer C-terminal peptide (residues 890–907) of GluR-A and of two variant PDZ2 domains in unliganded state at 1.8–2.44 A ˚ resolutions. SAP97 PDZ2 folds to a compact globular domain comprising six b-strands and two a-helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C-terminal residues of the GluR- A are visible, and align as an antiparallel b-strand in the binding groove of SAP97 PDZ2 . The free carboxylate group and the aliphatic side chain of the C-terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR-A peptide are engaged in essential class I PDZ interactions. Compar- ison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The bA)bB loop moves away from the C-terminal end of aB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in bA)bB loop with Asp396 or Thr394 in bB. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif-destroying L907A mutation as well as the insertion of an extra alanine residue between the C-terminal Leu907 and the stop codon are also consistent with a ‘classical’ type I PDZ interaction between SAP97 and GluR-A C-terminus. Abbreviations AMPA, a-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid; GluR-A, ionotropic glutamate receptor subunit A; GST, glutathione S-transferase; Maguk, membrane-associated guanylate kinase homolog; PDZ, postsynaptic density )95 ⁄ Discs large ⁄ zona occludens-1; PSD-93, postsynaptic density )93; PSD-95, postsynaptic density )95; SAP97, synapse-associated protein 97; SAP102, synapse-associated protein 102. FEBS Journal 273 (2006) 5219–5229 ª 2006 The Authors Journal compilation ª 2006 FEBS 5219 The GluR-A C-terminus contains a class I PDZ ligand motif which is necessary for the stimulated synaptic incorporation of GluR-A subunit in an acute fashion [7–9]. Interestingly, it was recently reported that trans- genic mice expressing GluR-A variant lacking seven C-terminal residues display apparently normal synaptic plasticity and basal GluR-A localization [10], suggest- ing developmental plasticity and ⁄ or existence of multi- ple parallel pathways in GluR-A transport. To date, four PDZ domain-containing proteins have been reported to associate with GluR-A via its C-terminus: SAP97 [11], mLin-10 [12], Shank3 [13], and RIL [14], although the binding of RIL to GluR-A is not PDZ domain-mediated. From these candidates, the multi- domain scaffolding protein SAP97 emerges as a strong candidate to subserve synaptic delivery of GluR-A AMPA receptors. Overexpression of SAP97 drives GluR-A to synapses and increases AMPA receptor mediated synaptic currents [15,16], and concomitantly occludes long-term potentiation [16]. In a converse manner, RNAi block of SAP97 expression in cultured neurons inhibits expression of surface GluR-A and AMPA receptor mediated synaptic currents [16]. SAP97 may also play a role in the endocytosis of GluR-A AMPA receptors, based on identification of a ternary complex between SAP97, GluR-A and myosin VI, a minus-end directed actin motor [17,18]. Detailed information on the molecular mechanism of SAP97–GluR-A interaction would help us to under- stand its physiological roles and regulation. In glu- tathione S-transferase (GST) pulldown assay, GluR-A C-terminus binds to the second PDZ domain of SAP97 (SAP97 PDZ2 ). The binding is dependent on the PDZ binding motif, but is also strongly affected by sequences upstream of the C-terminus of GluR-A [19,20], includ- ing formation of a disulfide-linked complex between synthetic GluR-A C-terminal peptide and SAP97 PDZ2 [19]. In an attempt to understand the structural basis of GluR-A–SAP97 interaction, we have determined the crystal structure of SAP97 PDZ2 in the presence and absence of a GluR-A C-terminal 18-mer peptide ligand. The structure reveals an archetypical class I PDZ inter- action that involves the last four residues of GluR-A with no apparent contribution by other residues. Results Binding of GluR-A C-terminal domain to SAP97 in cultured cells To complement and extend our earlier GST pulldown analysis of GluR-A–SAP97 PDZ interaction, we first examined the interaction of the C-terminal domain (CTD; residues 827–907) of GluR-A with SAP97 under cellular conditions. We created three different GFP- tagged GluR-A CTDs: the wild-type with the C-term- inal residues Ala904-Thr905-Gly906-Leu907 and two point-mutated versions, a L907A substitution which eliminates the class I PDZ binding motif, and ‘XA908’ in which an extra alanine residue was inserted between the C-terminal Leu907 and the stop codon. These were cotransfected with myc-tagged SAP97 in HEK293 cells. All proteins were expressed at roughly equal levels as indicated by the intensities of anti-GFP and anti-myc immunoblots, but only the wild-type CTD associated with myc-tagged SAP97 (Fig. 1A). This result is in agreement with our earlier in vitro binding analysis [20], which confirms the importance of the canonical class I PDZ binding motif. As SAP97 is endogenously present in HEK293 cells, we also analyzed anti-SAP97 immuno- precipitates of cells transfected only with GFP-GluR- A CTD expression plasmids. Consistent with the above results, only the wild-type CTD associated with the native SAP97 (Fig. 1B). In vitro binding of GluR-A C-terminus to SAP97 PDZ domains To further examine the canonical class I PDZ binding motif of the GluR-A C-terminus, we used a microplate A Lysate Blot: anti-GFP 37 25 Lysate Blot: anti-myc 100 150 IP: anti-GFP Blot: anti-myc 100 150 wt L907A XA908 myc-SAP97 + GFP-GluRA CTD kDa B wt L907A XA908 25 37 37 25 Lysate IP: Anti-SAP97 N GFP-GluRA CTD Blot: anti-GFP kDa Fig. 1. Coimmunoprecipitation of GluR-A CTD with SAP97. HEK293 cells were transfected for expression of wild-type (‘wt’) GFP-GluR- A CTD, and mutated CTDs L907A and ‘XA908’ with (A) or without (B) N-terminally myc-tagged SAP97. The cells were subjected to immunoprecipitation and immunoblotting by using anti-myc or anti- GFP as indicated. Molecular size markers are indicated on the left. IP, immunoprecipitation. SAP97 PDZ2–GluR-A peptide complex I. von Ossowski et al. 5220 FEBS Journal 273 (2006) 5219–5229 ª 2006 The Authors Journal compilation ª 2006 FEBS binding assay to analyze the interaction between puri- fied SAP97 PDZ proteins. Purified His-tagged PDZ1, PDZ2, and PDZ3 domains of SAP97 were adsorbed on microwells, followed by blocking of nonspecific protein adsorption sites by an excess of BSA. The binding of wild-type or mutated GluR-A C-terminal 11-mer pep- tides (GST-A 11 ) produced as GST fusions was deter- mined by using anti-GST-horseradish peroxidase conjugate as an enzymatic marker (Fig. 2A). Short C-terminal peptides were used instead of ‘full-length’ CTDs because of the higher stability of the former in bacterial expression. GST-A 11 bound consistently stron- gest to the PDZ2 domain, whereas the L907A mutation led to significantly decreased binding (Fig. 2B). More- over, GST-A 11 showed somewhat weaker, but detect- able PDZ motif-dependent binding to the first but not to the third PDZ domain of SAP97 (Fig. 2B). PDZ motif-independent binding of GST fusions was consis- tently higher to PDZ2 coated wells than to PDZ1 and PDZ3 wells. Control experiments indicate that both ele- vated background adsorption of the anti-GST-peroxi- dase conjugate to SAP97 PDZ2 and nonspecific formation of disulfide links between GST proteins and SAP97 con- tribute to the higher background (results not shown). Crystal structure of SAP97 PDZ2 In order to obtain detailed structural information on the binding, we set up crystallization screens for purified PDZ1 and PDZ2 domains and PDZ1-3 segment of SAP97. Attempts to crystallize SAP97 PDZ1 or SAP97 PDZ1-3 were unsuccessful, but crystals were obtained from SAP97 PDZ2 both alone and complexed with GluR-A C-terminal 18-mer peptide (A 18 ). During initial purification of SAP97 PDZ2 , we noticed the occa- sional formation of disulfide-linked dimers due to the presence of a single cysteine residue at position 378. The corresponding residue in the homologous PDZ2 domain of PSD-95 is Gly219 (Fig. 3), which is located on the protein surface 15 A ˚ from the binding pocket in the solution NMR structure of PSD-95 PDZ2 [21]. To avoid problems arising from the potential heterogeneity of dimeric and monomeric PDZ domains and from disul- fide-linked PDZ domain–peptide ligand complexes observed previously in in vitro peptide binding studies [19], we expressed and purified two variants in which Cys378 was replaced either by glycine (C378G) or the isosteric serine residue (C378S). While crystals of wild-type SAP97 PDZ2 never diffracted in the absence of the GluR-A 18 peptide, well-diffracting crystals, belonging to space group P2 1 , were obtained from the C378S (resolution 1.8 A ˚ ) and C378G (2.44 A ˚ ) variants A B Fig. 2. Binding of GluR-A wild-type and mutated GluR-A C-termini to SAP97 PDZ domain-coated microwells. (A) Schematic outline of the assay. The immobilized PDZ domains and GST fusion protein con- taining 11-residue C-terminal segment from GluR-A are illustrated. (B) Binding of wild-type and L907A variant GST-GluR-A fusion pep- tides to immobilized PDZ1, PDZ2, and PDZ3 domains of SAP97. Fig. 3. Amino acid sequence similarities of PDZ domain proteins. Multiple sequence alignment of PDZ domains from SAP97, PSD-95, PSD93, SAP102, Shank3, and mLin-10 proteins. The secondary structural elements of SAP97 PDZ2 are included at the top of the sequence alignment. The residues (Gly328-Leu329-Gly330-Phe331) of the carboxylate binding loop and the critical histidine residue (His384) in aB are highlighted by green and blue, respectively. The single cysteine residue (Cys378) in SAP97 is indicated by red color. The numbers refer to amino acid sequence of the corresponding full-length proteins. Identity and similarity of the aligned residues are indicated by asterisks, semi- colons (high similarity), and dots (low similarity), respectively. I. von Ossowski et al. SAP97 PDZ2–GluR-A peptide complex FEBS Journal 273 (2006) 5219–5229 ª 2006 The Authors Journal compilation ª 2006 FEBS 5221 (Tables 1 and 2). The structures were solved by molecu- lar replacement (see Experimental procedures). There are two PDZ domains in the asymmetric unit, each with a typical PDZ domain folding topology with six b-strands (bAtobF) and two a-helices (aA and aB) (Fig. 4). The structure of the C378G variant of SAP97 PDZ2 was practically identical to that of C378S variant, except for the mutated residue, and because of its better resolution, only the structure of the C378S var- iant will be discussed below. The first two N-terminal residues are not visible in either of the two chains, and 11 (chain A) or 13 (chain B) C-terminal residues includ- ing the hexahistidinyl tags also appear unstructured. The binding site region of chain B contains additional electron density, modeled tentatively as two histidines, packed into the binding groove between bB and aB, which may arise from binding of the C-terminus of a neighboring PDZ domain in the crystal. We therefore used chain A, which does not contain anything in the ligand binding site, to compare with the peptide-bound structure. The serine and glycine residues, used to replace Cys378 in the native PDZ domain in the C378S and C378G variants are located on the outer surface and the serine side chain pointing outwardly into solu- tion (Fig. 4). Comparison to closely related PDZ domain structures The solution structure of the second PDZ domain of PSD-95 has been determined by NMR [21], and, quite recently, coordinates of the PDZ2 domain of SAP102 crystallized in the absence of ligand have been depos- ited in the PDB (access code: 2FE5). As these domains Table 1. Data collection and processing statistics. Data set SAP97 PDZ2 wt + GluR-A 18 SAP97 PDZ2 C378G + GluR-A 18 SAP97 PDZ2 C378G SAP97 PDZ2 C378S Space group P2 1 P2 1 P2 1 P2 1 Unit cell parameters a 34.8 A ˚ 34.4 A ˚ 32.2 A ˚ 32.3 A ˚ b 54.4 A ˚ 54.3 A ˚ 52.1 A ˚ 52.4 A ˚ c 55.1 A ˚ 54.6 A ˚ 52.6 A ˚ 52.9 A ˚ a 90° 90° 90° 90° b 94.0° 84.0° 102.7° 102.7° c 90° 90° 90° 90° Wavelength (A ˚ ) 0.934 0.934 1.519 0.931 Resolution (A ˚ ) a 20–2.35 (2.41–2.35) 34.1–2.2 (2.33–2.2) 20–2.44 (2.59–2.44) 20–1.8 (1.91–1.8) Completeness (%) a 97.5 (96.6) 99.7 (99.3) 96.6 (90.8) 97.7 (97.3) Redundancy a 7.5 (7.6) 5.0 (5.0) 7.4 (7.2) 2.5 (2.5) R merge a,b 0.081 (0.517) 0.054 (0.40) 0.025 (0.08) 0.055 (0.40) I ⁄ r a 16.8 (4.1) 16.6 (3.5) 37.2 (17.8) 14.1 (2.8) a Values for the highest resolution shell are given in parentheses. b R merge ¼ S hkl |I(hkl) – <I(hkl)>| ⁄S hkl I(hkl) where <I(hkl)> is the mean of the symmetry equivalent reflections of I(hkl). Table 2. Refinement statistics. Data set SAP97 PDZ2 wt + GluR-A 18 SAP97 PDZ2 C378G + GluR-A 18 SAP97 PDZ2 C378G SAP97 PDZ2 C378S Total unique reflections ⁄ test set (% size of the test set) 8005 ⁄ 422 (5) 9658 ⁄ 509 (5) 5908 ⁄ 311 (5) 14964 ⁄ 788 (5) R work ⁄ R free a,b 0.218 ⁄ 0.295 (0.319 ⁄ 0.350) 0.242 ⁄ 0.326 (0.350 ⁄ 0.443) 0.180 ⁄ 0.295 (0.209 ⁄ 0.380) 0.188 ⁄ 0.254 (0.245 ⁄ 0.374) Bond rmsd from ideality (A ˚ ) 0.017 0.017 0.015 0.012 Angle rmsd from ideality (°) 1.78 1.75 1.56 1.97 Residues in favored Ramachandran plot regions (%) 91.0 92.8 94.5 97.2 Residues in allowed Ramachandran plot regions (%) 98.9 98.3 98.9 100 a Values for the highest resolution shell are given in parentheses. b R work ¼ S hkl (|F o (hkl)| - |F c (hkl)|) ⁄S hkl |F o (hkl)| for F(hkl) not belonging to the test set; R free ¼ S hkl (|F o (hkl)| ) |F c (hkl)|) ⁄S hkl |F o (hkl)| for F(hkl) in the test set. SAP97 PDZ2–GluR-A peptide complex I. von Ossowski et al. 5222 FEBS Journal 273 (2006) 5219–5229 ª 2006 The Authors Journal compilation ª 2006 FEBS show 90% sequence similarity (80–89% identity) to SAP97 PDZ2 , it was interesting to compare the three unliganded PDZ domain structures. Figure 5 shows a superposition of SAP97 PDZ2 C378S structure with the NMR structure of PSD-95 PDZ2 and the crystal struc- ture of SAP102. The backbone structures are highly similar, except for some of the surface loops. The loop bA-bB is packed closer to the C-terminus of aBin SAP97 than in PSD-95 or SAP102. In addition, bB-bC loop is extended outwards to the solution in SAP97 and SAP102, whereas it bends towards the C-terminus in PSD-95. Ligand binding interactions of SAP97 PDZ2 Well-diffracting crystals belonging to the same space group as the free PDZ domains were obtained for the wild-type and C378G variant of SAP97 PDZ2 cocrystal- lized with a synthetic peptide representing the 18 C-terminal residues of GluR-A (A 18 ). The two struc- tures were solved and turned out to be highly similar to each other (Table 3). Only the last four C-terminal residues of A 18 peptide are visible in the complex as an extended b-strand sandwiched between aB and bB and antiparallel to the b-strand (Fig. 6A). The term- inal carboxylate group of Leu907 forms hydrogen bonds with main chain amide nitrogens of Leu329, Gly330, and Phe331. The aliphatic side chain of the C-terminal leucine is accommodated in a hydrophobic pocket formed by the apolar side chains of Phe331, Ile333, and Leu391 (Fig. 6B). The threonine at posi- tion P )2 forms an essential hydrogen bond with the His384 at the N-terminal end of aB. These results are in agreement with our previous mutagenesis study which indicated that the ability of point-mutated GluR-A C-terminal domains to bind to PDZ1-3 seg- ment of SAP97 is not sensitive to residue changes at positions P )1 and P )3 , while P )2 and the C-terminal P 0 are critical [20]. The side chain of Cys378 points out into solution in the complex of wild-type SAP97 and αA C N C378S βA βF βC βB βD αB Fig. 4. Ribbon diagram of the SAP97 PDZ2 C378S variant structure. Helices and strands are named according to Doyle et al. [22]. Ser378 is shown in sticks. α A αA αB αB βF βF βA βA βD βD βB βB βC βC Loop βA-βB Loop βA-βB Loop βB-βC Loop βB-βC Fig. 5. Stereoscopic representation of superposed backbone structures for SAP97 PDZ2 C378S (blue), SAP102 PDZ2 (orange; 2FE5), and PSD-95 PDZ2 (magenta; 1QLC) [21]. Secondary structural elements and loops are labeled accordingly. I. von Ossowski et al. SAP97 PDZ2–GluR-A peptide complex FEBS Journal 273 (2006) 5219–5229 ª 2006 The Authors Journal compilation ª 2006 FEBS 5223 A 18 peptide, consistent with a potential reactivity in disulfide bond formation. Conformational differences between the free and peptide bound SAP97 PDZ2 Superposition of the a-carbon traces of the complex structure and apo PDZ domain (C378S) reveals differ- ences that may arise from ligand-induced conforma- tional changes in the PDZ domain (Fig. 7A). The major differences can be seen in areas which are close to the C-terminus of the bound peptide: in the unli- ganded PDZ domain, the bA-bB loop is closer to the C-terminal end of the aB than in the complex struc- ture, suggesting that peptide binding is accompanied by slight opening of the structure, also including slight alterations in the aB-bF loop. The peptide binding cavity in the uncomplexed PDZ domain is partially filled by five water molecules that occupy similar posi- tions to each of the polar atoms, both main chain and side chain, in the bound peptide. In addition, there is a water molecule occupying the hydrophobic pocket for the terminal leucine. Moreover, there is an intriguing switch involving Lys324 in the bA-bB loop. In the unliganded structure, it forms a tight ion-pair (2.6 A ˚ ) with Asp396 between aB and bF, while in the liganded structure, Asp396 moves to the outside and Lys324 forms hydrogen-bonds to Thr394 Oc2, Thr394 O and Ser395 O (Fig. 7B). Unlike the interactions already described in Doyle et al. [22] (see Discussion), such Table 3. Superpositions of PDZ domains. No. of aligned residues ⁄ Ca rmsd (A ˚ ) ⁄ Sequence identity (%) Stationary Stationary II Moving SAP97 PDZ2 C378S (chain A) SAP97 PDZ2 + GluR-A 18 (chain A) SAP97 PDZ2 C378G + GluR-A 18 (chain A) 90 ⁄ 0.69 ⁄ 98 90 ⁄ 0.33 ⁄ 98 SAP102 PDZ2 (2FE5) 89 ⁄ 0.73 ⁄ 80 90 ⁄ 0.71 ⁄ 80 PSD95 PDZ3 (1BFE) 83 ⁄ 0.99 ⁄ 37 83 ⁄ 1.03 ⁄ 37 PSD95 PDZ3 + CRIPT peptide (1BE9) 83 ⁄ 1.14 ⁄ 37 83 ⁄ 1.03 ⁄ 37 AB Fig. 6. Ligand binding interactions between GluR-A C-terminal (A 18 ) peptide and SAP97 PDZ2 . (A) A Fo-Fc omit electron density map of the last four C-terminal residues of the GluR-A 18 peptide contoured at 3.0 r and the residues Gly328-Leu329-Gly330-Phe331-Ser332-Ile333 of the carboxylate binding loop. The peptide backbone is shown in green and the carboxylate binding loop in grey. Hydrogen bonds between GluR-A C-terminus and the carboxylate binding loop are indicated as dotted lines. (B) Schematic presentation produced by LIGPLOT of the polar and hydrophobic interactions between the C-terminal Leu907 of GluR-A 18 peptide and the PDZ domain. Carbon, nitrogen and oxygen atoms are shown as black, blue and red spheres, respectively. Hydrogen bond are indicated as dotted lines (including distances in A ˚ ) and hydrophobic interactions as arcs with radial spokes. SAP97 PDZ2–GluR-A peptide complex I. von Ossowski et al. 5224 FEBS Journal 273 (2006) 5219–5229 ª 2006 The Authors Journal compilation ª 2006 FEBS bond movements could be the switch between the two conformations for facilitating better accommodation of the peptide in the PDZ binding groove. Recently, it was reported [23] that residues within the bB-bC loop of ZO1-PDZ1 rearrange in the liganded structure to produce a deep pocket not present in the unliganded form, for binding of the residue at P )6 . However, in our study, no conformational change in the extended bB-bC loop was observed (Fig. 7A), and consequently this region is less likely to contribute to ligand recogni- tion in SAP97–GluR-A interaction. Discussion Leonard et al. [11] originally identified an interaction between GluR-A and SAP97, and showed that the extreme C-terminus of GluR-A binds to the PDZ1-3 segment of SAP97 in vitro. This PDZ interaction is probably solely responsible for the association, because GluR-A subunit lacking seven C-terminal residues expressed in transgenic mice did not form an immuno- precipitable complex with SAP97 unlike its wild-type counterpart [10]. In vitro binding of SAP97 PDZ1-3 to GluR-A C-terminal domain showed an absolute requirement for a large hydrophobic side chain at the C-terminus and a side chain hydroxyl group at the P )2 position, in agreement with a typical class I PDZ inter- action [20]. Of the three PDZ domains in SAP97, only PDZ2 bound to the GluR-A C-terminus in our earlier GST pulldown assay [20]. However, in our present study, the use of microplate assay with immobilized PDZ domains to analyze the interaction revealed that the PDZ1 domain is also active, albeit less so than PDZ2. The ability of SAP97 PDZ1 to bind to GluR-A is in agreement with some earlier findings [24,25] and with reports indicating that the PDZ1 and PDZ2 domains of PSD-95 family membrane-associated gua- nylate kinase homologs (Maguks) display similar pre- ferences for their peptide ligands, and differ in this respect from PDZ3 [26]. The crystal structure of SAP97 PDZ2 with bound GluR-A peptide, determined in the present study, is consistent with the archetypical features of class I PDZ domains. The interaction with bound peptide occurs at positions P 0 (C-terminus) and P )2 , but not at P )1 and P )3 other than through main chain hydrogen bonds with bB. In most class I PDZ domain-ligand pairs, additional interactions engaging the amino acid residues at P )1 and P )3 of the peptide in multiple polar and non- polar contacts with the PDZ domain impart increased affinity and sequence specificity, important for physio- logical interactions. The absence of such supplementary interactions from the GluR-A–SAP97 PDZ2 complex may not be surprising considering that in GluR-A, ala- nine and glycine residues occupy at P )3 and P )1 , which is unusual for a class I PDZ ligand. These two residues do not show any unusual structural features in the pep- tide complex, and can be mutated to glycine ⁄ alanine without affecting binding in GST pulldown assay [20]. Therefore, it seems probable that additional interac- tions or mechanisms are required to provide further specificity and affinity. Previously, we had demonstrated with both in vitro peptide binding assays and NMR spectroscopy that under nonreducing conditions a disulfide bond may form between the Cys893 of a GluR-A 18 peptide and the Cys378 of SAP97 PDZ2 domain, and consequently we suggested this covalent coupling could be the basis for a redox-regulated binding mechanism [19]. Formation of a disulfide bond would be consistent with the finding that in GST pulldown experiments, A B Fig. 7. Structural changes in PDZ domain upon peptide binding. (A) Superposition of the wild-type SAP97 PDZ2 (green) in the peptide complex with the unliganded structure of the C378S variant (blue), highlighting the potential movements of the bA-bB loop and aB upon peptide binding. (B) Conformational switch in the bA-bB car- boxylate binding loop between apo (blue) and liganded (green) structures. Relevant amino acids and secondary structural elements are labeled. I. von Ossowski et al. SAP97 PDZ2–GluR-A peptide complex FEBS Journal 273 (2006) 5219–5229 ª 2006 The Authors Journal compilation ª 2006 FEBS 5225 replacement of a tripeptide sequence Ser897-Ser898- Gly899 in GluR-A C-terminal domain by three alanine residues leads to loss of binding, possibly due to decreased flexibility of the C-terminus [20]. However, in our crystal structure of wild-type SAP97 PDZ2 cocrys- tallized with the GluR-A 18 peptide, there was no dis- cernible electron density for bound peptide beyond the last four residues of the PDZ-motif and therefore the structure does not provide an explanation for the above-mentioned contributions by upstream sequence elements. Whether this is due to insufficient length of the peptide can definitely be answered only through determination of the larger structures including the entire C-terminal domain. Our attempts to form crys- tals of SAP97 PDZ2 in complex with the GluR-A CTD have so far remained unsuccessful due to the poor solubility and stability of purified full-length GluR- A CTD protein. Notwithstanding the remaining questions concerning the structural basis for the exquisite specificity of the interaction between SAP97 and GluR-A, the present crystal structures provide interesting new findings. Of the PSD-95 family Maguk proteins, only the structure of PSD-95 PDZ3 has been solved in the presence of a peptide ligand [22]. In addition, nonliganded structures have been solved for the third PDZ domain of human SAP97 [27], and for the second PDZ domain of SAP102 (2FE5; Structural Genomics Consortium, unpublished results). In the PSD)95 PDZ3 peptide com- plex structure, peptide binding was accompanied by only minor conformational changes limited to a few amino acid side chains [22]. In their structure, the authors also saw a small shift between liganded and nonliganded forms, but ascribed it to crystal contacts, a viewpoint we do not entirely share. While it is true that there are some crystal contacts at the bottom of the ligand-binding pocket, there is a correlation between binding of ligand, changes in cell dimensions, and the opening and closing of the pocket; whenever the ligand is bound, the b-angle, for instance, is 84–93°, and whenever it is not, the b-angle is 103° (Table 1). It seems clear that the rearrangement of the interactions around Lys324 provides two different but stable con- formations that correlate with ligand being bound or not. In contrast, in the structure of PSD-95 PDZ3 pep- tide ligand complex, the residue equivalent to Lys324, Arg318, binds the terminal carboxylate through a water molecule. As described above, the interactions we see are quite different and may help explain some of the differences in binding properties between PSD-95 PDZ3 and SAP97 PDZ2 . Furthermore, although it may be a coincidence, we find it interesting that both the PSD-95 and our structure make crystal contacts in the same region (at the bottom of the peptide-binding groove) even though the space group is different and the sequence identity is low in this region. We wonder if this may indicate some of the regions of allosteric speci- ficity, and the possibility that other proteins interact with this region as well. Direct structural evidence exists for an analogous situation for non-PDZ protein inter- action domains in PSD-95: an intramolecular interac- tion between a ‘hook’ domain and guanylate kinase domain keeps the molecule in a closed state [28,29]. In conclusion, our crystallographic analysis shows a canonical class I interaction between SAP97 PDZ2 and GluR-A 18 peptide. The discrepancy between the relative scarcity of interactions in the complex struc- ture and the high affinity and specificity evident from in vitro binding experiments [19,20] suggests the existence of additional, but as yet unclear, mechanisms in this interaction. An unexpected and novel feature of SAP97 PDZ2 structure is the conformational shift in bA-bB loop between the free and liganded structures. Experimental procedures Bacterial expression and protein purification C-terminally His-tagged SAP97 PDZ2 (residues 315–410 in SwissProt Q911D0) was expressed in Escherichia coli BL21(DE3)pLysS and purified by immobilized metal chela- tion affinity chromatography on Ni 2+ -charged NTA-Agar- ose (Qiagen, Hilden, Germany) as described previously [19,20]. SAP97 PDZ2 C378S and C378G mutations were gen- erated by PCR, and the mutated proteins were expressed and purified as the wild-type domain. His-tagged PDZ1 (residues 213–313), PDZ3 (residues 459–450) and PDZ1-3 (213–450) segment were expressed and purified as above. All PDZ constructs contained an eight amino acid residue seg- ment SRHHHHHH at their C-termini. GST fusion proteins of wild-type GluR-A C-terminal 11-mer peptide (GST-A 11 ) has been described previously [20]. GST fusion of C-term- inal 11-mer containing a L907A mutation was produced by PCR mutagenesis. GST fusion proteins were expressed in E. coli BL21 and purified by GSH-Sepharose chromatogra- phy (Amersham Biotech, Uppsala, Sweden) [20]. Mammalian cell expression Expression plasmids encoding GFP-tagged GluR-A CTD (resi- dues 827–907 in rat GluR-A; M36418) and GluR-A CTD var- iants L907A and ‘XA908’ (carrying an extra alanine residue after C-terminal Leu907) were generated by subcloning appropriate PCR fragments between EcoRI and HindIII sites of a derivative of pEGFP-C1 (Clontech, Palo Alto, CA, USA), which harbored a hexahistidinyl tag linker (HHHHHHEF) between GFP and the C-terminal insert. SAP97 PDZ2–GluR-A peptide complex I. von Ossowski et al. 5226 FEBS Journal 273 (2006) 5219–5229 ª 2006 The Authors Journal compilation ª 2006 FEBS HEK293 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 2 mm glutamine, 10% (v ⁄ v) fetal calf serum, penicillin (50 units ⁄ mL), and streptomycin (50 units ⁄ mL) at 37 °C under 5% CO 2 . Cells were transfected with wild-type and point-mutated GFP-GluR-A CTD fusion plasmids either alone or together with expression plasmid encoding myc-tagged SAP97 [30] by using calcium phosphate coprecipitation, and harvested 36–48 h after the transfection and used for immunoprecipitations as described below. Immunoprecipitations Detergent extracts were prepared from transfected HEK293 cells by homogenizing the cells in 10 volumes of 50 mm Tris ⁄ HCl, pH 8.0, 140 mm NaCl, 1 mm EDTA, 1 mm Na 3 VO 4 ,1mm NaF, and 1 mm phenylmethylsulfonyl fluor- ide (TNE buffer). Triton X-100 was added to a final con- centration of 1% (w ⁄ v), and the suspension was mixed at 4 °C for 2 h, followed by centrifugation at 20 000 g for 15 min. Aliquots (1-mL) of supernatant were incubated with anti-GFP IgG (2 lg; Abcam, Cambridge, UK) or SAP97 N antiserum (2 lL) [20] overnight at 4 °C. Immuno- complexes were bound to GammaBind G-Sepharose (Amersham Biosciences; 20 lL) for 2 h under gentle rota- tion. Sepharose beads were collected by brief centrifugation, washed three times with TNE, and twice with NaCl ⁄ P i , and finally suspended in 20 lL of SDS sample buffer, heated at 95 °C for 5 min and analyzed by SDS ⁄ PAGE and immuno- blotting with anti-GFP or anti-myc antibodies [30]. Microplate binding assay In vitro binding of wild-type and mutated GluR-A C-ter- mini to SAP97 PDZ domains was analyzed by using a plate binding assay. Briefly, microtiter plates were coated with purified PDZ1, PDZ2, and PDZ3 domains (0.5 lm in NaCl ⁄ P i ; overnight at 4 °C), blocked with bovine serum albumin, and incubated with GST fusion proteins (5 lgÆmL )1 in NaCl ⁄ P i ) for 5–10 min at room temperature, conditions determined to yield the highest signal-to-back- ground ratio. The wells were washed three times with NaCl ⁄ P i ⁄ 0.5% Tween 20 and incubated with anti-GST- IgG–horseradish peroxidase conjugate for 1 h at room tem- perature. After further washes, peroxidase substrate was added, and the absorbance values at 450 nm were recorded after 30–60 min from substrate addition. Binding of GST fusion proteins to bovine serum albumin-coated control wells, generally representing <15% of total, was subtracted from the absorbance values as nonspecific binding. Protein crystallization SAP97 PDZ2 domain proteins were crystallized at room temperature using the sitting drop vapor diffusion method equilibrated against a reservoir solution of 30% polyethy- lene glycol (PEG 6000), 0.1 m Tris ⁄ HCl, pH 8.8, and 0.2 m sodium acetate. Crystals of the C378G and C378S variant proteins (1.3 and 0.85 mm in 10 mm Bis-Tris pH 6.5, respectively) were grown in a mixture of 1 lL protein and 1 lL reservoir solution. Both the wild-type (2.3 mm) and the C378G variant (1.3 mm) proteins were cocrystallized with the GluRA 18 peptide (SIPCMSHSSGMPLGATGL) by mixing 1 lL of protein and 1 lL of peptide (5 mm in 10 mm Bis-Tris pH 6.5) with 2 lL of the reservoir solution. All crystals grew as rod clusters within 1–3 days. Crystallographic structure determination The data were collected at the ESRF (European Synchro- tron Radiation Facility) beamlines ID29 for a 1.3 A ˚ reso- lution data set of C378G, ID14-1 for wild-type and C378G with peptide and ID14-3 for C378S. The data for C378G variant were collected at EMBL ⁄ DESY beamline BW7A. All data were collected at 100 K and processed with the program xds [31] (Table 1). Attempts to use the NMR structures of the PDZ1 [32] and PDZ2 [21] domains of PSD-95 with 53% and 89% sequence iden- tity, respectively, to SAP97 PDZ2 as search models yielded no useful solutions for molecular replacement, probably due to the rather high rmsd ⁄ Ca (Table 3). A molecular replacement solution could be obtained for the high reso- lution data set of the C378G variant using the crystal structure of PDZ3 domain of SAP97 ⁄ hDlg, PDB-ID 1PDR [27] as a model. The molecular replacement was performed with the program molrep [33] in the CCP4 suite [34]. Automatic model building and refinement was carried out with the program arp ⁄ warp [35]. The model thus obtained could not be refined satisfactorily by man- ual model building and refinement, but using one mole- cule from partly refined structure as a molecular replacement model, the other data sets could be phased by molecular replacement, although no solution was obtained with 1PDR as a model. The program refmac5 was used for refinement and the programs o [36] and coot [37] for manual model building. Structure validation was done with the molprobity web server [38]. The superpositions were carried out using the SSM algorithm [39]. Figures were prepared with pymol [40] and ligplot [41]. Protein data bank accession numbers X-ray structural data and the derived atomic coordinates have been deposited with the Protein Data Bank (http:// www.rcsb.org/pdb) under the accession codes: 2G2L (wild- type SAP97 PDZ2 with GluR-A 18 peptide), 2AWX (C378S variant), 2AWW (C378G variant with GluR-A 18 peptide), and 2AWU (C378G variant). I. von Ossowski et al. SAP97 PDZ2–GluR-A peptide complex FEBS Journal 273 (2006) 5219–5229 ª 2006 The Authors Journal compilation ª 2006 FEBS 5227 Acknowledgements We acknowledge the European Synchrotron Radiation Facility MX beamlines and The EMBL BW7A beam- line at the DORIS storage ring, DESY, Hamburg for provision of synchrotron radiation facilities and we would like to thank the beamline personnel for assis- tance in data collection. We thank D. Bansfield for technical assistance. This work was supported by grants from the Academy of Finland to AG (1105157), and to KK (202892). 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(2002) The PyMOL Molecular Graphics System DeLano Scientific, San Carlos, CA, USA 41 Wallace AC, Laskowski RA & Thornton JM (1995) LIGPLOT: a program to generate schematic diagrams of protein–ligand interactions Protein Eng 8, 127–134 FEBS Journal 273 (2006) 5219–5229 ª 2006 The Authors Journal compilation ª 2006 FEBS 5229 . C-terminal residues of the GluR- A are visible, and align as an antiparallel b-strand in the binding groove of SAP97 PDZ2 . The free carboxylate group and the aliphatic side chain of the C-terminal. the native SAP97 (Fig. 1B). In vitro binding of GluR -A C-terminus to SAP97 PDZ domains To further examine the canonical class I PDZ binding motif of the GluR -A C-terminus, we used a microplate A Lysate Blot:. (2.44 A ˚ ) variants A B Fig. 2. Binding of GluR -A wild-type and mutated GluR -A C-termini to SAP97 PDZ domain- coated microwells. (A) Schematic outline of the assay. The immobilized PDZ domains and