Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide Ingemar von Ossowski1, Esko Oksanen2, Lotta von Ossowski1, Chunlin Cai1, Maria Sundberg1, Adrian Goldman2,3 and Kari Keinanen1�� 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. Keinanen, �� 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 (SAP97PDZ2). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97PDZ2 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. SAP97PDZ2 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 SAP97PDZ2. 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 �� 2006 The Authors Journal compilation �� 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 (SAP97PDZ2). 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 SAP97PDZ2 [19]. In an attempt to understand the structural basis of GluR-A���SAP97 interaction, we have determined the crystal structure of SAP97PDZ2 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- ACTD 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-GluRACTD kDa B wt L907A XA908 25 37 37 25 Lysate IP: Anti-SAP97N GFP-GluRACTD 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 �� 2006 The Authors Journal compilation �� 2006 FEBS