November 1, 2003Open Access

Crystal Structure of the Shank PDZ-Ligand Complex Reveals a Class I PDZ Interaction and a Novel PDZ-PDZ Dimerization

YIYoung Jun ImChonnam National University JLJun Hyuck LeeKorea Polar Research Institute SPSeong Ho ParkUlsan College

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Abstract

The Shank/proline-rich synapse-associated protein family of multidomain proteins is known to play an important role in the organization of synaptic multiprotein complexes. For instance, the Shank PDZ domain binds to the C termini of guanylate kinase-associated proteins, which in turn interact with the guanylate kinase domain of postsynaptic density-95 scaffolding proteins. Here we describe the crystal structures of Shank1 PDZ in its peptide free form and in complex with the C-terminal hexapeptide (EAQTRL) of guanylate kinase-associated protein (GKAP1a) determined at 1.8- and 2.25-Å resolutions, respectively. The structure shows the typical class I PDZ interaction of PDZ-peptide complex with the consensus sequence -X-(Thr/Ser)-X-Leu. In addition, Asp-634 within the Shank1 PDZ domain recognizes the positively charged Arg at –1 position and hydrogen bonds, and salt bridges between Arg-607 and the side chains of the ligand at –3 and –5 positions contribute further to the recognition of the peptide ligand. Remarkably, whether free or complexed, Shank1 PDZ domains form dimers with a conserved βB/βC loop and N-terminal βA strands, suggesting a novel model of PDZ-PDZ homodimerization. This implies that antiparallel dimerization through the N-terminal βA strands could be a common configuration among PDZ dimers. Within the dimeric structure, the two-peptide binding sites are arranged so that the N termini of the bound peptide ligands are in close proximity and oriented toward the 2-fold axis of the dimer. This configuration may provide a means of facilitating dimeric organization of PDZ-target assemblies. The Shank/proline-rich synapse-associated protein family of multidomain proteins is known to play an important role in the organization of synaptic multiprotein complexes. For instance, the Shank PDZ domain binds to the C termini of guanylate kinase-associated proteins, which in turn interact with the guanylate kinase domain of postsynaptic density-95 scaffolding proteins. Here we describe the crystal structures of Shank1 PDZ in its peptide free form and in complex with the C-terminal hexapeptide (EAQTRL) of guanylate kinase-associated protein (GKAP1a) determined at 1.8- and 2.25-Å resolutions, respectively. The structure shows the typical class I PDZ interaction of PDZ-peptide complex with the consensus sequence -X-(Thr/Ser)-X-Leu. In addition, Asp-634 within the Shank1 PDZ domain recognizes the positively charged Arg at –1 position and hydrogen bonds, and salt bridges between Arg-607 and the side chains of the ligand at –3 and –5 positions contribute further to the recognition of the peptide ligand. Remarkably, whether free or complexed, Shank1 PDZ domains form dimers with a conserved βB/βC loop and N-terminal βA strands, suggesting a novel model of PDZ-PDZ homodimerization. This implies that antiparallel dimerization through the N-terminal βA strands could be a common configuration among PDZ dimers. Within the dimeric structure, the two-peptide binding sites are arranged so that the N termini of the bound peptide ligands are in close proximity and oriented toward the 2-fold axis of the dimer. This configuration may provide a means of facilitating dimeric organization of PDZ-target assemblies. Multidomain Shank, proline-rich synapse-associated protein, and somatostatin receptor-interacting protein scaffold proteins bind to various membrane and cytoplasmic proteins within the PSDs 1The abbreviations used are: PSDpostsynaptic densityPDZPSD/discs large/ZO-1GKAPguanylate kinase-associated proteinSH3Src homology 3SAMsterile α-motifEVH1Ena/VSAP homology 1MADmultiple anomalous dispersionGRIPglutamate receptor-interacting proteinNHERFNa+/H+ exchanger regulatory factorr.m.s.root mean square. in excitatory synapses (1Sheng M. Kim E. J. Cell Sci. 2000; 113: 1851-1856Crossref PubMed Google Scholar, 2Boeckers T.M. Bockmann J. Kreutz M.R. Gundelfinger E.D. J. Neurochem. 2002; 81: 903-910Crossref PubMed Scopus (280) Google Scholar). It has been suggested that Shank links N-methyl-d-aspartate receptor-PSD-95 complexes to the actin cytoskeleton, thereby playing a critical role in the organization of cytoskeletal signaling complexes at excitatory synapses (1Sheng M. Kim E. J. Cell Sci. 2000; 113: 1851-1856Crossref PubMed Google Scholar, 2Boeckers T.M. Bockmann J. Kreutz M.R. Gundelfinger E.D. J. Neurochem. 2002; 81: 903-910Crossref PubMed Scopus (280) Google Scholar). The three known members of the Shank family (Shank1–3) all contain multiple sites for alternative splicing and show distinct tissue distributions (2Boeckers T.M. Bockmann J. Kreutz M.R. Gundelfinger E.D. J. Neurochem. 2002; 81: 903-910Crossref PubMed Scopus (280) Google Scholar). Although shank proteins vary in molecular mass, they share a common domain organization consisting of seven N-terminal ankyrin repeats followed by an SH3 domain, a PDZ domain, a long proline-rich region, and a SAM domain. All of these motifs are potentially involved in protein-protein interactions. For instance, the proline-rich region commonly acts as a binding site for SH3, EVH1, and WW domains and SAM domains can bind to each other in homomeric and heteromeric fashion, enabling oligomerization of Shank and its interacting proteins (3Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). postsynaptic density PSD/discs large/ZO-1 guanylate kinase-associated protein Src homology 3 sterile α-motif Ena/VSAP homology 1 multiple anomalous dispersion glutamate receptor-interacting protein Na+/H+ exchanger regulatory factor root mean square. PDZs are globular domains containing ∼80–100 amino acids (4Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1044) Google Scholar). The Shank PDZ domain is a class I PDZ recognizing the C-terminal sequence X-(Thr/Ser)-X-Leu (where X represents any amino acid), which enables it to bind a variety of integral membrane proteins; however, it most specifically binds to the C terminus of GKAP, which in turn interacts with the guanylate kinase domain of PSD-95 (5Yao I. Hata Y. Hirao K. Deguchi M. Ide N. Takeuchi M. Takai Y. J. Biol. Chem. 1999; 274: 27463-27466Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). These interactions may be involved in the synaptic targeting and cytoskeletal attachment of receptors, linking them physically and functionally to the appropriate intracellular signaling pathways (5Yao I. Hata Y. Hirao K. Deguchi M. Ide N. Takeuchi M. Takai Y. J. Biol. Chem. 1999; 274: 27463-27466Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In addition, an interaction between the Shank PDZ and the C-terminal PDZ binding motif and leucine zipper domain of βPIX was reported recently (6Park E. Na M. Choi J. Kim S. Lee J.R. Yoon J. Park D. Sheng M. Kim E. J. Biol. Chem. 2003; 278: 19220-19229Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). βPIX is a guanidine nucleotide exchange factor that binds p21-activated kinase and promotes the functional coupling of Rac1 and Cdc42 small GTPases with downstream effector kinases (7Manser E. Loo T.H. Koh C.G. Zhao Z.S. Chen X.Q. Tan L. Tan I. Leung T. Lim L. Mol. Cell. 1998; 1: 183-192Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar, 8Bagrodia S. Taylor S.J. Jordon K.A. Van Aelst L. Cerione R.A. J. Biol. Chem. 1998; 273: 23633-23636Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 9Oh W.K. Yoo J.C. Jo D. Song Y.H. Kim M.G. Park D. Biochem. Biophys. Res. Commun. 1997; 235: 794-798Crossref PubMed Scopus (58) Google Scholar). The aim of the present study was to better understand the structural mechanism of the interaction between the Shank1 PDZ and its target protein. To that end, we determined the crystal structures of the Shank1 PDZ domain in its peptide-free form and in complex with the C-terminal hexapeptide of GKAP to resolutions of 1.8 and 2.25 Å, respectively. Protein Purification and Crystallization—Recombinant Shank1 PDZ (residues 584–690) from Rattus novergicus with a cleavable glutathione S-transferase tag was expressed in Escherichia coli strain BL21(DE3) and then purified and crystallized as described previously (10Park S.H. Im Y.J. Rho S.H. Lee J.H. Yang S. Kim E. Eom S.H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1353-1355Crossref PubMed Scopus (3) Google Scholar). The C-terminal hexapeptide (EAQTRL) of GKAP used for PDZ-peptide cocrystallization was chemically synthesized (Anygen). Data Collection—A MAD data set was collected from a peptide-free frozen crystal to a resolution of 1.8 Å using an ADSC Quantum 4R CCD detector at beamline 18B at the Photon Factory. Because bromine is a convenient anomalous scatterer for MAD phasing, a bromine MAD data set was collected from a single crystal crystallized in a solution containing 200 mm NaBr (11Dauter Z. Dauter M. Rajashankar K.R. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 232-237Crossref PubMed Scopus (285) Google Scholar). The diffraction data from a PDZ-peptide complex to a resolution of 2.25 Å were collected at 100 K at beamline 6B of the Pohang Accelerator Laboratory. Structure Determination and Refinement—The structure of the peptide-free PDZ domain was determined by MAD phasing using bromine as an anomalous scatterer. The positions of three bromines within the asymmetric unit were located and refined using the program SOLVE (12Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The initial phases were improved by solvent flattening using the program RESOLVE (overall figure of merit, 0.52) (13Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1863-1871Crossref PubMed Scopus (199) Google Scholar). The resultant map, showing a dimer in an asymmetric unit, was readily interpretable. Model building then proceeded using the program O (14Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar), after which the structure was refined using the program CNS (15Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges N. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar). The final crystallographic R value for the peptide-free PDZ model, calculated using data from 27.7–1.8 Å, was 23.4% (Rfree = 25.1%). Using the structure of the peptide-free PDZ dimer served as a starting model, the structure of the PDZ-hexapeptide complex was determined by molecular replacement methods with the program MOLREP (16Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4153) Google Scholar). There were two PDZ domains related by non-crystallographic 2-fold symmetry within the asymmetric unit. After applying a simulated annealing procedure using data to 2.25 Å, the locations of the two bound peptides were determined from a Fo – Fc difference electron density map. The electron densities for all of the six residues of the peptide ligands were obvious, indicating that they were well ordered within the structure. The hexapeptide was modeled using program O, after which the ligand-bound PDZ domain was refined to a final crystallographic R value of 25.1% and a free R value of 28.0%. The refined model of the peptide-bound PDZ domain consisted of a PDZ dimer related by a non-crystallographic 2-fold axis, two hexapeptides, and 44 water molecules. The stereochemistry of the model was analyzed with PROCHECK (17Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). No residues were found in the disallowed regions of the Ramachandran plot. Data collection and refinement statistics are summarized in Table I.Table IData collection and refinement statisticsData setPeptide-free (Br-MAD)aDerivatized by crystallizing the protein in solution containing 200 mm NaBr.PDZ-peptide complexCrystal formP21P41212X-ray sourceBL-18B (Photon Factory)6B (PAL)Wavelengthλ1λ2λ31.0000.92020.91920.900Resolution (Å)27.7-1.830-2.25RsymbRsym = \〈I〉 - I\/〈I〉. (%)4.8 (37.7)4.8 (32.6)4.9 (33.7)4.1 (25.8)Data coverage total/final shell (%)99.0 (99.0)99.0 (99.0)99.0 (99.0)96.2 (95.6)Phasing (34.5-1.9 Å)0.52 (RESOLVE)Overall figure of meritRefinement RcrystcRcryst = \|Fo| - |Fc|\/|Fo|. total (%)23.425.1 RfreedRfree calculated using 5% of all reflections excluded from the refinement stages. total (%)25.128.0 R.m.s. bond length (Å)0.0060.007 R.m.s. bond angle (°)1.21.3 Average B-value (Å2)32.243.7a Derivatized by crystallizing the protein in solution containing 200 mm NaBr.b Rsym = \〈I〉 - I\/〈I〉.c Rcryst = \|Fo| - |Fc|\/|Fo|.d Rfree calculated using 5% of all reflections excluded from the refinement stages. Open table in a new tab Overall Structure of the Shank1 PDZ Domain—The Shank1 PDZ monomer is a compact, globular domain containing eight segments of secondary structure: six β strands that form an antiparallel β barrel and two α helices (Fig. 1A). The amino acid sequences of all of the Shank protein PDZ domains are nearly identical but differ significantly from other PDZ domains (Fig. 1B) (18Boeckers T.M. Kreutz M.R. Winter C. Zuschratter W. Smalla K.H. Sanmarti-Vila L. Wex H. Langnaese K. Bockmann J. Garner C.C. Gundelfinger E.D. J. Neurosci. 1999; 19: 6506-6518Crossref PubMed Google Scholar, 19Lim S. Naisbitt S. Yoon J. Sheng M. Kim E. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The amino acid sequence of the Shank1 PDZ domain (residues used in study is identical to the domain and and sequence with the PDZ domains of and respectively. The of the Shank1 PDZ shows the of structural of any known PDZ (Fig. In addition, the Shank1 PDZ long N-terminal βA and C-terminal strands, which residues each and in antiparallel β interactions. The βA is and in dimeric interaction by an antiparallel β with the βA of the other monomer (Fig. The PDZ domain that the most is the loop the and The Shank1 PDZ the known βB/βC the in the sequence and length of βB/βC the sequence is conserved among Shank PDZ is typical of most PDZ the peptide ligand is within a between the and the oriented as an antiparallel to Within the crystal structure of the all six amino acid residues of the peptide ligand were well as by the difference electron density calculated of the of the structures of the peptide-free and peptide-bound PDZ domain shows that is ligand The root mean between two is for the of the Shank1 domains bind to the residues at the C terminus of interacting proteins. Shank PDZ domains are known to bind to the C-terminal six residues of which in turn with the guanylate kinase domain of PSD-95 E. Naisbitt S. A. A. Sheng M. J. Cell Biol. 1997; PubMed Scopus Google Scholar). Although they the typical consensus sequence X-(Thr/Ser)-X-Leu and are as I PDZ Shank PDZ domains show for the GKAP C-terminal The interaction with GKAP is for the Shank (residues with the other class I PDZ domains of or (3Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). the C-terminal of bind the Shank it interacts with the PDZ domains of PSD-95 (3Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). The Shank PDZ domain is for recognition of GKAP, it binds other ligands the consensus Within the crystal structure, the peptide ligand the binding antiparallel to the an of hydrogen and interactions (Fig. and The sequence in the binding loop of the Shank1 PDZ hydrogen with the of the GKAP peptide C terminus in a to that previously in complexes. The of and of the binding loop are involved in hydrogen with the C terminus of the the side of the by and of the side of the of the ligand oriented toward the the Shank1 PDZ interacts with the –1 position of the ligand (Fig. The of located at the of the a salt with the of To the long side of at the toward shows that is conserved among all of the Shank proteins so and Shank1 and all of the known GKAP family proteins protein with the amino acids M. Hata Y. Hirao K. A. M. Takai Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). This that a positively charged is at ligand position –1 it to the of the with that with significantly the of the interaction (3Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). The for a positively charged at the –1 position was previously reported in the interaction a to that of in the Shank PDZ S. Leung T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, PDZ domain ligands with a side of at the –1 the of residues to the binding of the ligands K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In class I PDZ or at the position of the ligand a hydrogen bond with the side of residues at the of within the Shank the at ligand position to the by hydrogen with at (Fig. The ligand residues at the –3 and –5 positions to contribute to the and of the of the residues at the –3 position in the interaction was reported in of PDZ domains S. Leung T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. 2001; PubMed Scopus Google Scholar, Lee A. J. Kim E. Sheng M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In Shank PDZ domain, the of in the is within the by the of the side chains of and The of hydrogen with the and interacts with the side of and the of This means that the of may further the interaction by hydrogen with the residues at positions –3 and interactions at positions are for ligand of PDZ well scaffold proteins, PSD-95 E. A. Sheng M. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, Kim E. Sheng M. Neuron. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), the family of proteins B. Tu J.C. A. A. R.J. Worley P.F. Neuron. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and protein S. P. L. B. C. S. Valtschanoff Weinberg R.J. Neuron. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), the to form or in is by the PDZ domains A. C. J. Cell Biol. 1998; PubMed Scopus (199) Google Scholar, R.A. 2001; PubMed Scopus Google Scholar, H. P. Song I. D. J. Neurosci. 1999; 19: PubMed Google Scholar). For was recently in the crystal structure of which shows a dimer Y.J. Park S.H. Rho S.H. Lee J.H. Sheng M. Kim E. Eom S.H. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). In Shank proteins, the SAM domain which is for of Shank proteins; however, the present study shows that the PDZ domain to the of Shank proteins, suggesting structural of a dimerization by PDZ The crystal structures dimers in the asymmetric of the peptide free and peptide bound which to the and (Fig. asymmetric unit two a dimer with non-crystallographic 2-fold and the Shank1 PDZ to be a dimer in solution (Fig. D and the Shank1 PDZ domain has the to form a dimer whether it is free or with a ligand the dimer may the functional of the Shank PDZ domain. The between dimeric PDZ domains a βA and a βB/βC loop from each monomer (Fig. and The βA strands form antiparallel the of a 2-fold axis so that the N and C termini of each PDZ domain in The of dimer is or of the total of each which implies the of between the two that may The dimeric is of of and of Within the the are by six hydrogen bonds, hydrogen bonds, and interactions. All of the six hydrogen from in the βA strands the antiparallel In side in the βB/βC which of the in the interactions at the residues in the loop are conserved in all of the Shank proteins so implies a role for the βB/βC loop in dimerization and protein-protein interactions. the crystal structure of complex the important of the loop in ligand the that the loop could be for the of the PDZ domain J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). to Shank by antiparallel with N-terminal βA strands (Fig. and the of the βA strands differ in the two dimers Y.J. Park S.H. Rho S.H. Lee J.H. Sheng M. Kim E. Eom S.H. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Shank PDZ has a βA and of the is involved in the interaction the peptide binding to be oriented In the peptide binding are located at the of the complex and oriented in antiparallel In the Shank PDZ however, the N termini of the ligands are oriented toward the so that they are in close proximity the 2-fold axis of the dimer (Fig. of Shank PDZ proteins a SAM domain at C which can oligomerization of the proteins. which interacts with Shank has a domain that to the of the complex S. Sala C. Yoon J. Park S. S. Sheng M. Kim E. Mol. Cell Neurosci. 2001; PubMed Scopus Google Scholar). the crystal structure of the Shank1 PDZ domain that the dimeric configuration of the PDZ domain may organization of Shank proteins. Although of GKAP and Shank PDZ has to be the interaction of βPIX and Shank PDZ to the of the Shank PDZ dimer. The Shank1 PDZ domain interacts with the C-terminal domain of and the leucine zipper domain the of βPIX (6Park E. Na M. Choi J. Kim S. Lee J.R. Yoon J. Park D. Sheng M. Kim E. J. Biol. Chem. 2003; 278: 19220-19229Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, S. Lee S.H. Park D. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). This means that the C-terminal PDZ binding motif and the leucine zipper domain of βPIX are involved in the interaction and that an interaction that at the peptide binding of the PDZ domain is involved in the binding of βPIX (6Park E. Na M. Choi J. Kim S. Lee J.R. Yoon J. Park D. Sheng M. Kim E. J. Biol. Chem. 2003; 278: 19220-19229Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The leucine zipper motif commonly a of two at C termini with a 2-fold of βPIX secondary structure that six amino acid residues the C-terminal PDZ binding motif and the leucine zipper (Fig. the ligands binding to the PDZ are in close proximity the 2-fold axis of the dimer. the of the βB/βC loop among Shank proteins, are to the that in we a model of the Shank C-terminal complex in which the C-terminal root of the leucine zipper domain interacts with the at the of PDZ dimer and the C-terminal are the peptide binding (Fig. This model that the novel interacting with the leucine zipper domain is the conserved βB/βC a configuration is it dimeric PDZ domains to with dimeric target proteins. N. and M. and N. for with data collection at beamline of Photon H. S. Lee and H. Kim at the of Pohang Accelerator

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