An isoform of kalirin, a brain-specific GDP/GTP exchange factor, is enriched in the postsynaptic density fraction

Communication between membranes and the actin cytoskeleton is an important aspect of neuronal function. Regulators of actin cytoskeletal dynamics include the Rho-like small GTP-binding proteins and their exchange factors. Kalirin is a brain-specific protein, first identified through its interaction with peptidylglycine-alpha-amidating monooxygenase. In this study, we cloned rat Kalirin-7, a 7-kilobase mRNA form of Kalirin. Kalirin-7 contains nine spectrin-like repeats, a Dbl homology domain, and a pleckstrin homology domain. We found that the majority of Kalirin-7 protein is associated with synaptosomal membranes, but a fraction is cytosolic. We also detected higher molecular weight Kalirin proteins. In rat cerebral cortex, Kalirin-7 is highly enriched in the postsynaptic density fraction. In primary cultures of neurons, Kalirin-7 is detected in spine-like structures, while other forms of Kalirin are visualized in the cell soma and throughout the neurites. Kalirin-7 and its Dbl homology-pleckstrin homology domain induce formation of lamellipodia and membrane ruffling, when transiently expressed in fibroblasts, indicative of Rac1 activation. Using Rac1, the Dbl homology-pleckstrin homology domain catalyzed the in vitro exchange of bound GDP with GTP. Kalirin-7 is the first guanine-nucleotide exchange factor identified in the postsynaptic density, where it is positioned optimally to regulate signal transduction pathways connecting membrane proteins and the actin cytoskeleton.     

The actin binding domain of βI-spectrin regulates the morphological and functional dynamics of dendritic spines

Actin microfilaments regulate the size, shape and mobility of dendritic spines and are in turn regulated by actin binding proteins and small GTPases. The βI isoform of spectrin, a protein that links the actin cytoskeleton to membrane proteins, is present in spines. To understand its function, we expressed its actin-binding domain (ABD) in CA1 pyramidal neurons in hippocampal slice cultures. The ABD of βI-spectrin bundled actin in principal dendrites and was concentrated in dendritic spines, where it significantly increased the size of the spine head. These effects were not observed after expression of homologous ABDs of utrophin, dystrophin, and α-actinin. Treatment of slice cultures with latrunculin-B significantly decreased spine head size and decreased actin-GFP fluorescence in cells expressing the ABD of α-actinin, but not the ABD of βI-spectrin, suggesting that its presence inhibits actin depolymerization. We also observed an increase in the area of GFP-tagged PSD-95 in the spine head and an increase in the amplitude of mEPSCs at spines expressing the ABD of βI-spectrin. The effects of the βI-spectrin ABD on spine size and mEPSC amplitude were mimicked by expressing wild-type Rac3, a small GTPase that co-immunoprecipitates specifically with βI-spectrin in extracts of cultured cortical neurons. Spine size was normal in cells co-expressing a dominant negative Rac3 construct with the βI-spectrin ABD. We suggest that βI-spectrin is a synaptic protein that can modulate both the morphological and functional dynamics of dendritic spines, perhaps via interaction with actin and Rac3.     

Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin.

NMDA receptors are linked to intracellular cytoskeletal and signaling molecules via the PSD-95 protein complex. We report a novel family of postsynaptic density (PSD) proteins, termed Shank, that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton.     

The glutamate transporter GLT1b interacts with the scaffold protein PSD-95

The glutamate transporter (GLT1) regulates glutamate concentrations in glutamatergic synapses and it is expressed in at least two isoforms, GLT1a and GLT1b. In this work, we show that the C-terminus of GLT1b is able to interact with the PDZ domains of a number of proteins. Notably, one of them might be the scaffold protein post-synaptic density (PSD-95). GLT1b formed co-immunoprecipitable complexes with PSD-95 in solubilizated rat brain extracts, complexes that also contained NMDA receptors. Co-transfection of GLT1b, PSD-95, and NMDA receptor subunits in heterologous expression systems recapitulated in vitro the interactions among these proteins that had been observed in the rat brain extracts and revealed the importance of the GLT1b C-terminal PDZ binding motif in tethering this transporter to PSD-95. Significantly, co-expression of GLT1b and PSD-95 increased the V _max of the transporter by decreasing the rate of GLT1b endocytosis. Moreover, GLT1b transfected into primary cultured neurons or glia formed protein clusters that co-localized with co-transfected PSD-95, clusters that in these neurons accumulated preferentially in dendritic spines. We hypothesize that the GLT1b/PSD-95 interaction, characterized here in vitro , might anchor this transporter close to the post-synaptic glutamate receptors, thereby permitting the fine regulation of glutamate concentrations in this microenvironment. This tight association might also facilitate the regulation of GLT1b through the signaling pathways initiated by the activation of glutamate receptors.     

Regulation of dendritic spine morphology by SPAR, a PSD-95-associated RapGAP

The PSD-95/SAP90 family of scaffold proteins organizes the postsynaptic density (PSD) and regulates NMDA receptor signaling at excitatory synapses. We report that SPAR, a Rap-specific GTPase-activating protein (RapGAP), interacts with the guanylate kinase-like domain of PSD-95 and forms a complex with PSD-95 and NMDA receptors in brain. In heterologous cells, SPAR reorganizes the actin cytoskeleton and recruits PSD-95 to F-actin. In hippocampal neurons, SPAR localizes to dendritic spines and causes enlargement of spine heads, many of which adopt an irregular appearance with putative multiple synapses. Dominant negative SPAR constructs cause narrowing and elongation of spines. The effects of SPAR on spine morphology depend on the RapGAP and actin-interacting domains, implicating Rap signaling in the regulation of postsynaptic structure.     

SPIN90/WISH interacts with PSD-95 and regulates dendritic spinogenesis via an N-WASP-independent mechanism

SPIN90/WISH (SH3 protein interacting with Nck, 90 kDa/Wiskott-Aldrich syndrome protein (WASP) interacting SH3 protein) regulates actin polymerization through its interaction with various actin-regulating proteins. It is highly expressed in the brain, but its role in the nervous system is largely unknown. We report that it is expressed in dendritic spines where it associates with PSD-95. Its overexpression increased the number and length of dendritic filopodia/spines via an N-WASP-independent mechanism, and knock down of its expression with small interfering RNA reduced dendritic spine density. The increase in spinogenesis is accompanied by an increase in synaptogenesis in contacting presynaptic neurons. Interestingly, PSD-95-induced dendritic spinogenesis was completely abolished by knock down of SPIN90/WISH. Finally, in response to chemically induced long-term potentiation, SPIN90/WISH associated with PSD-95 and was redistributed to dendritic spines. Our results suggest that SPIN90/WISH associates with PSD-95, and so becomes localized to dendritic spines where it modulates actin dynamics to control dendritic spinogenesis. They also raise the possibility that SPIN90/WISH is a downstream effector of PSD-95-dependent synaptic remodeling.     

Opposite effects of PSD-95 and MPP3 PDZ proteins on serotonin 5-hydroxytryptamine2C receptor desensitization and membrane stability

PSD-95/Disc large/Zonula occludens 1 (PDZ) domain-containing proteins (PDZ proteins) play an important role in the targeting and the trafficking of transmembrane proteins. Our previous studies identified a set of PDZ proteins that interact with the C terminus of the serotonin 5-hydroxytryptamine (5-HT)(2C) receptor. Here, we show that the prototypic scaffolding protein postsynaptic density-95 (PSD-95) and another membrane-associated guanylate kinase, MAGUK p55 subfamily member 3 (MPP3), oppositely regulate desensitization of the receptor response in both heterologous cells and mice cortical neurons in primary culture. PSD-95 increased desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response, whereas MPP3 prevented desensitization of the Ca(2+) response. The effects of the PDZ proteins on the desensitization of the Ca(2+) response were correlated with a differential regulation of cell surface expression of the receptor. Additional experiments were performed to assess how PDZ proteins globally modulate desensitization of the 5-HT(2C) receptor response in neurons, by using a peptidyl mimetic of the 5-HT(2C) receptor C terminus fused to the human immunodeficiency virus type-1 Tat protein transduction domain, which disrupts interaction between the 5-HT(2C) receptor and PDZ proteins. Transduction of this peptide inhibitor into cultured cortical neurons increased the desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response. This indicates that, overall, interaction of 5-HT(2C) receptors with PDZ proteins inhibits receptor desensitization in cortical neurons.     

KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX

Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor β-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses.     

Expression and potential function of Rho family small G proteins in cells of the mammalian seminiferous epithelium

Dynamic cellular rearrangements involving the actin cytoskeleton are required of both Sertoli and germ cells during spermatogenesis. Rho family small G proteins have been implicated in the control of the actin cytoskeleton in numerous cell types. Therefore, RhoA and Rac1 were investigated in Sertoli and germ cells. RhoA and Rac1 have been detected at both the mRNA and protein levels in these cells. In addition, Sertoli cell L-selectin is shown to interact with actin binding proteins, potentially providing a link between L-selectin and Rac1 signaling. Finally, inactivation of Sertoli cell Rho family proteins yields disruption of the actin cytoskeleton.     

Trafficking of the NMDAR2B receptor subunit distal cytoplasmic tail from endoplasmic reticulum to the synapse

NMDA receptor NR2A/B subunits have PDZ-binding domains on their extreme C-termini that are known to interact with the PSD-95 family and other PDZ proteins. We explore the interactions between PSD-95 family proteins and the NR2A/B cytoplasmic tails, and the consequences of these interactions, from the endoplasmic reticulum (ER) through delivery to the synapse in primary rat hippocampal and cortical cultured neurons. We find that the NR2A/B cytoplasmic tails cluster very early in the secretory pathway and interact serially with SAP102 beginning at the intermediate compartment, and then PSD-95. We further establish that colocalization of the distal C-terminus of NR2B and PSD-95 begins at the trans-Golgi Network (TGN). Formation of NR2B/PSD-95/SAP102 complexes is dependent on the PDZ binding domain of NR2B subunits, but association with SAP102 and PSD-95 plays no distinguishable role in cluster pre-formation or initial targeting to the vicinity of the synapse. Instead the PDZ binding domain plays a role in restricting cell-surface clusters to postsynaptic targets.     

MAGUIN, a novel neuronal membrane-associated guanylate kinase-interacting protein

Postsynaptic density (PSD)-95/Synapse-associated protein (SAP) 90 and synaptic scaffolding molecule (S-SCAM) are neuronal membrane-associated guanylate kinases. Because PSD-95/SAP90 and S-SCAM function as synaptic scaffolding proteins, identification of ligands for these proteins is important to elucidate the structure of synaptic junctions. Here, we report a novel protein interacting with the PDZ domains of PSD-95/SAP90 and S-SCAM and named it MAGUIN-1 (membrane-associated guanylate kinase-interacting protein-1). MAGUIN-1 has one sterile alpha motif, one PDZ, and one plekstrin homology domain. MAGUIN-1 is localized at the plasma membrane via the plekstrin homology domain and the C-terminal region and interacts with PSD-95/SAP90 and S-SCAM via a C-terminal PDZ domain-binding motif. MAGUIN-1 has a short isoform, MAGUIN-2, which lacks a PDZ domain-binding motif. MAGUINs are expressed in neurons and localized in the cell body and neurites and are coimmunoprecipitated with PSD-95/SAP90 and S-SCAM from rat crude synaptosome. MAGUIN-1 may play an important role with PSD-95/SAP90 and S-SCAM to assemble the components of synaptic junctions.     

Sema4c, a transmembrane semaphorin, interacts with a post-synaptic density protein, PSD-95

Semaphorins are known to act as chemorepulsive molecules that guide axons during neural development. Sema4C, a group 4 semaphorin, is a transmembrane semaphorin of unknown function. The cytoplasmic domain of Sema4C contains a proline-rich region that may interact with some signaling proteins. In this study, we demonstrate that Sema4C is enriched in the adult mouse brain and associated with PSD-95 isoforms containing PDZ (PSD-95/DLG/ZO-1) domains, such as PSD-95/SAP90, PSD-93/chapsin110, and SAP97/DLG-1, which are concentrated in the post-synaptic density of the brain. In the neocortex, S4C is enriched in the synaptic vesicle fraction and Triton X-100 insoluble post-synaptic density fraction. Immunostaining for Sema4C overlaps that for PSD-95 in superficial layers I-IV of the neocortex. In neocortical culture, S4C is colocalized with PSD-95 in neurons, with a dot-like pattern along the neurites. Sema4C thus may function in the cortical neurons as a bi-directional transmembrane ligand through interacting with PSD-95.     

Interaction of neuronal nitric-oxide synthase with alpha1-syntrophin in rat brain

Neuronal nitric-oxide synthase (nNOS) has a PSD-95/Dlg/ZO-1 (PDZ) domain that can interact with multiple proteins. nNOS has been known to interact with PSD-95 and a related protein, PSD-93, in brain and with alpha1-syntrophin in skeletal muscle in mammals. In this study, we have purified an nNOS-interacting protein from bovine brain using an affinity column made of Sepharose conjugated with glutathione S-transferase-rat nNOS fusion protein and identified it as alpha1-syntrophin by microsequencing. Immunostaining of primary cultures of rat embryonic brain neuronal cells with antibodies against these proteins showed that nNOS and alpha1-syntrophin were colocalized in neuronal cell bodies and neurites. Immunohistochemical analysis indicated that the nNOS- and alpha1-syntrophin-like immunoreactive substances were highly expressed in the rat hypothalamic suprachiasmatic nucleus (SCN) and paraventricular nucleus. In the SCN, nNOS- and alpha1-syntrophin-like immunoreactive substances were colocalized in the same neurons as detected by confocal microscopy. These results indicate that nNOS in brain interacts with alpha1-syntrophin in specific neurons of the SCN and paraventricular nucleus and that this interaction might play a physiological role in functions of these neurons.     

Proteomic analysis revealed a novel synaptic proline-rich membrane protein (PRR7) associated with PSD-95 and NMDA receptor

Proteomic analyses have revealed a novel synaptic proline-rich membrane protein: PRR7 (proline rich 7), in the postsynaptic density (PSD) fraction of rat forebrain. PRR7 is 269 amino acid residues long, and displays a unique architecture, composed of a very short N-terminal extracellular region, a single membrane spanning domain, and a cytoplasmic domain possessing a proline-rich sequence and a C-terminal type-1 PDZ binding motif. A fraction of PRR7 accumulates in spines along with synapse maturation, and colocalizes with PSD-95 in a punctate pattern in rat hippocampal neural cultures. Immunoprecipitation and GST pull-down assays demonstrated that PRR7 binds to the third PDZ domain of PSD-95. In addition, the NMDA receptor subunits, NR1 and NR2B, specifically co-immunoprecipitated with PRR7. These results suggest that PRR7 is involved in modulating neural activities via interactions with the NMDA receptor and PSD-95, and PSD core formation.     

Scaffold Protein X11α Interacts with Kalirin-7 in Dendrites and Recruits It to Golgi Outposts

Pyramidal neurons in the mammalian forebrain receive their synaptic inputs through their dendritic trees, and dendritic spines are the sites of most excitatory synapses. Dendritic spine structure is important for brain development and plasticity. Kalirin-7 is a guanine nucleotide-exchange factor for the small GTPase Rac1 and is a critical regulator of dendritic spine remodeling. The subcellular localization of kalirin-7 is thought to be important for regulating its function in neurons. A yeast two-hybrid screen has identified the adaptor protein X11α as an interacting partner of kalirin-7. Here, we show that kalirin-7 and X11α form a complex in the brain, and this interaction is mediated by the C terminus of kalirin-7. Kalirin-7 and X11α co-localize at excitatory synapses in cultured cortical neurons. Using time-lapse imaging of fluorescence recovery after photobleaching, we show that X11α is present in a mobile fraction of the postsynaptic density. X11α also localizes to Golgi outposts in dendrites, and its overexpression induces the removal of kalirin-7 from spines and accumulation of kalirin-7 in Golgi outposts. In addition, neurons overexpressing X11α displayed thinner spines. These data support a novel mechanism of regulation of kalirin-7 localization and function in dendrites, providing insight into signaling pathways underlying neuronal plasticity. Dissecting the molecular mechanisms of synaptic structural plasticity will improve our understanding of neuropsychiatric and neurodegenerative disorders, as kalirin-7 has been associated with schizophrenia and Alzheimer disease.     

Role of actin cytoskeleton in dendritic spine morphogenesis

Dendritic spines are the postsynaptic receptive regions of most excitatory synapses, and their morphological plasticity play a pivotal role in higher brain functions, such as learning and memory. The dynamics of spine morphology is due to the actin cytoskeleton concentrated highly in spines. Filopodia, which are thin and headless protrusions, are thought to be precursors of dendritic spines. Drebrin, a spine-resident side-binding protein of filamentous actin (F-actin), is responsible for recruiting F-actin and PSD-95 into filopodia, and is suggested to govern spine morphogenesis. Interestingly, some recent studies on neurological disorders accompanied by cognitive deficits suggested that the loss of drebrin from dendritic spines is a common pathognomonic feature of synaptic dysfunction. In this review, to understand the importance of actin-binding proteins in spine morphogenesis, we first outline the well-established knowledge pertaining to the actin cytoskeleton in non-neuronal cells, such as the mechanism of regulation by small GTPases, the equilibrium between globular actin (G-actin) and F-actin, and the distinct roles of various actin-binding proteins. Then, we review the dynamic changes in the localization of drebrin during synaptogenesis and in response to glutamate receptor activation. Because side-binding proteins are located upstream of the regulatory pathway for actin organization via other actin-binding proteins, we discuss the significance of drebrin in the regulatory mechanism of spine morphology through the reorganization of the actin cytoskeleton. In addition, we discuss the possible involvement of an actin-myosin interaction in the morphological plasticity of spines.     

Characterization of the delta2 glutamate receptor-binding protein delphilin: Splicing variants with differential palmitoylation and an additional PDZ domain

The glutamate receptor delta2 (GluRdelta2) is predominantly expressed at parallel fiber-Purkinje cell postsynapses and plays crucial roles in synaptogenesis and synaptic plasticity. Although the mechanism by which GluRdelta2 functions remains unclear, its lack of channel activity and its role in controlling the endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors have suggested that GluRdelta2 may convey signals by interacting with intracellular signaling molecules. Among several proteins that interact with GluRdelta2, delphilin is unique in that it is selectively expressed at parallel fiber-Purkinje cell synapses and that, in addition to a single PDZ domain, it contains a formin homology domain that is thought to regulate actin dynamics. Here, we report a new isoform of delphilin, designated as L-delphilin, that has alternatively spliced N-terminal exons encoding an additional PDZ domain. Although original delphilin, designated S-delphilin, was palmitoylated at the N terminus, this region was spliced out in L-delphilin. As a result, S-delphilin was associated with plasma membranes in COS cells and dendritic spines in hippocampal neurons, whereas L-delphilin formed clusters in soma and dendritic shafts. In addition, S-delphilin, but not L-delphilin, facilitated the expression of GluRdelta2 on the cell surface. These results indicate that, like PSD-95 and GRIP/ABP, delphilin isoforms with differential palmitoylation and clustering capabilities may provide two separate intracellular and surface GluRdelta2 pools and may control GluRdelta2 signaling in Purkinje cells.     

Depolarization drives beta-Catenin into neuronal spines promoting changes in synaptic structure and function

Activity-induced changes in adhesion molecules may coordinate presynaptic and postsynaptic plasticity. Here, we demonstrate that beta-catenin, which mediates interactions between cadherins and the actin cytoskeleton, moves from dendritic shafts into spines upon depolarization, increasing its association with cadherins. beta-catenin's redistribution was mimicked or prevented by a tyrosine kinase or phosphatase inhibitor, respectively. Point mutations of beta-catenin's tyrosine 654 altered the shaft/spine distribution: Y654F-beta-catenin-GFP (phosphorylation-prevented) was concentrated in spines, whereas Y654E-beta-catenin-GFP (phosphorylation-mimic) accumulated in dendritic shafts. In Y654F-expressing neurons, the PSD-95 or associated synapsin-I clusters were larger than those observed in either wild-type-beta-catenin or also Y654E-expressing neurons. Y654F-expressing neurons exhibited a higher minifrequency. Thus, neural activity induces beta-catenin's redistribution into spines, where it interacts with cadherin to influence synaptic size and strength.     

Ligand binding of the second PDZ domain regulates clustering of PSD-95 with the Kv1.4 potassium channel.

The molecular mechanisms underlying the protein assembly at synaptic junctions are thought to be important for neural functions. PSD-95, one of the major postsynaptic density proteins, is composed of three PDZ domains (PDZ1, PDZ2, and PDZ3), an SH3 domain, and a GK (guanylate kinase ) domain. It binds to the N-methyl-D-aspartate glutamate receptor NR2 subunit or to the Shaker-type K(+) channel, Kv1.4, via the PDZ1 or PDZ2 domain, whereas PDZ3 binds to distinct partners. The intramolecular interaction of these multiple domains has been implicated in efficient protein clustering. We introduced missense and deletion mutations into PDZ1 (PDZ1mDelta) and/or PDZ2 (PDZ2mDelta) of the full-length PSD-95 to disrupt the association of each domain with the target proteins, while preserving the overall structure. The ion channel clustering activities of the PSD-95 mutants were analyzed in COS-1 cells coexpressing each mutant and Kv1.4. The mutant bearing the dysfunctional PDZ2 (PSD-95:1-2mDelta) showed significantly reduced clustering efficiency, whereas the mutant with the dysfunctional PDZ1 (PSD-95:1mDelta-2) exhibited activity comparable with the wild-type activity. Furthermore, we also examined the requirements for the position of PDZ2 in full-length PSD-95 by constructing a series of PDZ1-PDZ2 inversion mutants. Surprisingly, the clustering activity of PSD-95:2-1mDelta was severely defective. Taken together, these findings show that PDZ2, which is endowed with the highest affinity for Kv1.4, is required for efficient ligand binding. In addition, the ligand binding at the position of the second PDZ domain in full-length PSD-95 is prerequisite for efficient and typical cluster formation. This study suggests that the correct placement of the multiple domains in the full-length PSD-95 protein is necessary for the optimal protein activity.     

Solution structure and backbone dynamics of the second PDZ domain of postsynaptic density-95

The second PDZ domain of postsynaptic density-95 (PSD-95 PDZ2) plays a critical role in coupling N-methyl-D-aspartate receptors to neuronal nitric oxide synthase (nNOS). In this work, the solution structure of PSD-95 PDZ2 was determined to high resolution by NMR spectroscopy. The structure of PSD-95 PDZ2 was compared in detail with that of alpha1-syntrophin PDZ domain, as the PDZ domains share similar target interaction properties. The interaction of the PSD-95 PDZ2 with a carboxyl-terminal peptide derived from a cytoplasmic protein CAPON was studied by NMR titration experiments. Complex formation between PSD-95 PDZ2 and the nNOS PDZ was modelled on the basis of the crystal structure of the alpha1-syntrophin PDZ/nNOS PDZ dimer. We found that the prolonged loop connecting the betaB and betaC strands of PSD-95 PDZ2 is likely to play a role in both the binding of the carboxyl-terminal peptide and the nNOS beta-finger. Finally, the backbone dynamics of the PSD-95 PDZ2 in the absence of bound peptide were studied using a model-free approach. The "GLGF"-loop and the loop connecting alphaB and betaF of the protein display some degree of flexibility in solution. The rest of the protein is rigid and lacks detectable slow time-scale (microseconds to milliseconds) motions. In particular, the loop connecting betaB and betaC loop adopts a well-defined, rigid structure in solution. It appears that the loop adopts a pre-aligned conformation for the PDZ domain to interact with its targets.