Formation of nNOS/PSD-95 PDZ dimer requires a preformed beta-finger structure from the nNOS PDZ domain

PDZ domains are modular protein units that play important roles in organizing signal transduction complexes. PDZ domains mediate interactions with both C-terminal peptide ligands and other PDZ domains. Here, we used PDZ domains from neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) to explore the mechanism for PDZ-dimer formation. The nNOS PDZ domain terminates with a approximately 30 residue amino acid beta-finger peptide that is shown to be required for nNOS/PSD-95 PDZ dimer formation. In addition, formation of the PDZ dimer requires this beta-finger peptide to be physically anchored to the main body of the canonical nNOS PDZ domain. A buried salt bridge between the beta-finger and the PDZ domain induces and stabilizes the beta-hairpin structure of the nNOS PDZ domain. In apo-nNOS, the beta-finger peptide is partially flexible and adopts a transient beta-strand like structure that is stabilized upon PDZ dimer formation. The flexibility of the NOS PDZ beta-finger is likely to play a critical role in supporting the formation of nNOS/PSD-95 complex. The experimental data also suggest that nNOS PDZ and the second PDZ domain of PSD-95 form a "head-to-tail" dimer similar to the nNOS/syntrophin complex characterized by X-ray crystallography.     

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.     

Single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands

PDZ domains are modular protein-protein interaction domains that bind to specific C-terminal sequences of membrane proteins and/or to other PDZ domains. Certain PDZ domains in PSD-95 and syntrophins interact with C-terminal peptide ligands and heterodimerize with the extended nNOS PDZ domain. The capacity to interact with nNOS correlates with the presence of a Lys residue in the carboxylate- binding loop of these PDZ domains. Here, we report that substitution of an Arg for Lys-165 in PSD-95 PDZ2 disrupted its interaction with nNOS, but not with the C terminus of the Shaker-type K(+) channel Kv1.4. The same mutation affected nNOS binding to alpha1- and beta1-syntrophin PDZ domains to a lesser extent, due in part to the stabilizing effect of tertiary interactions with the canonical nNOS PDZ domain. PDZ domains with an Arg in the carboxylate-binding loop do not bind nNOS; however, substitution with Lys or Ala was able to confer nNOS binding. Our results indicate that the carboxylate-binding loop Lys or Arg is a critical determinant of nNOS binding and that the identity of this residue can profoundly alter one mode of PDZ recognition without affecting another. We also analyzed the effects of mutating Asp-143, a residue in the alphaB helix of alpha1-syntrophin that forms a tertiary contact with the nNOS PDZ domain. This residue is important for both nNOS and C-terminal peptide binding and confers a preference for peptides with a positively charged residue at position -4. On this basis, we have identified the C terminus of the Kir2.1 channel as a possible binding partner for syntrophin PDZ domains. Together, our results demonstrate that single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands.     

PSD-95 assembles a ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain.

Nitric oxide (NO) biosynthesis in cerebellum is preferentially activated by calcium influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors, suggesting that there is a specific link between these receptors and neuronal NO synthase (nNOS). Here, we find that PSD-95 assembles a postsynaptic protein complex containing nNOS and NMDA receptors. Formation of this complex is mediated by the PDZ domains of PSD-95, which bind to the COOH termini of specific NMDA receptor subunits. In contrast, nNOS is recruited to this complex by a novel PDZ-PDZ interaction in which PSD-95 recognizes an internal motif adjacent to the consensus nNOS PDZ domain. This internal motif is a structured "pseudo-peptide" extension of the nNOS PDZ that interacts with the peptide-binding pocket of PSD-95 PDZ2. This asymmetric interaction leaves the peptide-binding pocket of the nNOS PDZ domain available to interact with additional COOH-terminal PDZ ligands. Accordingly, we find that the nNOS PDZ domain can bind PSD-95 PDZ2 and a COOH-terminal peptide simultaneously. This bivalent nature of the nNOS PDZ domain further expands the scope for assembly of protein networks by PDZ domains.     

Defining the minimal interacting regions of the tight junction protein MAGI-1 and HPV16 E6 oncoprotein for solution structure studies

The oncoprotein E6 produced by tumorigenic high-risk genital human papillomaviruses targets a number of cellular proteins containing PDZ domains for proteasome-mediated degradation. In particular, E6 targets the tight junction protein MAGI-1 by binding to its PDZ1 domain. Using light scattering and NMR, we explored different fragments of both the HPV16 E6 and the MAGI-1 PDZ1 domain to define the best-behaving complex for solution structure studies. We showed that the 70-residue HPV16 E6 C-terminal domain (E6C) can be efficiently substituted by a peptide spanning the 11 C-terminal residues of E6. The construct of MAGI-1 PDZ1 best suited for solution structure analysis presents a 14-residue N-terminal extension and a 26-residue C-terminal extension as compared to the construct used for the recently solved X-ray structure of a MAGI-1 PDZ1/HPV18 E6 complex. These data suggest a stabilizing role for the interdomain linker regions which separate the PDZ1 domain from its neighboring domains.     

Solution structure of the extended neuronal nitric oxide synthase PDZ domain complexed with an associated peptide.

The PDZ domain of neuronal nitric oxide synthase (nNOS) functions as a scaffold for organizing the signal transduction complex of the enzyme. The NMR structure of a complex composed of the nNOS PDZ domain and an associated peptide suggests that a two-stranded beta-sheet C-terminal to the canonical PDZ domain may mediate its interaction with the PDZ domains of postsynaptic density-95 and alpha-syntrophin. The structure also provides the molecular basis of recognition of Asp-X-Val-COOH peptides by the nNOS PDZ domain. The role of the C-terminal extension in Asp-X-Val-COOH peptide binding is investigated. Additionally, NMR studies further show that the Asp-X-Val-COOH peptide and a C-terminal peptide from a novel cytosolic protein named CAPON bind to the same pocket of the nNOS PDZ domain.     

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.     

Supramodular structure and synergistic target binding of the N-terminal tandem PDZ domains of PSD-95

PDZ domain proteins play critical roles in binding, clustering and subcellular targeting of membrane receptors and ion channels. PDZ domains in multi-PDZ proteins often are arranged in groups with highly conserved spacing and intervening sequences; however, the functional significance of such tandem arrangements of PDZs is unclear. We have solved the three-dimensional structure of the first two PDZ domains of postsynaptic density protein-95 (PSD-95 PDZ1 and PDZ2), which are closely linked to each other in the PSD-95 family of scaffold proteins. The two PDZs have limited freedom of rotation and their C-terminal peptide-binding grooves are aligned with each other with an orientation preference for binding to pairs of C termini extending in the same direction. Increasing the spacing between PDZ1 and PDZ2 resulted in decreased binding between PDZ12 and its dimeric targets. The same mutation impaired the functional ability of PSD-95 to cluster Kv1.4 potassium channels in heterologous cells. The data presented provide a molecular basis for preferential binding of PSD-95 to multimeric membrane proteins with appropriate C-terminal sequences.     

Detecting Protein–Protein Interactions in Living Cells: Development of a Bioluminescence Resonance Energy Transfer Assay to Evaluate the PSD-95/NMDA Receptor Interaction

The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein–protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat-conjugated peptide for its ability to disrupt the PSD-95/NMDA receptor interaction in living cells.     

Flavonoids from Radix Scutellariae as potential stroke therapeutic agents by targeting the second postsynaptic density 95 (PSD-95)/disc large/zonula occludens-1 (PDZ) domain of PSD-95.

Excessive activation of N-methyl-D-aspartate receptors (NMDARs) and subsequent production of nitric oxide by neuronal nitric oxide synthase (nNOS) contribute to neuronal damage resulting from hypoxic and ischemic insults. NMDARs and nNOS are coupled together at the postsynaptic membrane through their interaction with postsynaptic density protein (PSD) 95 via PSD-95/disc large/zonula occludens-1 (PDZ) domains. We used NMR (nuclear magnetic resonance) spectroscopy to screen medicinal herbs used in traditional Chinese medicine (TCM) stroke therapy for compounds binding to the second PDZ domain (PDZ2) of PSD-95, the domain linking nNOS and PSD-95. Aqueous extract of Huangqin, the root of Scutellaria baicalensis Georgi (Labiatae), showed significant binding to PDZ2 of PSD-95. The binding site of the active components in the extract overlapped with the nNOS/NR2B-binding pocket of PDZ2 of PSD-95. Four flavones, baicalin, norwogonoside, oroxylin A-glucuronide (oroxyloside), and wogonoside were isolated and found to account for the PDZ-binding activity of the extract. NMR titration experiments showed that baicalin and norwogonoside displayed the highest PDZ2 binding affinity, while oroxylin A-glucuronide and wogonoside showed 4-5 fold less potency in binding to the PDZ domain. Identification of the PDZ binding activity of these compounds will allow investigating whether or not it contributes to the observed clinical effects of Radix Scutellariae. Furthermore, these molecules might provide leads for the development of drugs targeting the signaling pathways mediated by PDZ domains.     

Solution structure of the RIM1alpha PDZ domain in complex with an ELKS1b C-terminal peptide

PDZ domains are widespread protein modules that commonly recognize C-terminal sequences of target proteins and help to organize macromolecular signaling complexes. These sequences usually bind in an extended conformation to relatively shallow grooves formed between a beta-strand and an alpha-helix in the corresponding PDZ domains. Because of this binding mode, many PDZ domains recognize primarily the C-terminal and the antepenultimate side-chains of the target protein, which commonly conform to motifs that have been categorized into different classes. However, an increasing number of PDZ domains have been found to exhibit unusual specificities. These include the PDZ domain of RIMs, which are large multidomain proteins that regulate neurotransmitter release and help to organize presynaptic active zones. The RIM PDZ domain binds to the C-terminal sequence of ELKS with a unique specificity that involves each of the four ELKS C-terminal residues. To elucidate the structural basis for this specificity, we have determined the 3D structure in solution of an RIM/ELKS C-terminal peptide complex using NMR spectroscopy. The structure shows that the RIM PDZ domain contains an unusually deep and narrow peptide-binding groove with an exquisite shape complementarity to the four ELKS C-terminal residues in their bound conformation. This groove is formed, in part, by a set of side-chains that is conserved selectively in RIM PDZ domains and that hence determines, at least in part, their unique specificity.     

Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs

During synaptic development, proteins aggregate at specialized pre- and postsynaptic structures. Mechanisms that mediate protein clustering at these sites remain unknown. To investigate this process, we analyzed synaptic targeting of a postsynaptic density protein, PSD-95, by expressing green fluorescent protein- (GFP-) tagged PSD-95 in cultured hippocampal neurons. We find that postsynaptic clustering relies on three elements of PSD-95: N-terminal palmitoylation, the first two PDZ domains, and a C-terminal targeting motif. In contrast, disruptions of PDZ3, SH3, or guanylate kinase (GK) domains do not affect synaptic targeting. Palmitoylation is sufficient to target the diffusely expressed SAP-97 to synapses, and palmitoylation cannot be replaced with alternative membrane association motifs, suggesting that a specialized synaptic lipid environment mediates postsynaptic clustering. The requirements for PDZ domains and a C-terminal domain of PSD-95 indicate that protein-protein interactions cooperate with lipid interactions in synaptic targeting.     

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.     

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.     

Post-synaptic density perturbs insulin-induced Kv1.3 channel modulation via a clustering mechanism involving the SH3 domain

The olfactory bulb (OB) contains the highest concentration of the insulin receptor (IR) kinase in the central nervous system; however, its functional role and modulation in this region remains poorly understood. IR kinase contains a number of proline-rich motifs, making it an excellent candidate for modulation by SH₃ domain-containing adaptor proteins. Kv1.3, a voltage-gated Shaker potassium channel and tyrosine phosphorylation substrate of IR kinase, contains several proline-rich sequences and a canonical post-synaptic density 95 (PSD-95)/discs large/zO-1 domain (PDZ) recognition motif common to most Shaker family members. We sought to determine if a functional relationship existed between Kv1.3, IR kinase, and the SH₃/PDZ adaptor protein PSD-95. Through patch-clamp electrophysiology, immunochemistry, and co-immunoprecipitation, we found that while Kv1.3 and PSD-95 alone interact via the canonical C-terminal PDZ recognition motif of the channel, this molecular site of interaction acts to cluster the channels but the PSD-95 SH₃-guanylate kinase domain functionally modulates Kv1.3 activity via two proline-rich domains in its N- and C-terminal. Therefore, these data suggest that adaptor domains responsible for ion-channel clustering and functional modulation are not necessarily coupled. Moreover, IR kinase and Kv1.3 can only be co-immunoprecipitated in the presence of PSD-95 as the adapting linker. Functionally, insulin-dependent Kv1.3 phosphorylation that causes channel current suppression is blocked via interaction with the PSD-95 SH₃-guanylate kinase domain. Because all the three proteins co-localize in multiple lamina of the OB that are known to be rich in synaptic connections, membrane excitability and synaptic transmission at critical locations in the OB have the capacity to be finely regulated.     

Tamalin is a scaffold protein that interacts with multiple neuronal proteins in distinct modes of protein-protein association

Tamalin is a scaffold protein that comprises multiple protein-interacting domains, including a 95-kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain, a leucine-zipper region, and a carboxyl-terminal PDZ binding motif. Tamalin forms a complex with metabotropic glutamate receptors and guanine nucleotide exchange factor cytohesins and promotes intracellular trafficking and cell surface expression of group 1 metabotropic glutamate receptors. In the present study, using several different approaches we have shown that tamalin interacts with multiple neuronal proteins through its distinct protein-binding domains. The PDZ domain of tamalin binds to the PDZ binding motifs of SAP90/PSD-95-associated protein and tamalin itself, whereas the PDZ binding motif of tamalin is capable of interacting with the PDZ domain of S-SCAM. In addition, tamalin forms a complex with PSD-95 and Mint2/X11beta/X11L by mechanisms different from the PDZ-mediated interaction. Tamalin has the ability to assemble with these proteins in vivo; their protein complex with tamalin was verified by coimmunoprecipitation of rat brain lysates. Interestingly, the distinct protein-interacting domains of tamalin are evolutionarily conserved, and mRNA expression is developmentally up-regulated at the postnatal period. The results indicate that tamalin exists as a key element that forms a protein complex with multiple postsynaptic and protein-trafficking scaffold proteins.     

Phosphorylation of a PDZ domain extension modulates binding affinity and interdomain interactions in postsynaptic density-95 (PSD-95) protein, a membrane-associated guanylate kinase (MAGUK)

Postsynaptic density-95 is a multidomain scaffolding protein that recruits glutamate receptors to postsynaptic sites and facilitates signal processing and connection to the cytoskeleton. It is the leading member of the membrane-associated guanylate kinase family of proteins, which are defined by the PSD-95/Discs large/ZO-1 (PDZ)-Src homology 3 (SH3)-guanylate kinase domain sequence. We used NMR to show that phosphorylation of conserved tyrosine 397, which occurs in vivo and is located in an atypical helical extension (α3), initiates a rapid equilibrium of docked and undocked conformations. Undocking reduced ligand binding affinity allosterically and weakened the interaction of PDZ3 with SH3 even though these domains are separated by a ~25-residue linker. Additional phosphorylation at two linker sites further disrupted the interaction, implicating α3 and the linker in tuning interdomain communication. These experiments revealed a novel mode of regulation by a detachable PDZ element and offer a first glimpse at the dynamic interaction of PDZ and SH3-guanylate kinase domains in membrane-associated guanylate kinases.     

An allosteric intramolecular PDZ-PDZ interaction modulates PTP-BL PDZ2 binding specificity

PDZ (acronym of the synapse-associated protein PSD-95/SAP90, the septate junction protein Discs-large, and the tight junction protein ZO-1) domains are abundant small globular protein interaction domains that mainly recognize the carboxyl termini of their target proteins. Detailed knowledge on PDZ domain binding specificity is a prerequisite for understanding the interaction networks they establish. We determined the binding preference of the five PDZ domains in the protein tyrosine phosphatase PTP-BL by screening a random C-terminal peptide lambda phage display library. Interestingly, the potential of PDZ2 to interact with class III-type ligands was found to be modulated by the presence of PDZ1. Structural studies revealed a direct and specific interaction of PDZ1 with a surface on PDZ2 that is opposite the peptide binding groove. Long-range allosteric effects that cause structural changes in the PDZ2 peptide binding groove thus explain the altered PDZ2 binding preference. Our results experimentally corroborate that the molecular embedding of PDZ domains is an important determinant of their ligand binding specificity.     

The interaction between PSD-95 and Ca2+/calmodulin is enhanced by PDZ-binding proteins

In this study, we evaluate the interaction between the postsynaptic scaffolding protein, PSD-95, and calmodulin. Surface plasmon resonance spectroscopy was used to characterize the binding of PSD-95 to calmodulin that had been immobilized on a sensor chip. Additionally, soluble calmodulin was found to inhibit the binding of PSD-95 to immobilized calmodulin. The HOOK region of PSD-95, which is located between the src homology 3 domain and the guanylate kinase-like domain, was determined to be involved in the binding of PSD-95 to calmodulin. We also found that C-terminal peptides from proteins such as CRIPT and the N-methyl-d-aspartate receptor NR2B subunit, which associate with the PDZ domain of PSD-95, enhanced the affinity of PSD-95 for calmodulin. The binding of ligands to the PDZ domain may change the conformation of PSD-95 and affect the interaction between PSD-95 and calmodulin.     

Energetic determinants of internal motif recognition by PDZ domains

PDZ domains are protein-protein interaction modules that organize intracellular signaling complexes. Most PDZ domains recognize specific peptide motifs followed by a required COOH-terminus. However, several PDZ domains have been found which recognize specific internal peptide motifs. The best characterized example is the syntrophin PDZ domain which, in addition to binding peptide ligands with the consensus sequence -E-S/T-X-V-COOH, also binds the neuronal nitric oxide synthase (nNOS) PDZ domain in a manner that does not depend on its precise COOH-terminal sequence. In the structure of the syntrophin-nNOS PDZ heterodimer complex, the two PDZ domains interact in a head-to-tail fashion, with an internal sequence from the nNOS PDZ domain binding precisely at the peptide binding groove of the syntrophin PDZ domain. To understand the energetic basis of this alternative mode of PDZ recognition, we have undertaken an extensive mutagenic and biophysical analysis of the nNOS PDZ domain and its interaction with the syntrophin PDZ domain. Our data indicate that the presentation of the nNOS internal motif within the context of a rigid beta-hairpin conformation is absolutely essential to binding; amino acids crucial to the structural integrity of the hairpin are as important or more important than residues that make direct contacts. The results reveal the general rules of PDZ recognition of diverse ligand types.