The interaction of phospholipase C-beta3 with Shank2 regulates mGluR-mediated calcium signal

Phospholipase C-beta isozymes that are activated by G protein-coupled receptors (GPCR) and heterotrimeric G proteins carry a PSD-95/Dlg/ZO-1 (PDZ) domain binding motif at their C terminus. Through interactions with PDZ domains, this motif may endow the PLC-beta isozyme with specific roles in GPCR signaling events that occur in compartmentalized regions of the plasma membrane. In this study, we identified the interaction of PLC-beta3 with Shank2, a PDZ domain-containing multimodular scaffold in the postsynaptic density (PSD). The C terminus of PLC-beta3, but not other PLC-beta isotypes, specifically interacts with the PDZ domain of Shank2. Homer 1b, a Shank-interacting protein that is linked to group I metabotropic glutamate receptors and IP3 receptors, forms a multiple complex with Shank2 and PLC-beta3. Importantly, microinjection of a synthetic peptide specifically mimicking the C terminus of PLC-beta3 markedly reduces the mGluR-mediated intracellular calcium response. These results demonstrate that Shank2 brings PLC-beta3 closer to Homer 1b and constitutes an efficient mGluR-coupled signaling pathway in the PSD region of neuronal synapses.     

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.     

Crystal structure of the Shank PDZ-ligand complex reveals a class I PDZ interaction and a novel PDZ-PDZ dimerization

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-A 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 beta B/beta C loop and N-terminal beta A strands, suggesting a novel model of PDZ-PDZ homodimerization. This implies that antiparallel dimerization through the N-terminal beta A 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 family of postsynaptic density proteins interacts with and promotes synaptic accumulation of the beta PIX guanine nucleotide exchange factor for Rac1 and Cdc42

The Shank/ProSAP family of multidomain proteins is known to play an important role in organizing synaptic multiprotein complexes. Here we report a novel interaction between Shank and beta PIX, a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases. This interaction is mediated by the PDZ domain of Shank and the C-terminal leucine zipper domain and the PDZ domain-binding motif at the extreme C terminus of beta PIX. Shank colocalizes with beta PIX at excitatory synaptic sites in cultured neurons. In brain, Shank forms a complex with beta PIX and beta PIX-associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42. Importantly, overexpression of Shank in cultured neurons promotes synaptic accumulation of beta PIX and PAK. Considering the involvement of Rac1 and PAK in spine dynamics, these results suggest that Shank recruits beta PIX and PAK to spines for the regulation of postsynaptic structure.     

The PDZ Domain of the LIM Protein Enigma Binds to β-Tropomyosin

PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein alpha-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal beta-TM). The interaction between Enigma and skeletal beta-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal beta-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal beta-TM in transfected cells. The association of Enigma with skeletal beta-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells.     

Autoinhibition of X11/Mint scaffold proteins revealed by the closed conformation of the PDZ tandem

Members of the X11/Mint family of multidomain adaptor proteins are composed of a divergent N terminus, a conserved PTB domain and a pair of C-terminal PDZ domains. Many proteins can interact with the PDZ tandem of X11 proteins, although the mechanism of such interactions is unclear. Here we show that the highly conserved C-terminal tail of X11alpha folds back and inserts into the target-binding groove of the first PDZ domain. The binding of this tail occludes the binding of other target peptides. This autoinhibited conformation of X11 requires that the two PDZ domains and the entire C-terminal tail be covalently connected to form an integral structural unit. The autoinhibited conformation of the X11 PDZ tandem provides a mechanistic explanation for the unique target-binding properties of the protein and hints at potential regulatory mechanisms for the X11-target interactions.     

Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells

Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaP_i cotransporter (NaP_i-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaP_i-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E coimmunoprecipitated with NaP_i-IIa from rat renal cortex tissue and HA-NaP_i-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP_i-IIa and truncation of the COOH-terminal TRL motif from NaP_i-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP_i-IIa interaction occurred directly between the two proteins. NaP_i-IIa activity is regulated by moderating its abundance in the apical membrane. High-P_i conditions induce NaP_i-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P_i conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaP_i-IIa via PDZ interactions, and undergoes P_i-induced internalization. PDZ domains; endocytosis; degradation; epithelia     

ProSAP-interacting protein 1 (ProSAPiP1), a novel protein of the postsynaptic density that links the spine-associated Rap-Gap (SPAR) to the scaffolding protein ProSAP2/Shank3

ProSAPs/Shanks are a family of proteins that have a major scaffolding function for components of the postsynaptic density (PSD) of excitatory brain synapses. Members of the family harbor a variety of domains for protein-protein interactions, one of which is a unique PDZ domain that differs significantly from those of other proteins. We have identified a novel binding partner for this PDZ domain, termed ProSAPiP1, that is highly enriched in the PSD and shares significant sequence homology with the PSD protein PSD-Zip70. Both molecules code for a Fez1 domain that can be found in a total of four related proteins. ProSAPiP1 is widely expressed in rat brain and co-localizes with ProSAP2/Shank3 in excitatory spines and synapses. ProSAP2/Shank3 co-immunoprecipitates with ProSAPiP1 but not with PSD-Zip70. Both proteins, however, bind and recruit SPAR to synapses with a central coiled-coil region that harbors a leucine zipper motif. This region is also responsible for homo- and heteromultimerization of ProSAPiP1 and PSD-Zip70. Thus, ProSAPiP1 and PSD-Zip70 are founders of a novel family of scaffolding proteins, the "Fezzins," which adds further complexity to the organization of the PSD protein network.     

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.     

Unusual binding interactions in PDZ domain crystal structures help explain binding mechanisms

PDZ domains most commonly bind the C‐terminus of their protein targets. Typically the C‐terminal four residues of the protein target are considered as the binding motif, particularly the C‐terminal residue (P0) and third‐last residue (P‐2) that form the major contacts with the PDZ domain's “binding groove”. We solved crystal structures of seven human PDZ domains, including five of the seven PDLIM family members. The structures of GRASP, PDLIM2, PDLIM5, and PDLIM7 show a binding mode with only the C‐terminal P0 residue bound in the binding groove. Importantly, in some cases, the P‐2 residue formed interactions outside of the binding groove, providing insight into the influence of residues remote from the binding groove on selectivity. In the GRASP structure, we observed both canonical and noncanonical binding in the two molecules present in the asymmetric unit making a direct comparison of these binding modes possible. In addition, structures of the PDZ domains from PDLIM1 and PDLIM4 also presented here allow comparison with canonical binding for the PDLIM PDZ domain family. Although influenced by crystal packing arrangements, the structures nevertheless show that changes in the positions of PDZ domain side‐chains and the αB helix allow noncanonical binding interactions. These interactions may be indicative of intermediate states between unbound and fully bound PDZ domain and target protein. The noncanonical “perpendicular” binding observed potentially represents the general form of a kinetic intermediate. Comparison with canonical binding suggests that the rearrangement during binding involves both the PDZ domain and its ligand.     

Crystal structure of GRIP1 PDZ6-peptide complex reveals the structural basis for class II PDZ target recognition and PDZ domain-mediated multimerization

PDZ domains bind to short segments within target proteins in a sequence-specific fashion. Glutamate receptor-interacting protein (GRIP)/ABP family proteins contain six to seven PDZ domains and interact via the sixth PDZ domain (class II) with the C termini of various proteins including liprin-alpha. In addition the PDZ456 domain mediates the formation of homo- and heteromultimers of GRIP proteins. To better understand the structural basis of peptide recognition by a class II PDZ domain and PDZ-mediated multimerization, we determined the crystal structures of the GRIP1 PDZ6 domain alone and in complex with a synthetic C-terminal octapeptide of human liprin-alpha at resolutions of 1.5 and 1.8 A, respectively. Remarkably, unlike other class II PDZ domains, Ile-736 at alphaB5 rather than conserved Leu-732 at alphaB1 makes a direct hydrophobic contact with the side chain of the Tyr at the -2 position of the ligand. Moreover, the peptide-bound structure of PDZ6 shows a slight reorientation of helix alphaB, indicating that the second hydrophobic pocket undergoes a conformational adaptation to accommodate the bulkiness of the Tyr side chain, and forms an antiparallel dimer through an interface located at a site distal to the peptide-binding groove. This configuration may enable formation of GRIP multimers and efficient clustering of GRIP-binding proteins.     

RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.     

Characterization of the Shank family of synaptic proteins. Multiple genes, alternative splicing, and differential expression in brain and development.

Shank1, Shank2, and Shank3 constitute a family of proteins that may function as molecular scaffolds in the postsynaptic density (PSD). Shank directly interacts with GKAP and Homer, thus potentially bridging the N-methyl-D-aspartate receptor-PSD-95-GKAP complex and the mGluR-Homer complex in synapses (Naisbitt, S., Kim, E., Tu, J. C. , Xiao, B., Sala, S., Valtschanoff, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999) Neuron 23, 569-582; Tu, J. C., Xiao, B., Naisbitt, S., Yuan, J. P., Petralia, R. S., Brakeman, P., Doan, A., Aakalu, V. K., Lanahan, A. A., Sheng, M., and Worley, P. F. (1999) Neuron 23, 583-592). Shank contains multiple domains for protein-protein interaction including ankyrin repeats, an SH3 domain, a PSD-95/Dlg/ZO-1 domain, a sterile alpha motif domain, and a proline-rich region. By characterizing Shank cDNA clones and RT-PCR products, we found that there are four sites for alternative splicing in Shank1 and another four sites in Shank2, some of which result in deletion of specific domains of the Shank protein. In addition, the expression of the splice variants is differentially regulated in different regions of rat brain during development. Immunoblot analysis of Shank proteins in rat brain using five different Shank antibodies reveals marked heterogeneity in size (120-240 kDa) and differential spatiotemporal expression. Shank1 immunoreactivity is concentrated at excitatory synaptic sites in adult brain, and the punctate staining of Shank1 is seen in developing rat brains as early as postnatal day 7. These results suggest that alternative splicing in the Shank family may be a mechanism that regulates the molecular structure of Shank and the spectrum of Shank-interacting proteins in the PSDs of adult and developing brain.     

The binding affinities of proteins interacting with the PDZ domain of PICK1

Protein interacting with C kinase (PICK1) is well conserved throughout evolution and plays a critical role in synaptic plasticity by regulating the trafficking and posttranslational modification of its interacting proteins. PICK1 contains a single PSD95/DlgA/Zo-1 (PDZ) protein-protein interaction domain, which is promiscuous and shown to interact with over 60 proteins, most of which play roles in neuronal function. Several reports have suggested the role of PICK1 in disorders such as epilepsy, pain, brain trauma and stroke, drug abuse and dependence, schizophrenia and psychosis. Importantly, lead compounds that block PICK1 interactions are also now becoming available. Here, a new modeling approach was developed to investigate binding affinities of PDZ interactions. Using these methods, the binding affinities of all major PICK1 interacting proteins are reported and the effects of PICK1 mutations on these interactions are described. These modeling methods have important implications in defining the binding properties of proteins interacting with PICK1 as well as the general structural requirements of PDZ interactions. The study also provides modeling methods to support in the drug design of ligands for PDZ domains, which may further aid in development of the family of PDZ domains as a drug target.     

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.     

Binding of PTEN to specific PDZ domains contributes to PTEN protein stability and phosphorylation by microtubule-associated serine/threonine kinases

The tumor suppressor phosphatase PTEN is a key regulator of cell growth and apoptosis that interacts with PDZ domains from regulatory proteins, including MAGI-1/2/3, hDlg, and MAST205. Here we identified novel PTEN-binding PDZ domains within the MAST205-related proteins, syntrophin-associated serine/threonine kinase and MAST3, characterized the regions of PTEN involved in its interaction with distinctive PDZ domains, and analyzed the functional consequences on PTEN of PDZ domain binding. Using a panel of PTEN mutations, as well as PTEN chimeras containing distinct domains of the related protein TPTE, we found that the PTP and C2 domains of PTEN do not affect PDZ domain binding and that the C-terminal tail of PTEN (residues 350-403) provides selectivity to recognize specific PDZ domains from MAGI-2, hDlg, and MAST205. Binding of PTEN to the PDZ-2 domain from MAGI-2 increased PTEN protein stability. Furthermore, binding of PTEN to the PDZ domains from microtubule-associated serine/threonine kinases facilitated PTEN phosphorylation at its C terminus by these kinases. Our results suggest an important role for the C-terminal region of PTEN in the selective association with scaffolding and/or regulatory molecules and provide evidence that PDZ domain binding stabilizes PTEN and targets this tumor suppressor for phosphorylation by microtubule-associated serine/threonine kinases.     

Distinct ligand specificity of the Tiam1 and Tiam2 PDZ domains

Guanine nucleotide exchange factor proteins of the Tiam family are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These proteins are modular and contain a variety of interaction domains, including a single post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Previous studies suggest that the specificities of the Tiam1 and Tiam2 PDZ domains are distinct. Here, we sought to conclusively define these specificities and determine their molecular origin. Using a combinatorial peptide library, we identified a consensus binding sequence for each PDZ domain. Analysis of these consensus sequences and binding assays with peptides derived from native proteins indicated that these two PDZ domains have overlapping but distinct specificities. We also identified residues in two regions (S(0) and S(-2) pockets) of the Tiam1 PDZ domain that are important determinants of ligand specificity. Site-directed mutagenesis of four nonconserved residues in these two regions along with peptide binding analyses confirmed that these residues are crucial for ligand affinity and specificity. Furthermore, double mutant cycle analysis of each region revealed energetic couplings that were dependent on the ligand being investigated. Remarkably, a Tiam1 PDZ domain quadruple mutant had the same specificity as the Tiam2 PDZ domain. Finally, analysis of Tiam family PDZ domain sequences indicated that the PDZ domains segregate into four distinct families based on the residues studied here. Collectively, our data suggest that Tiam family proteins have highly evolved PDZ domain-ligand interfaces with distinct specificities and that they have disparate PDZ domain-dependent biological functions.     

ZRP-1, a zyxin-related protein, interacts with the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E.

Protein-protein interactions play an important role in the specificity of cellular signaling cascades. By using the yeast two-hybrid system, a specific interaction was identified between the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E and a novel protein, which was termed ZRP-1 to indicate its sequence similarity to the Zyxin protein family. The mRNA encoding this protein is distributed widely in human tissues and contains an open reading frame of 1428 base pairs, predicting a polypeptide of 476 amino acid residues. The deduced protein displays a proline-rich amino-terminal region and three double zinc finger LIM domains at its carboxyl terminus. The specific interaction of this novel protein with the second PDZ domain of hPTP1E was demonstrated both in vitro, using bacterially expressed proteins, and in vivo, by co-immunoprecipitation studies. Deletion analysis indicated that an intact carboxyl terminus is required for its interaction with the second PDZ domain of hPTP1E in the yeast two-hybrid system and suggested that other sequences, including the LIM domains, also participate in the interaction. The genomic organization of the ZRP-1 coding sequence is identical to that of the lipoma preferred partner gene, another Zyxin-related protein, suggesting that the two genes have evolved from a recent gene duplication event.     

Role of electrostatic interactions in PDZ domain ligand recognition

PDZ domains are protein-protein interaction modules that normally recognize short C-terminal peptides. The apparent requirement for a ligand with a free terminal carboxylate group has led to the proposal that electrostatic interactions with the terminus play a significant role in recognition. However, this model has been called into question by the more recent finding that PDZ domains can recognize some internal peptide motifs that occur within a specific secondary structure context. Although these motifs bind at the same interface, they lack a terminal charge. Here we have investigated the role of electrostatics in PDZ-mediated recognition in the mouse alpha1-syntrophin PDZ domain by examining the salt dependence of binding to both terminal and internal ligands and the effects of mutating a conserved basic residue previously proposed to play a role in electrostatic recognition. These studies indicate that direct electrostatic interactions with the peptide terminus do not play a significant energetic role in binding. Additional chemical modification studies of the peptide terminus support a model in which steric and hydrogen bonding complementarity play a primary role in recognition specificity. Peptides with a free carboxy terminus, or presented within a specific structural context, can satisfy these requirements.