Bivalent Peptides as Models for Multimeric Targets of PDZ Domains

Paduch, Marcin; Biernat, Monika; Stefanowicz, Piotr; Derewenda, Zygmunt S.; Szewczuk, Zbigniew; Otlewski, Jacek

doi:10.1002/cbic.200600389

Cited by 18 publications

(18 citation statements)

References 91 publications

(42 reference statements)

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“…2A). Tat-N-dimer is thereby by far the most potent PDZ domain inhibitor described (15,(24)(25)(26), and provides a 1,000-fold increase in affinity relative to monomeric Tat-NR2B9c (K i ; mean AE SEM: 4;600 AE 300 nM, Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

Bach

Clausen²,

Møller³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

174

244

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Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4ðIETDVÞ 2 (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol∕g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-Ndimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.drug discovery | ischemic stroke | protein-protein interactions P rotein-protein interactions mediated by postsynaptic density protein-95 (PSD-95)/Discs-large/ZO-1 (PDZ) domains are important for intracellular signaling events, and several PDZ domains are potential drug targets for neuronal diseases and cancer (1, 2). The postsynaptic scaffolding protein PSD-95 simultaneously binds the N-methyl-D-aspartate (NMDA)-type of ionotropic glutamate receptors and the enzyme neuronal nitric oxide synthase (nNOS) through its PDZ1 and PDZ2 domains (3). Activation of the NMDA receptor causes influx of Ca 2þ , which activates nNOS thereby leading to nitric oxide generation (4), a key facilitator of glutamate-mediated excitotoxicity (5, 6). Ligands that bind to the first two PDZ domains of PSD-95 inhibit the formation of the ternary nNOS/PSD-95/NMDA receptor complex and uncouple the harmful production of nitric oxide from NMDA receptor activity (Fig. 1A). As PSD-95 inhibition does not affect ion-flux (7) or prosurvival signaling pathways (8) mediated by the NMDA receptor, it is believed that compounds targeting PDZ1 and PDZ2 of PSD-95 can provide an efficient and safe treatment of ischemic brain damage (9), where excitotoxicity is known to dominate in the acute poststroke period, as well as other NMDA receptor-related disorders such as chronic pain and Alzheimer's disease (10-14).The shallow and elongated binding pocket of PDZ domains generally favor binding of peptides or peptide analogues and so far no drug-like small-molecule inhibitors of PDZ domains with affinities below 5 μM have been identified (15). Accordingly, the most advanced PSD-95 inhibitor is a 20-mer peptide, Tat-NR2B9c (7, 8, 16), composed of nine amino acids corresponding to the C-terminal of the GluN2B subunit of the NMDA receptor, fused to the HIV-1 Tat peptide (17). This peptide has shown promising effects against ischemic brain damage in rats (...

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Section: Resultsmentioning

confidence: 99%

A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

Bach

Clausen²,

Møller³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

174

244

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“…By using fluorescence-based methods or isothermal titration calorimetry (ITC), several studies have determined the K d value between isolated C-terminal peptides from PlexinB1 and the PDZ domain of PDZ-RhoGEF in the range of 30-36 μM under various buffer conditions (24,26). The PDZ domain of LARG shows similar affinity to the tail peptide from PlexinB1 (24)(25)(26). The peptides used in the studies of PDZ-RhoGEF included no more than the C-terminal six residues of human PlexinB1, which are identical to those in mouse PlexinB2.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, isolated C-terminal peptides from class B plexins and the PDZ domains of PDZRhoGEF/LARG only exhibit modest affinity, with the dissociation constant (K d ) in the range of 10-40 μM (24)(25)(26). This Significance Protein interactions mediated by modular domains, such as PDZ and SH2 domains, play critical roles in biology.…”

mentioning

confidence: 99%

Secondary PDZ domain-binding site on class B plexins enhances the affinity for PDZ–RhoGEF

Pascoe

Gutowski

Chen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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PDZ domains are abundant protein interaction modules and typically recognize a short motif at the C terminus of their ligands, with a few residues in the motif endowing the binding specificity. The sequence-based rules, however, cannot fully account for the specificity between the vast number of PDZ domains and ligands in the cell. Plexins are transmembrane receptors that regulate processes such as axon guidance and angiogenesis. Two related guanine nucleotide exchange factors (GEFs), PDZ-RhoGEF and leukemiaassociated RhoGEF (LARG), use their PDZ domains to bind class B plexins and play critical roles in signaling. Here, we present the crystal structure of the full-length cytoplasmic region of PlexinB2 in complex with the PDZ domain of PDZ-RhoGEF. The structure reveals that, in addition to the canonical C-terminal motif/PDZ interaction, the 3D domain of PlexinB2 forms a secondary interface with the PDZ domain. Our biophysical and cell-based assays show that the secondary interface contributes to the specific interaction between plexin and PDZ-RhoGEF and to signaling by plexin in the cell. Formation of secondary interfaces may be a general mechanism for increasing affinity and specificity of modular domain-mediated interactions.PDZ | plexin | signaling | protein interaction module | specificity P lexins are cell surface receptors for semaphorins, extracellular cues that control essential processes such as neuronal axon guidance and vasculature development (1). Binding of semaphorin to the extracellular region of plexin induces formation of the active dimer of the cytoplasmic region, which transduces signal to downstream pathways (2-7). The plexin cytoplasmic region contains a juxtamembrane segment (JM-segment), a RhoGTPase binding domain (RBD), and a GTPase activating protein (GAP) domain (8-10). The GAP domain, activated by the dimerization, transduces signal through converting its substrate GTPase Rap from the GTP-bound active to the GDP-bound inactive state (2, 3). The RBD regulates plexin activity in response to binding of Rho family GTPases, such as Rac1 (reviewed in ref. 11).In addition to the common signaling pathways through the domains shared by all plexins, class B plexins (B1, B2, and B3) mediate a pathway through their unique C terminus. The conserved "VTDL" motif at the C terminus of these plexins binds to the N-terminal PDZ (PSD-95/Discs-large/ZO-1) domains of two related guanine nucleotide exchange factors (GEFs), PDZ-RhoGEF, and leukemia-associated RhoGEF (LARG) (12)(13)(14)(15)(16)(17). This interaction recruits PDZ-RhoGEF and LARG to the plasma membrane, where they promote the exchange of GDP for GTP on RhoA. GTP-bound RhoA binds its downstream effectors and contributes to plexin signaling (13)(14)(15)18). A recent study has shown that deletion of the C terminus of PlexinB2 causes defects in the development of the liver vasculature in mice, highlighting the critical role of the PDZ-RhoGEF/LARG-RhoA pathway in plexin function in vivo (19).More than 250 PDZ domains exist in the human proteome, con...

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“…33 Moreover, it has been discussed that PDZ domain/peptide analyses may have oversimplified the understanding of these interactions in vivo, because two-site interactions that are likely a critical determinant of physiological PDZ binding are often overlooked. 34 Indeed, multimerization of PDZ-containing proteins and their receptor targets might constitute physiological regulatory mechanisms, and so the enhancement of avidity by multiple interactions of inherently weak ligands may be a common theme in BIRs and PDZs.…”

Section: Discussionmentioning

confidence: 99%

The mechanism of peptide-binding specificity of IAP BIR domains

et al. 2008

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We describe the peptide-binding specificity of the baculoviral IAP repeat (BIR) domains of the human inhibitor of apoptosis (IAP) proteins, X-linked IAP, cellular IAP1 and neuronal apoptosis inhibitory protein (NAIP). Synthetic peptide libraries were used to profile each domain, and we distinguish two types of binding specificity, which we refer to as type II and type III BIR domains. Both types have a dominant selectivity for Ala in the first position of the four N-terminal residues of the peptide ligands, which constitute a core recognition motif. Our analysis allows us to define the signature of type III BIRs that demonstrate a preference for Pro in the third residue of the ligand, resembling the classic IAP-binding motif (IBM). The signature of the type II BIRs was similar to type III, but with a striking absence of specificity for Pro in the third position, suggesting that the definition of an IBM must be modified depending on the type of BIR in question. These findings explain how subtle changes in the peptide-binding groove of IAP BIR domains can significantly alter the target protein selectivity. Our analysis allows for prediction of BIR domain protein-binding preferences, provides a context for understanding the mechanism of peptide selection and heightens our knowledge of the specificity of IAP antagonists that are being developed as cancer therapeutics. Cell Death and Differentiation (2008) 15, 920-928; doi:10.1038/cdd.2008 ; published online 1 February 2008Apoptosis is a highly regulated cellular signaling pathway that results in the elimination of unwanted cells from multicellular animals. The apoptotic signaling cascades can be initiated by various extracellular (extrinsic pathway) or intracellular (intrinsic pathway) stimuli and ultimately converge on the activation of a family of proteases known as the caspasesreviewed in Fuentes-Prior and Salvesen. 1 Once active, caspases cleave a limited number of substrates that results in the destruction of the cell and its eventual disposal by phagocytic cells.Members of the inhibitor of apoptosis (IAP) protein family have the unique ability to attenuate apoptosis induced through both intrinsic and extrinsic stimuli. It is well established that the X-linked IAP (XIAP) is capable of blocking apoptosis by direct inhibition of caspase-3, -7, and -9 -reviewed in Salvesen and Duckett 2 and Eckelman et al. 3 The means by which the other members of the IAP family inhibit apoptosis is less understood. Many IAPs have the capacity to bind caspases, yet lack the ability to directly inhibit the proteolytic activity of these enzymes -reviewed in Eckelman et al. 3 The defining feature of the IAP family is the presence of 1-3 baculoviral IAP repeat (BIR) domains. BIRs are small, B70-residue zinc-coordinated domains that have been identified as the regions essential to the IAP-caspase interaction. The BIR domain(s) are necessary for the antiapoptotic activity of most IAPs. Often a surface groove on the BIR domain endows it with an affinity toward extreme N-terminal epitopes...

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Bivalent Peptides as Models for Multimeric Targets of PDZ Domains

Cited by 18 publications

References 91 publications

A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

Secondary PDZ domain-binding site on class B plexins enhances the affinity for PDZ–RhoGEF

The mechanism of peptide-binding specificity of IAP BIR domains

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