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 (...

High-density lipoprotein (HDL) particles are cholesterol and lipid transport containers. Mature HDL particles destined for the liver develop through the formation of intermediate discoidal HDL particles, which are the primary acceptors for cholesterol. Here we present the three-dimensional structure of reconstituted discoidal HDL (rdHDL) particles, using a shortened construct of human apolipoprotein A-I, determined from a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and transmission electron microscopy (TEM) data. The rdHDL particles feature a protein double belt surrounding a lipid bilayer patch in an antiparallel fashion. The integrity of this structure is maintained by up to 28 salt bridges and a zipper-like pattern of cation-π interactions between helices 4 and 6. To accommodate a hydrophobic interior, a gross 'right-to-right' rotation of the helices after lipidation is necessary. The structure reflects the complexity required for a shuttling container to hold a fluid lipid or cholesterol interior at a protein:lipid ratio of 1:50.

Protein-protein interactions mediated by modular protein domains are critical for cell scaffolding, differentiation, signaling, and ultimately, evolution. Given the vast number of ligands competing for binding to a limited number of domain families, it is often puzzling how specificity can be achieved. Selectivity may be modulated by intradomain allostery, whereby a remote residue is energetically connected to the functional binding site via side chain or backbone interactions. Whereas several energetic pathways, which could mediate intradomain allostery, have been predicted in modular protein domains, there is a paucity of experimental data to validate their existence and roles. Here, we have identified such functional energetic networks in one of the most common protein-protein interaction modules, the PDZ domain. We used double mutant cycles involving site-directed mutagenesis of both the PDZ domain and the peptide ligand, in conjunction with kinetics to capture the fine energetic details of the networks involved in peptide recognition. We performed the analysis on two homologous PDZ-ligand complexes and found that the energetically coupled residues differ for these two complexes. This result demonstrates that amino acid sequence rather than topology dictates the allosteric pathways. Furthermore, our data support a mechanism whereby the whole domain and not only the binding pocket is optimized for a specific ligand. Such cross-talk between binding sites and remote residues may be used to fine tune target selectivity.Several studies on protein-protein interaction modules (1), such as SH2, SH3, and PDZ 5 domains, have highlighted the apparent problem of how selectivity is achieved in a cellular context (2-4). For example, the synapse contains several PDZ proteins (5) with apparently overlapping specificity, and yet they have distinct roles in scaffolding and signaling (6). One mechanism that could modulate specificity is intradomain allostery (7). Multisubunit proteins such as hemoglobin are well known for their allosteric behavior whereby the oxygen binding is cooperative because ligation of one subunit will modulate the affinity in the other subunits of the tetramer (8). But allostery is also present within monomeric proteins (9), and it may be invoked even in the absence of a detectable conformational change (10 -15).Previous studies on monomeric globular proteins have predicted allosteric pathways on an amino acid residue level based on analysis of co-evolution (16, 17), molecular dynamics simulations (18), or NMR-constrained molecular dynamics (19). Although the PDZ domain family has served as a prime model system for these studies, experimental data that detect intramolecular allosteric pathways are scarce and sometimes conflicting (16,20,21).In protein folding studies, it proved illuminating to compare homologous proteins to unravel basic determinants of the underlying mechanism (22). Here, we employ the same strategy to assess the structural pattern of intradomain allostery by looking at the coupling fr...

Understanding the molecular principles that govern allosteric communication is an important goal in protein science. One way allostery could be transmitted is via sparse energetic networks of residues, and one such evolutionary conserved network was identified in the PDZ domain family of proteins by multiple sequence alignment [Lockless SW, Ranganathan R (1999) Science 286:295-299]. We have reassessed the energetic coupling of these residues by double mutant cycles together with ligand binding and stability experiments and found that coupling is not a special property of the coevolved network of residues in PDZ domains. The observed coupling for ligand binding is better explained by a distance relationship, where residues close in space are more likely to couple than distal residues. Our study demonstrates that statistical coupling from sequence analysis is not necessarily a reporter of energetic coupling and allostery.allostery ͉ coupling energy ͉ dynamics ͉ energetic network of residues ͉ PDZ domain P redicting allosteric mechanisms from protein sequence is an important goal, yet very difficult because the molecular basis for allostery in proteins is still not fully understood. Allosteric regulation is the alteration of protein function through the binding of an effector molecule elsewhere within the same protein. This definition can be expanded to include amino acid point mutations distal from the active site that can effectively alter protein function (1). Allosteric regulation is well established in multidomain proteins (2, 3), but allosteric behavior in single protein domains is less documented (for example, ref. 4), and its mechanisms unclear. Nevertheless, the idea of allostery without a (well defined) conformational change (5) has become popular, and it has even been proposed that all dynamic proteins are allosteric (6).Lockless and Ranganathan (7) set out to investigate whether allostery could be predicted from sequence conservation. They found a coevolved network of residues in the PDZ domain family of proteins, and these statistically coupled residues were confirmed by experiments on PSD95 PDZ3 to be energetically coupled (7). This study has served as a classic example of allostery in a single protein domain, with a ''sparse network'' of energetically linked positions that could affect function, in this case ligand binding. However, pathways of energetic connectivity inferred from statistical analysis have been questioned because the correlated mutation algorithm that was used finds pairs of residues that are close in physical space in the protein structure and it is therefore not surprising that the residues actually do couple (8). Furthermore, other issues regarding the validity of the statistical method used by Lockless and Ranganathan have been raised: the algorithm used is not symmetric (9), does not incorporate evolutionary noise (10), and performs less well than other algorithms (8,9,11). From an experimental point of view, previous work on PSD95 PDZ3 in our laboratory suggested that this PDZ ...

The protein-protein interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. An undecapeptide corresponding to the C-terminal of the NMDA was used as a template for finding lead candidates for the inhibition of the PSD-95/NMDA receptor interaction. Initially, truncation and alanine scan studies were carried out, which resulted in a pentapeptide with wild-type affinity, as examined in a fluorescence polarization assay. Further examination was performed by systematic substitutions with natural and unnatural amino acids, which disclosed a tripeptide with micromolar affinity and N-methylated tetrapeptides with improved affinities. Molecular modeling studies guided further N-terminal modifications and introduction of a range of N-terminal substitutions dramatically improved affinity. The best compound, N-cyclohexylethyl-ETAV (56), demonstrated up to 19-fold lower K i value ( K i = 0.94 and 0.45 microM against PDZ1 and PDZ2 of PSD-95, respectively) compared to wild-type values, providing the most potent inhibitors of this interaction reported so far. These novel and potent inhibitors provide an important basis for development of small molecule inhibitors of the PSD-95/NMDA receptor interaction.