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 (...

Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 106 V/m, and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein.

Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.

Intrinsically disordered proteins are abundant in the eukaryotic proteome, and they are implicated in a range of different diseases. However, there is a paucity of experimental data on molecular details of the coupled binding and folding of such proteins. Two interacting and relatively well studied disordered protein domains are the activation domain from the p160 transcriptional co-activator ACTR and the nuclear co-activator binding domain (NCBD) of CREB binding protein. We have analyzed the transition state for their coupled binding and folding by protein engineering and kinetic experiments (Φ-value analysis) and found that it involves weak native interactions between the N-terminal helices of ACTR and NCBD, but is otherwise "disordered-like". Most native hydrophobic interactions in the interface between the two domains form later, after the rate-limiting barrier for association. Linear free energy relationships suggest a cooperative formation of native interactions, reminiscent of the nucleation-condensation mechanism in protein folding.

PostprintThis is the accepted version of a paper published in Biophysical Chemistry. This paper has been peerreviewed but does not include the final publisher proof-corrections or journal pagination.Citation for the original published paper (version of record):Gianni, S., Dogan, J., Jemth, P. (2014) Distinguishing induced fit from conformational selection.