Vet Behavior Consults, Ithaca, NY 14850, U Department of Molecular Medicine, Corn E-mail: reo1@cornell.edu † Any opinions or views expressed in the f authors and do not represent the views Processes & Impacts, or The Royal Society ‡ The opinions expressed in this commun represent the views of Cornell University i be considered official or sanctioned organization with which they are affiliated Cite this: Environ. Sci.: Processes Impacts, 2014, 16, 1860

Cdc42Hs(F28L) is a single-point mutant of Cdc42Hs, a member of the Ras superfamily of GTP-binding proteins, that facilitates cellular transformation brought about by an increased rate of cycling between GTP and GDP [Lin, R., et al. (1997) Curr. Biol. 7, 794-797]. Dynamics studies of Cdc42Hs(F28L)-GDP have shown increased flexibility for several residues at the nucleotide-binding site [Adams, P. D., et al. (2004) Biochemistry 43, 9968-9977]. The solution structure of Cdc42Hs-GDP (wild type) has previously been determined by NMR spectroscopy [Feltham, J. L., et al. (1997) Biochemistry 36, 8755-8766]. Here, we describe the solution structure of Cdc42Hs(F28L)-GDP, which provides insight into the structural basis for the change in affinity for GDP. Heteronuclear NMR experiments were performed to assign resonances in the protein, and distance, hydrogen bonding, residual dipolar coupling, and dihedral angle constraints were used to calculate a set of low-energy structures using distance geometry and simulated annealing refinement protocols. The overall structure of Cdc42Hs(F28L)-GDP is very similar to that of wild-type Cdc42Hs, consisting of a centrally located six-stranded beta-sheet structure surrounding the C-terminal alpha-helix [Feltham, J. L., et al. (1997) Biochemistry 36, 8755-8766]. In addition, the same three regions in wild-type Cdc42Hs that show structural disorder (Switch I, Switch II, and the Insert region) are disordered in F28L as well. Although the structure of Cdc42Hs(F28L)-GDP is very similar to that of the wild type, interactions with the nucleotide and hydrogen bonding within the nucleotide binding site are altered, and the region surrounding L28 is substantially more disordered.

High-resolution studies of ionotropic glutamate receptor (iGluR) extracellular domains are beginning to bridge the gap between structure and function. Crystal structures have defined the ligand binding pocket well beyond what was suggested by mutational analysis and homology models alone, providing initial suggestions about the mechanisms of channel gating and desensitization. NMR-derived backbone dynamics and molecular dynamics simulations have added further insights into the role of protein dynamics in receptor function. As a whole, the current knowledge of iGluR structure in conjunction with new advances in the understanding of K+ channels provides a vastly improved understanding of iGluR function. This review focuses on structural and dynamic studies of the extracellular ligand binding domain of iGluRs and the pore region of K+ channels that have contributed to mechanistic insights into the processes of iGluR gating and desensitization

Ionotropic glutamate receptors are the major excitatory neurotransmitters in mammalian brain but are found throughout the animal kingdom as well as in plants and bacteria. A great deal of progress in understanding the structure of these essential neurotransmitter receptors has been made since the first examples were cloned and sequenced in 1989. The atomic structure of the ligand-binding domain of several ionotropic glutamate receptors has been determined, and a great deal of progress has been made in relating the structural properties of the binding site to the function of the intact receptor. In addition, the identification of glutamate receptors from a wide variety of organisms ranging from several types of bacteria to Arabidopsis to a range of animal species has made glutamate receptors a molecular laboratory for studying the evolution of proteins. The fact that glutamate receptors are a particularly ancient intercellular signaling molecule suggests a potential role in the transition from single celled to multicellular organisms. This review focuses on the structure and dynamics of ionotropic glutamate receptors and their relation to the function and evolution of these proteins.

Cdc42Hs, a member of the Ras superfamily of GTP-binding signal transduction proteins, binds guanine nucleotides, and acts as a molecular-timing switch in multiple signal transduction pathways. The structure of the wild-type protein has been solved (Feltham et al. (1997) Biochemistry 36, 8755-8766), and the backbone dynamics have been characterized by NMR spectroscopy (Loh et al. (1999) Biochemistry 38, 12547-12557). The F28L mutation of Cdc42Hs is characterized by an increased rate of cycling between the GTP and GDP-bound forms leading to cell transformation (Lin et al. (1997) Curr. Biol. 7, 794-797). Here, we describe the backbone dynamics of Cdc42Hs(F28L)-GDP using 1H-15N NMR measurements of T1, T1rho, and steady-state NOE at two magnetic field strengths. Residue-specific values of the generalized order parameters (Ss2 and Sf2), local correlation time (tau(e)), and exchange rate (R(ex)) were obtained using the Lipari-Szabo formalism. Chemical-shift perturbation analysis suggested that very little structural change was evident outside of the nucleotide-binding site. However, residues comprising the nucleotide-binding site, as well as the nucleotide itself, exhibit increased dynamics over a wide range of time scales in Cdc42Hs(F28L) relative to the wild type. In addition to changes in dynamics measured by relaxation methods, hydrogen-deuterium exchange indicated a substantial disruption of the hydrogen-bonding network within the nucleotide-binding site. Thus, local dynamic changes introduced by a single-point mutation can affect important aspects of signaling processes without disrupting the conformation of the whole protein.

GluR6 is an ionotropic glutamate receptor subunit of the kainate subtype. It plays an essential role in synaptic plasticity and epilepsy. We expressed this recombinant receptor in HEK-293 cells and characterized the glutamate-induced channel-opening reaction, using a laser-pulse photolysis technique with the caged glutamate (gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate). This technique permits glutamate to be liberated photolytically from the caged glutamate with a time constant of approximately 30 micros. Prior to laser photolysis, the caged glutamate did not activate the GluR6 channel, nor did it inhibit or potentiate the glutamate response. At the transmembrane voltage of -60 mV, pH 7.4 and 22 degrees C, the channel-opening and -closing rate constants were determined to be (1.1 +/- 0. 4) x 10(4) and (4.2 +/- 0.2) x 10(2) s(-1), respectively. The intrinsic dissociation constant of glutamate and the channel-opening probability were found to be 450 +/- 200 microM and 0.96, respectively. These constants are derived from a minimal kinetic mechanism of the channel activation involving the binding of two glutamate molecules. This mechanism describes the time course of the open-channel form of the receptor as a function of glutamate concentration. On the basis of the channel-opening rate constants obtained, the shortest rise time (20-80% of the receptor current response) or the fastest time by which the GluR6Q channel can open is predicted to be 120 micros. The open-channel form of the receptor determines the transmembrane voltage change, which in turn controls synaptic signal transmission between two neurons. The comparison of the channel-opening kinetic rate constants between GluR6Q and GluR2Q(flip), reported in the companion paper, suggests that at a glutamate concentration of 100 microM, for instance, the integrated neuronal signal will be dominated by a slower GluR6Q receptor response, as compared to the GluR2Q(flip) component.

Ionotropic glutamate receptors play important roles in a variety of neuronal processes and have been implicated in multiple neurodegenerative diseases. The extracellular ligand-binding (S1S2) core of the GluR2 subtype can be expressed in bacteria as a soluble, monomeric protein with binding properties essentially identical to those of the intact receptor. The crystal structure of this protein has been determined in the presence and absence of various agonists and antagonists [Armstrong, N., Sun, Y., Chen, G. Q., and Gouaux, E. (1998) Nature 395, 913-917; Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165-181]. The protein consists of two lobes, with the S1 segment composing the majority of lobe 1 and the S2 segment composing most of lobe 2. A domain closure upon ligand binding has been postulated, but details of intradomain motions have not been investigated. In this paper, the backbone motions of the ligand-binding core of GluR2 bound to glutamate were studied using (15)N longitudinal (T1) and transverse (T2) relaxation measurements as well as [1H]-15N nuclear Overhauser effects at 500 and 600 MHz. Residues in the agonist-binding pocket exhibited two main classes of motion. Those contacting the alpha-substituents of the ligand glutamate exhibited minimal internal motion, while those contacting the gamma-constituents exhibited exchange dynamics, indicating two dynamically distinct portions of the binding pocket. Also, two residues in transdomain linkers between lobes 1 and 2 show exchange, lending new insight into the previously proposed domain closure hypothesis. Finally, concerted motion of helix F suggests a pathway for ligand dissociation without the necessity of domain reopening.

Mechanosensitive channels (MSCs) play key roles in sensory processing and have been implicated as primary transducers for a variety of cellular responses ranging from osmosensing to gene expression. This paper presents the first structures of any kind known to interact specifically with MSCs. GsMTx-4 and GsMtx-2 are inhibitor cysteine knot peptides isolated from venom of the tarantula,Grammostola spatulata (Suchyna, T. M., Johnson, J. H., Hamer, K., Leykam, J. F., Gage, D. A., Clemo, H. F., Baumgarten, C. M., and Sachs, F. (2000) J. Gen. Physiol. 115, 583–598). Inhibition of cationic MSCs by the higher affinity GsMtx-4 (K D ∼500 nm) reduced cell size in swollen and hypertrophic heart cells, swelling-activated currents in astrocytes, and stretch-induced arrhythmias in the heart. Despite the relatively low affinity, no cross-reactivity has been found with other channels. Using two-dimensional NMR spectroscopy, we determined the solution structure of GsMTx-4 and a lower affinity (GsMTx-2; K D ∼6 μm) peptide from the same venom. The dominant feature of the two structures is a hydrophobic patch, utilizing most of the aromatic residues and surrounded with charged residues. The spatial arrangement of charged residues that are unique to GsMTx-4 and GsMTx-2 may underlie the selectivity of these peptides.

Cdc42Hs is a member of the Ras superfamily of GTPases which, when active, initiates a cascade beginning, with the activation of several kinases, including P-21-activated kinase (PAK). We previously determined the structure of a complex between a 46 amino acid fragment peptide derived from the PAK binding, domain (PBD46) and Cdc42Hs(.)GMPPCP (Gizachew, D., Guo, W., Chohan, K. K.. Sutcliffe. M. J., and Oswald, R. E. (2000) Biochemistry 39, 3963-3971). Previous Studies (Loh, A. P.. Guo. W.. Nicholson, L. K., and Oswald, R. E. (1999) Biocheinistry, 38, 12547-12557) suo-est that the regions of Cdc42Hs that bind effectors and regulators have distinct dynamic properties from the remainder of the protein. Here. we describe the backbone dynamics of PBD46 bound to Cdc42Hs-GMPPCP. T-1. T-2. T-1p, and steady-state nuclear Overhauser effects were measured at 500 and 600 MHz. An extension of the Lipari-Szabo model-free analysis was used to determine the order parameters (S-2) and local correlation times (tau (c)) of the N-H bond vectors within PBD46. Both Cdc42Hs and PBD46 exhibit increased mobility in the free versus the bound state, suggesting that protein flexibility may be required for high-affinity PBD46 binding and, presumably, the activation or PAK. Different backbone dynamics were observed in different regions of the peptide. The beta -strand region of bound PBD46, which makes contacts with beta2 of Cdc42Hs. exhibits low mobility on the pico- to nanosecond timescale. However, the part of PBD46 that interacts with Switch I of Cdc42Hs exhibits greater mobility. Thus, PBD46 and Cdc42Hs form a tight complex that exhibits concerted dynamics.

Cdc42Hs is a signal transduction protein that is involved in cytoskeletal growth and organization. We describe here the methyl side chain dynamics of three forms of (2)H,(13)C,(15)N-Cdc42Hs [GDP-bound (inactive), GMPPCP-bound (active), and GMPPCP/PBD46-bound (effector-bound)] from (13)C-(1)H NMR measurements of deuterium T(1) and T(1 rho) relaxation times. A wide variation in flexibility was observed throughout the protein, with methyl axis order parameters (S(2)(axis)) ranging from 0.2 to 0.4 (highly disordered) in regions near the PBD46 binding site to 0.8--1.0 (highly ordered) in some helices. The side chain dynamics of the GDP and GMPPCP forms are similar, with methyl groups on the PBD46 binding surface experiencing significantly greater mobility (lower S(2)(axis)) than those not on the binding surface. Binding of PBD46 results in a significant increase in the disorder and a corresponding increase in entropy for the majority of methyl groups. Many of the methyl groups that experience an increase in mobility are found in residues that are not part of the PBD46 binding interface. This entropy gain represents a favorable contribution to the overall entropy of effector binding and partially offsets unfavorable entropy losses such as those that occur in the backbone.

Effects of cocaine and cocaine methiodide were evaluated on the homomeric alpha 7 neuronal nicotinic receptor (nAChR). Whereas cocaine itself is a general nAChR noncompetitive antagonist, we report here the characterization of cocaine methiodide, a novel selective agonist for the alpha 7 subtype of nAChR. Data from (125)I-alpha-bungarotoxin binding assays indicate that cocaine methiodide binds to alpha 7 nAChR with a K(i) value of approximately 200 nM while electrophysiology studies indicate that the addition of a methyl group at the amine moiety of cocaine changes the drug's activity profile from inhibitor to agonist. Cocaine methiodide activates alpha 7 nAChR with an EC(50) value of approximately 50 microM and shows comparable efficacy to ACh in oocyte experiments. While agonist effects are specific for the alpha 7 neuronal nAChR and are not observed with heteromeric neuronal or skeletal muscle nAChR, antagonist effects are present for heteromeric nAChR combinations. Studies of PC12 cells transiently transfected with human alpha 7 cDNA and expressing a variety of functional nicotinic receptor subtypes confirm the specificity of cocaine methiodide agonist effects. Our results indicate that a quaternary structural derivative of cocaine can be used as a specific agonist for the alpha 7 subtype of neuronal nicotinic receptor.

The extracellular, G protein-linked Ca(2+)-sensing receptor (CaSR), first identified in the parathyroid gland, is expressed in several tissues and cells and can be activated by Ca(2+) and some other inorganic cations and organic polycations. Calcimimetics such as NPS (R)-N-(3-phenylpropyl)-alpha-methyl-3-methoxybenzylamine hydrochloride (R-467), a phenylalkylamine, are thought to activate CaSR by allosterically increasing the affinity of the receptor for Ca(2+). When tested for its effect on insulin release in C57BL/6 mice, R-467 had no effect under basal conditions but enhanced both phases of glucose-stimulated release. The betaHC9 cell also responded to R-467 and to the enantiomer S-467 with a stimulation of insulin release. In subsequent studies with the betaHC9 cell, it was found that the stimulatory effect was due to activation of a nonspecific cation channel, depolarization of the beta-cell, and increased Ca(2+) entry. No other stimulatory mechanism was uncovered. The depolarization of the cell induced by the calcimimetic could be due to a direct action on the channel or via the CaSR. However, it appeared not to be mediated by G(i), G(o), G(q/11), or G(s). The novel mode of action of the calcimimetic, combined with the glucose-dependence of the stimulation on islets, raises the possibility of a totally new class of drugs that will stimulate insulin secretion during hyperglycemia but which will not cause hypoglycemia.

Cdc42Hs is a member of the Ras superfamily of GTPases and initiates a cascade that begins with the activation of several kinases, including p21-activated kinase (PAK). We have previously used a 46 amino acid fragment of PAK (PBD46) to define the binding surface on Cdc42Hs [Guo et al. (1998) Biochemistry 37, 14030-14037]. Here we describe the three-dimensional solution structure of the Cdc42Hs. GMPPCP-PBD46 complex. Heteronuclear NMR methods were used to assign resonances in the complex, and approximately 2400 distance and dihedral restraints were used to calculate a set of 20 structures using a combination of distance geometry, simulated annealing, and chemical shift and Ramachandran refinement. The overall structure of Cdc42Hs in the complex differs from the uncomplexed structure in two major aspects: (1) the first alpha helix is reoriented to accommodate the binding of the peptide and (2) the regions corresponding to switch I and switch II are less disordered. As suggested by our previous work (Guo et al., 1998) and similar to the complex between Cdc42Hs and fACK [Mott et al. (1999) Nature 399, 384-388], PBD46 forms an intermolecular beta-sheet with beta2 of Cdc42Hs and contacts both switch I and switch II. The extensive binding surface between PBD46 and Cdc42Hs can account for both the high affinity of the complex and the inhibition by PBD46 of GTP hydrolysis.