Structural Mobility of the Extracellular Ligand-Binding Core of an Ionotropic Glutamate Receptor. Analysis of NMR Relaxation Dynamics^†

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 15N 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 α-substituents of the ligand glutamate exhibited minimal internal motion, while those contacting the γ-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

Long-term impacts of unconventional drilling operations on human and animal health

<div><p>Public health concerns related to the expansion of unconventional oil and gas drilling have sparked intense debate. In 2012, we published case reports of animals and humans affected by nearby drilling operations. Because of the potential for long-term effects of even low doses of environmental toxicants and the cumulative impact of exposures of multiple chemicals by multiple routes of exposure, a longitudinal study of these cases is necessary. Twenty-one cases from five states were followed longitudinally; the follow-up period averaged 25 months. In addition to humans, cases involved food animals, companion animals and wildlife. More than half of all exposures were related to drilling and hydraulic fracturing operations; these decreased slightly over time. More than a third of all exposures were associated with wastewater, processing and production operations; these exposures increased slightly over time. Health impacts decreased for families and animals moving from intensively drilled areas or remaining in areas where drilling activity decreased. In cases of families remaining in the same area and for which drilling activity either remained the same or increased, no change in health impacts was observed. Over the course of the study, the distribution of symptoms was unchanged for humans and companion animals, but in food animals, reproductive problems decreased and both respiratory and growth problems increased. This longitudinal case study illustrates the importance of obtaining detailed epidemiological data on the long-term health effects of multiple chemical exposures and multiple routes of exposure that are characteristic of the environmental impacts of unconventional drilling operations.</p></div

Backbone Dynamics of Inactive, Active, and Effector-Bound Cdc42Hs from Measurements of ¹⁵N Relaxation Parameters at Multiple Field Strengths^†

Cdc42Hs, a member of the Ras superfamily of GTP-binding proteins, initiates a cascade that begins with the activation of several kinases, including p21-activated kinase (PAK). We have previously determined the structure of Cdc42Hs and found that the regions involved in effector (Switch I) and regulator (Switch II) actions are partially disordered [Feltham, J. L., et al. (1997) Biochemistry 36, 8755−8766]. Recently, we used a 46-amino acid fragment of PAK (PBD46) to define the binding surface on Cdc42Hs, which includes the β2 strand and a portion of Switch I [Guo, W., et al. (1998) Biochemistry 37, 14030−14037]. Here we describe the backbone dynamics of three constructs of [15N]Cdc42Hs (GDP-, GMPPCP-, and GMPPCP- and PBD46-bound) using 15N−1H NMR measurements of T1, T1ρ, and the steady-state NOE at three magnetic field strengths. Residue-specific values of the generalized order parameters (Ss2 and Sf2), local correlation time (τe), and exchange rate (Rex) were obtained using the Lipari−Szabo model-free formalism. Residues in Switch I were found to exhibit high-amplitude (low-order) motions on a nanosecond time scale, whereas those in Switch II experience low-amplitude motion on the nanosecond time scale and chemical (conformational) exchange on a millisecond time scale. The Insert region of Cdc42Hs-GDP exhibits high-order, nanosecond motions; the time scale of motion in the Insert is reduced in Cdc42Hs−GMPPCP and Cdc42Hs−PBD46. Overall, significant flexibility was observed mainly in the regions of Cdc42Hs that are involved in protein−protein interactions (Switch I, Switch II, and Insert), and flexibility was reduced upon interaction with a protein ligand. These results suggest that protein flexibility is important for high-affinity binding interactions

Role of Stoichiometry in the Dimer-Stabilizing Effect of AMPA Receptor Allosteric Modulators

Protein dimerization provides a mechanism for the modulation of cellular signaling events. In α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, the rapidly desensitizing, activated state has been correlated with a weakly dimeric, glutamate-binding domain conformation. Allosteric modulators can form bridging interactions that stabilize the dimer interface. While most modulators can only bind to one position with a one modulator per dimer ratio, some thiazide-based modulators can bind to the interface in two symmetrical positions with a two modulator per dimer ratio. Based on small-angle X-ray scattering (SAXS) experiments, dimerization curves for the isolated glutamate-binding domain show that a second modulator binding site produces both an increase in positive cooperativity and a decrease in the EC₅₀ for dimerization. Four body binding equilibrium models that incorporate a second dimer-stabilizing ligand were developed to fit the experimental data. The work illustrates why stoichiometry should be an important consideration during the rational design of dimerizing modulators

Thermodynamics and Mechanism of the Interaction of Willardiine Partial Agonists with a Glutamate Receptor: Implications for Drug Development

Understanding the thermodynamics of binding of a lead compound to a receptor can provide valuable information for drug design. The binding of compounds, particularly partial agonists, to subtypes of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor is, in some cases, driven by increases in entropy. Using a series of partial agonists based on the structure of the natural product, willardiine, we show that the charged state of the ligand determines the enthalpic contribution to binding. Willardiines have uracil rings with p<i>K</i>_a values ranging from 5.5 to 10. The binding of the charged form is largely driven by enthalpy, while that of the uncharged form is largely driven by entropy. This is due at least in part to changes in the hydrogen bonding network within the binding site involving one water molecule. This work illustrates the importance of charge to the thermodynamics of binding of agonists and antagonists to AMPA receptors and provides clues for further drug discovery

Mechanism of Partial Agonism at the GluR2 AMPA Receptor: Measurements of Lobe Orientation in Solution

The mechanism by which the binding of a neurotransmitter to a receptor leads to channel opening is a central issue in molecular neurobiology. The structure of the agonist binding domain of ionotropic glutamate receptors has led to an improved understanding of the changes in structure that accompany agonist binding and have provided important clues about the link between these structural changes and channel activation and desensitization. However, because the binding domain has exhibited different structures under different crystallization conditions, understanding the structure in the absence of crystal packing is of considerable importance. The orientation of the two lobes of the binding domain in the presence of a full agonist, an antagonist, and several partial agonists was measured using NMR spectroscopy by employing residual dipolar couplings. For some partial agonists, the solution conformation differs from that observed in the crystal. A model of channel activation based on the results is discussed

Modulation of AMPA Receptor Gating by the Anticonvulsant Drug, Perampanel

Postsynaptic AMPA/glutamate receptors, essential for neuronal excitability, are important targets for anticonvulsant therapy. This single channel study of the selective noncompetitive AMPA receptor antagonist, perampanel, was performed on homotetrameric GluA3 receptor-channels that open in a stepwise manner to four distinct conductance levels through independent subunit activation. Previous structural studies show that perampanel binds to four sites located within the extracellular/transmembrane boundary of closed AMPA receptor-channel subunits. We found that channels exposed to 1 or 2 μM perampanel opened mainly to the two lower conductance levels in a dose-dependent manner. Comparison of the single channel results in the structures of the full length AMPA receptor in the closed state bound to perampanel, and the open state provide insights into the mechanism of allosteric reduction of AMPA-receptor-mediated excitation in epilepsy

The Perturbation of Tryptophan Fluorescence by Phenylalanine to Alanine Mutations Identifies the Hydrophobic Core in a Subset of Bacterial Ig-like Domains

Many host–parasite interactions are mediated via surface-exposed proteins containing bacterial immunoglobulin-like (Big) domains. Here, we utilize the spectral properties of a conserved Trp to provide evidence that, along with a Phe, these residues are positioned within the hydrophobic core of a subset of Big_2 domains. The mutation of the Phe to Ala decreases Big_2 domain stability and impairs the ability of LigBCen2 to bind to the host protein, fibronectin

The Loss of an Electrostatic Contact Unique to AMPA Receptor Ligand Binding Domain 2 Slows Channel Activation

Ligand-gated ion channels undergo conformational changes that transfer the energy of agonist binding to channel opening. Within ionotropic glutamate receptor (iGluR) subunits, this process is initiated in their bilobate ligand binding domain (LBD) where agonist binding to lobe 1 favors closure of lobe 2 around the agonist and allows formation of interlobe hydrogen bonds. AMPA receptors (GluAs) differ from other iGluRs because glutamate binding causes an aspartate–serine peptide bond in a flexible part of lobe 2 to rotate 180° (flipped conformation), allowing these residues to form cross-cleft H-bonds with tyrosine and glycine in lobe 1. This aspartate also contacts the side chain of a lysine residue in the hydrophobic core of lobe 2 by a salt bridge. We investigated how the peptide flip and electrostatic contact (D655–K660) in GluA3 contribute to receptor function by examining pharmacological and structural properties with an antagonist (CNQX), a partial agonist (kainate), and two full agonists (glutamate and quisqualate) in the wildtype and two mutant receptors. Alanine substitution decreased the agonist potency of GluA3_i-D655A and GluA3_i-K660A receptor channels expressed in HEK293 cells and differentially affected agonist binding affinity for isolated LBDs without changing CNQX affinity. Correlations observed in the crystal structures of the mutant LBDs included the loss of the D655–K660 electrostatic contact, agonist-dependent differences in lobe 1 and lobe 2 closure, and unflipped D­(A)­655–S656 bonds. Glutamate-stimulated activation was slower for both mutants, suggesting that efficient energy transfer of agonist binding within the LBD of AMPA receptors requires an intact tether between the flexible peptide flip domain and the rigid hydrophobic core of lobe 2

Definition of the Switch Surface in the Solution Structure of Cdc42Hs^†^,^‡

Proteins of the rho subfamily of ras GTPases have been shown to be crucial components of pathways leading to cell growth and the establishment of cell polarity and mobility. Presented here is the solution structure of one such protein, Cdc42Hs, which provides insight into the structural basis for specificity of interactions between this protein and its effector and regulatory proteins. Standard heteronuclear NMR methods were used to assign the protein, and approximately 2100 distance and dihedral angle constraints were used to calculate a set of 20 structures using a combination of distance geometry and simulated annealing refinement. These structures show overall similarity to those of other GTP-binding proteins, with some exceptions. The regions corresponding to switch I and switch II in H-ras are disordered, and no evidence was found for an α-helix in switch II. The 13-residue insertion, which is only present in rho-subtype proteins and has been shown to be an important mediator of binding of regulatory and target proteins, forms a compact structure containing a short helix lying adjacent to the β4−α3 loop. The insert forms one edge of a “switch surface” and, unexpectedly, does not change conformation upon activation of the protein by the exchange of GTP analogs for GDP. These studies indicate the insert region forms a stable invariant “footrest” for docking of regulatory and effector proteins