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 pKa 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.

Mechanisms of antagonism of the GluR2 AMPA receptor: structure and dynamics of the complex of two willardiine antagonists with the glutamate binding domain.

Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission. The development of selective antagonists for glutamate receptor subtypes is of interest in the treatment of a variety of neurological disorders. This study presents the crystal structure of the binding domain of GluR2 bound to two antagonists (UBP277 and UBP282) that are derivatives of the natural product, willardiine. The antagonists bind to one lobe of the protein with interactions similar to agonists. Interaction with the second lobe differs between the two antagonists, resulting in a different position of the uracil ring and different orientations of the bilobed structure. UBP277 binding produces a stable lobe orientation that is similar to the apo state, but the binding of UBP282 produces the largest hyperextension of the lobes yet reported for an AMPA receptor. The carboxyethyl (UBP277) and carboxybenzyl (UBP282) substituents in the N(3) position keep the lobes separated by a "foot-in-the-door" mechanism and the internal dynamics are minimal compared to the CNQX-bound form of the protein (which makes minimal contacts with one of the two lobes). In contrast to the antagonists CNQX and DNQX, UBP277 and UBP282 produce complexes with higher thermal stability, but affinities that are more than 100-fold lower. These structures support the idea that antagonism is associated with the overall orientation of the lobes rather than with specific interactions, and antagonism can rise either from specific interactions with both lobes ("foot-in-the-door" mechanism) or from the lack of extensive interactions with one of the two lobes.

Structure of glutamate receptors.

Glutamate receptors mediate a vast array of processes in plants, animals and bacteria. In particular, the ionotropic glutamate receptors (iGluRs) are the most abundant excitatory neurotransmitter receptors in the mammalian central nervous system. Because these proteins are constructed from distinct folding domains, most of which can be traced to bacterial precursors, the analyses of these important receptor proteins has been performed on a variety of levels ranging from atomic structure and dynamics to behavioral studies. This review will focus on the structure and dynamics of iGluRs, with particular emphasis on the role that the glutamate-binding domain (S1S2) plays in receptor function.

Dynamics of the S1S2 glutamate binding domain of GluR2 measured using 19F NMR spectroscopy.

Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission. Although the structure of the GluR2 binding domain (S1S2) is well known (agonist binding site between two lobes), little is known about the time scales of conformational transitions or the relationship between dynamics and function. (19)F NMR ((19)F-labeled tryptophan) spectroscopy was used to monitor motions in the S1S2 domain bound to ligands with varying efficacy and in the apo state. One tryptophan (Trp-671) undergoes chemical exchange in some but not all agonists, consistent with mus-ms motion. The dynamics can be correlated to ligand affinity, and a likely source of the motion is a peptide bond capable of transiently forming hydrogen bonds across the lobe interface. Another tryptophan (Trp-767) appears to monitor motions of the relative positions of the lobes and suggests that the relative orientation in the apo- and antagonist-bound forms can exchange between at least two conformations on the ms time scale.

Backbone dynamics of an oncogenic mutant of Cdc42Hs shows increased flexibility at the nucleotide-binding site.

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.