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ABSTRACT: Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs), the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD), and of multiple conformations of the ligand-binding domain (LBD). Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves additional quaternary packing arrangements. To explore the conformational space available to iGluR channels, we report here a second, clearly distinct domain architecture of homotetrameric, calcium-permeable AMPA receptors, determined by single-particle electron microscopy of untagged and fluorescently tagged constructs in a ligand-free state. It reveals a novel packing of NTD dimers, and a separation of LBD dimers across a central vestibule. In this arrangement, which reconciles diverse functional observations, agonist-induced cleft closure across LBD dimers can be converted into a twisting motion that provides a basis for receptor activation.
Frontiers in Molecular Neuroscience 01/2012; 4:56.
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ABSTRACT: Human lysozyme is a key component of the innate immune system, and recombinant forms of the enzyme represent promising leads in the search for therapeutic agents able to treat drug-resistant infections. The wild type protein, however, fails to participate effectively in clearance of certain infections due to inherent functional limitations. For example, wild type lysozymes are subject to electrostatic sequestration and inactivation by anionic biopolymers in the infected airway. A charge engineered variant of human lysozyme has recently been shown to possess improved antibacterial activity in the presence of disease associated inhibitory molecules. Here, the 2.04 Å crystal structure of this variant is presented along with an analysis that provides molecular level insights into the origins of the protein's enhanced performance. The charge engineered variant's two mutated amino acids exhibit stabilizing interactions with adjacent native residues, and from a global perspective, the mutations cause no gross structural perturbations or loss of stability. Importantly, the two substitutions dramatically expand the negative electrostatic potential that, in the wild type enzyme, is restricted to a small region near the catalytic residues. The net result is a reduction in the overall strength of the engineered enzyme's electrostatic potential field, and it appears that the specific nature of this remodeled field underlies the variant's reduced susceptibility to inhibition by anionic biopolymers.
PLoS ONE 01/2011; 6(3):e16788. · 4.09 Impact Factor
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ABSTRACT: AMPA receptors (AMPARs) are tetrameric ligand-gated ion channels that couple the energy of glutamate binding to the opening of a transmembrane channel. Crystallographic and electrophysiological analysis of AMPARs has suggested a coupling between (1) cleft closure in the bilobate ligand-binding domain (LBD), (2) the resulting separation of transmembrane helix attachment points across subunit dimers, and (3) agonist efficacy. In general, more efficacious agonists induce greater degrees of cleft closure and transmembrane separation than partial agonists. Several apparent violations of the cleft-closure/efficacy paradigm have emerged, although in all cases, intradimer separation remains as the driving force for channel opening. Here, we examine the structural basis of partial agonism in GluA4 AMPARs. We find that the L651V substitution enhances the relative efficacy of kainate without increasing either LBD cleft closure or transmembrane separation. Instead, the conformational change relative to the wild-type:kainate complex involves a twisting motion with the efficacy contribution opposite from that expected based on previous analyses. As a result, channel opening may involve transmembrane rearrangements with a significant rotational component. Furthermore, a two-dimensional analysis of agonist-induced GluA2 LBD motions suggests that efficacy is not a linearly varying function of lobe 2 displacement vectors, but is rather determined by specific conformational requirements of the transmembrane domains.
Journal of Neuroscience 01/2010; 30(4):1463-70. · 7.11 Impact Factor
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ABSTRACT: AMPA receptors are glutamate-gated ion channels that are essential mediators of synaptic signals in the central nervous system. They form tetramers that are assembled as combinations of subunits GluR1-4, each of which contains a ligand-binding domain (LBD). Crystal structures of the GluR2 LBD have revealed an agonist-binding cleft, which is located between two lobes and which acts like a Venus flytrap. In general, agonist efficacy is correlated with the extent of cleft closure. However, recent observations show that cleft closure is not the sole determinant of the relative efficacy for glutamate receptors. In addition, these studies have focused on the GluR2 subunit, which is the specific target of a physiologically important RNA-editing modification in vivo. We therefore sought to test the generality of the cleft closure-efficacy correlation for other AMPA-R subunits. Here, we present crystal structures of the GluR4(flip) LBD in complex with both full and partial agonists. As for GluR2, both agonists stabilize a closed-cleft conformation, and the partial agonist induces a smaller cleft closure than the full agonist. However, a detailed analysis of LBD-kainate interactions reveals the importance of subtle backbone conformational changes in the ligand-binding pocket in determining the magnitude of agonist-associated conformational changes. Furthermore, the GluR4 subunit exhibits a different correlation between receptor activation and LBD cleft closure than does GluR2.
Biochemistry 01/2009; 47(52):13831-41. · 3.42 Impact Factor
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ABSTRACT: Glutamate is the major excitatory neurotransmitter in the brain. Among the cognate ionotropic glutamate receptors, the subfamily selective for AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) is responsible for most fast excitatory synaptic signaling and plays key roles in synaptic plasticity. AMPA receptors (AMPA-Rs) have also been implicated in a number of neurological disorders. To investigate subunit-specific differences in the ligand binding and activation of AMPA-Rs, the GluR4 AMPA-R ligand-binding domain (LBD) was crystallized in complex with full and partial agonists. This is the first non-GluR2 AMPA-R LBD available for structural analysis. Standard cryoprotection protocols yielded high-resolution diffraction from flash-cooled crystals of the complex with the full agonist glutamate. However, for cocrystals with the partial agonist kainate, systematic screening and optimization of cryoprotection conditions yielded at best mosaic, weak diffraction at 100 K. In contrast, room-temperature data collection from capillary-mounted kainate cocrystals exhibited reproducible diffraction to better than 3 A resolution. Together, these crystals lay the foundation for a structural comparison of LBD-agonist interactions in distinct AMPA-R subunits.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2008; 64(Pt 9):831-5. · 0.51 Impact Factor
