Agonist-induced isomerization in a glutamate receptor ligand-binding domain. A kinetic and mutagenetic analysis.
Agonist binding to glutamate receptor ion channels occurs within an extracellular domain (S1S2) that retains ligand affinity when expressed separately. S1S2 is homologous to periplasmic binding proteins, and it has been proposed that a Venus flytrap-style cleft closure triggers opening of glutamate receptor ion channels. Here we compare the kinetics of S1S2-agonist binding to those of the periplasmic binding proteins and show that the reaction involves an initial rapid association, followed by slower conformational changes that stabilize the complex: "docking" followed by "locking." The motion detected here reflects the mechanism by which the energy of glutamate binding is converted into protein conformational changes within S1S2 alone. In the intact channel, these load-free conformational changes are harnessed and possibly modified as the agonist binding reaction is used to drive channel opening and subsequent desensitization. Using mutagenesis, key residues in each step were identified, and their roles were interpreted in light of a published S1S2 crystal structure. In contrast to the Venus flytrap proposal, which focuses on motion between the two lobes as the readout for agonist binding, we argue that smaller, localized conformational rearrangements allow agonists to bridge the cleft, consistent with published hydrodynamic measurements.
Abele
R
R
Ion Channel Structure Research Group, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany.
Keinanen
K
K
Madden
D R
DR
D019943:Amino Acid Substitution; D001665:Binding Sites; D018690:Excitatory Amino Acid Agonists / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D007608:Kainic Acid / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D018091:Receptors, AMPA / Q000737:chemistry* / Q000378:metabolism*; D017470:Receptors, Glutamate / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D018690:Excitatory Amino Acid Agonists; D007473:Ion Channels; D008024:Ligands; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D011994:Recombinant Proteins; D018698:Glutamic Acid; D007608:Kainic Acid