Molecular structures of membrane protein assemblies

Division Of Basic Sciences - Nci

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Abstract

We have made significant progress towards using 3D cryo-electron microscopy to determine the structures of membrane proteins and ligand-induced conformational changes at resolutions of 20 Angstroms or better. Work on numerous membrane protein assemblies including chemotaxis receptors, multi-drug transporters and ion channels. The example below with glutamate receptors illustrates the strategy we are taking. Glutamate receptor ion channels are membrane proteins that mediate excitatory synaptic transmission in the central nervous system of vertebrates. Insight into molecular mechanisms underlying glutamate receptor gating is limited by lack of structural information for receptors trapped in different conformational states. Using single particle cryo-electron tomography, we determined the structures, at 20 Angstrom resolution, of full-length GluK2 kainate receptors trapped in antagonist-bound resting and agonist-bound desensitized states. The resting state, stabilized by the competitive antagonist LY466195, closely resembles the crystal structure of the AMPA receptor GluA2, with well-resolved proximal and distal subunits exhibiting crossover between the 2-fold symmetric amino terminal domain (ATD) and a 2-fold symmetric ligand binding domain (LBD) dimer of dimers assembly. In the desensitized state, the LBD undergoes a major rearrangement resulting in a separation of the four subunits by 25 Angstroms. However, the ATD, transmembrane and cytoplasmic regions of the receptor have similar conformations in the resting and desensitized states. The LBD rearrangement was not anticipated in prior models based on crystal structures for soluble LBD dimer assemblies, and we speculate that subunit separation allows a better match to the 4-fold symmetric ion channel domain. From fits of the ATD and LBD domains into the density map of the desensitized state, we have derived a structural model for differences in quaternary conformation between the resting and desensitized states.