Lummis, S.C. et al. Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel. Nature 438, 248-252

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.
Nature (Impact Factor: 41.46). 12/2005; 438(7065):248-52. DOI: 10.1038/nature04130
Source: PubMed

ABSTRACT 5-hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2-M3), can link binding to gating through a cis-trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis-trans energy gap of the proline analogue and the activation of the channel, suggesting that cis-trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2-M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2-M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.

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Available from: Richard W Broadhurst, Sep 28, 2015
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    • "Reorganization of transmembrane helices and rigid body rotation of an extracellular domain are the important changes observed in the open and closed state structures of pentameric ligand-gated ion channels [26]. Using a series of proline analogs, experiments have suggested that cis–trans isomerization of a single proline residue provides a molecular switch for inter-converting open and closed states in the channels formed by 5HT3 receptors which are members of Cys-loop receptor superfamily [27]. "
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    Biochimica et Biophysica Acta 03/2015; 1848(6). DOI:10.1016/j.bbamem.2015.03.013 · 4.66 Impact Factor
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    • "In the process of amyloid fibril formation they are important, both, in the nucleation and in fibril elongation [26] [27]. The cis/trans isomerization of the peptide bond preceding proline is considered to form an intrinsic molecular switch controlling several physiologically relevant processes, such as opening of the pore of a neurotransmitter-gated ion channel [28] [29]. Peptidyl prolyl cis/trans isomerases (PPIases) are enzymes that catalyze the cis/ trans isomerization of prolyl bonds. "
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    • "The fact that XLMR is associated with the elimination of a proline at this position in hCASK led us to more closely consider the critical role proline can play in protein structure. Proline cis-to-trans isomerism has previously been proposed as a gate-keeper for fibril formation [78], [79] and can also serve as a molecular switch, as is seen in the pores of some neurotransmitter-gated channels [80], [81]. It is possible that the isomerization state of P396 is an important regulator of protein-protein interactions that involve hCASK’s L27 domain and hence hCASK cellular function, and when a proline is lost at position 396, a yet-to-be-described function of hCASK is lost. "
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    ABSTRACT: The overwhelming amount of available genomic sequence variation information demands a streamlined approach to examine known pathogenic mutations of any given protein. Here we seek to outline a strategy to easily classify pathogenic missense mutations that cause protein misfolding and are thus good candidates for chaperone-based therapeutic strategies, using previously identified mutations in the gene CASK. We applied a battery of bioinformatics algorithms designed to predict potential impact on protein structure to five pathogenic missense mutations in the protein CASK that have been shown to underlie pathologies ranging from X-linked mental retardation to autism spectrum disorder. A successful classification of the mutations as damaging was not consistently achieved despite the known pathogenicity. In addition to the bioinformatics analyses, we performed molecular modeling and phylogenetic comparisons. Finally, we developed a simple high-throughput imaging assay to measure the misfolding propensity of the CASK mutants in situ. Our data suggests that a phylogenetic analysis may be a robust method for predicting structurally damaging mutations in CASK. Mutations in two evolutionarily invariant residues (Y728C and W919R) exhibited a strong propensity to misfold and form visible aggregates in the cytosolic milieu. The remaining mutations (R28L, Y268H, and P396S) showed no evidence of aggregation and maintained their interactions with known CASK binding partners liprin-α3 Mint-1, and Veli, indicating an intact structure. Intriguingly, the protein aggregation caused by the Y728C and W919R mutations was reversed by treating the cells with a chemical chaperone (glycerol), providing a possible therapeutic strategy for treating structural mutations in CASK in the future.
    PLoS ONE 02/2014; 9(2):e88276. DOI:10.1371/journal.pone.0088276 · 3.23 Impact Factor
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