Re-examining how complexin inhibits neurotransmitter release: SNARE complex insertion or electrostatic hindrance?
- Charité-Universitätsmedizin Berlin, Germany
- University of Texas Southwestern Medical Center, United States
Abstract
Complexins play activating and inhibitory functions in neurotransmitter release. The complexin accessory helix inhibits release and was proposed to insert into SNARE complexes to prevent their full assembly. This model was supported by 'superclamp' and 'poor-clamp' mutations that enhanced or decreased the complexin-I inhibitory activity in cell-cell fusion assays, and by the crystal structure of a superclamp mutant bound to a synaptobrevin-truncated SNARE complex. NMR studies now show that the complexin-I accessory helix does not insert into synaptobrevin-truncated SNARE complexes in solution, and electrophysiological data reveal that superclamp mutants have slightly stimulatory or no effects on neurotransmitter release, whereas a poor-clamp mutant inhibits release. Importantly, increasing or decreasing the negative charge of the complexin-I accessory helix inhibits or stimulates release, respectively. These results suggest a new model whereby the complexin accessory helix inhibits release through electrostatic (and perhaps steric) repulsion enabled by its location between the vesicle and plasma membranes.
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Ethics
Animal experimentation: Animals were handled according to the animal welfare committee of the Charit� Berlin, Germany. Time pregnant females were anesthetized and euthanized at E18 according to the german animal law ({section sign}4 section 1) and permit by the Berlin authorities (Landesamt f�r Gesundheit und Soziales, LaGeSo) under the permit number T0220/09.
Copyright
© 2014, Trimbuch et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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Re-examining how complexin inhibits neurotransmitter release: SNARE complex insertion or electrostatic hindrance?
eLife 3:e02391.
https://doi.org/10.7554/eLife.02391
Further reading
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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
We have previously proposed that complexin cross-links multiple pre-fusion SNARE complexes via a trans interaction to function as a clamp on SNARE-mediated neurotransmitter release. A recent NMR study was unable to detect the trans clamping interaction of complexin and therefore questioned the previous interpretation of the fluorescence resonance energy transfer and isothermal titration calorimetry data on which the trans clamping model was originally based. Here we present new biochemical data that underscore the validity of our previous interpretation and the continued relevancy of the trans insertion model for complexin clamping.
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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The SARS-CoV-2 main protease (Mpro or Nsp5) is critical for production of viral proteins during infection and, like many viral proteases, also targets host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 is recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes cellular protein synthesis and redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. Evolutionary analysis shows the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. TRMT1 proteolysis results in reduced tRNA binding and elimination of tRNA methyltransferase activity. We also determined the structure of an Mpro-TRMT1 peptide complex that shows how TRMT1 engages the Mpro active site in an uncommon substrate binding conformation. Finally, enzymology and molecular dynamics simulations indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis following substrate binding. Together, these data provide new insights into substrate recognition by SARS-CoV-2 Mpro that could help guide future antiviral therapeutic development and show how proteolysis of TRMT1 during SARS-CoV-2 infection impairs both TRMT1 tRNA binding and tRNA modification activity to disrupt host translation and potentially impact COVID-19 pathogenesis or phenotypes.
