Large-scale state-dependent membrane remodeling by a transporter protein
- National Heart, Lung and Blood Institute, National Institutes of Health, United States
- Children's National Medical Center, United States
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
Abstract
That channels and transporters can influence the membrane morphology is increasingly recognized. Less appreciated is that the extent and free-energy cost of these deformations likely varies among different functional states of a protein, and thus, that they might contribute significantly to defining its mechanism. We consider the trimeric Na+-aspartate symporter GltPh, a homolog of an important class of neurotransmitter transporters, whose mechanism entails one of the most drastic structural changes known. Molecular simulations indicate that when the protomers become inward-facing, they cause deep, long-ranged, and yet mutually-independent membrane deformations. Using a novel simulation methodology, we estimate that the free-energy cost of this membrane perturbation is in the order of 6-7 kcal/mol per protomer. Compensating free-energy contributions within the protein or its environment must thus stabilize this inward-facing conformation for the transporter to function. We discuss these striking results in the context of existing experimental observations for this and other transporters.
Data availability
Input and output files for 1 (out of 3) replica of each simulation system/condition in our study have been uploaded to Zenodo, a public repository free of charge, and is available at the DOI 10.5281/zenodo.3558957.
-
Simulation files for "Large-scale state-dependent membrane remodeling by a transporter protein"Zenodo, doi:10.5281/zenodo.3558957.
Article and author information
Author details
José D Faraldo-Gómez
Funding
National Heart, Lung, and Blood Institute
- Wenchang Zhou
- Giacomo Fiorin
- Claudio Anselmi
- José D Faraldo-Gómez
National Institute of Neurological Disorders and Stroke
- Hossein Ali Karimi-Varzaneh
- Horacio Poblete
- Lucy Forrest
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Metrics
-
- 3,403
- views
-
- 459
- downloads
-
- 44
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Large-scale state-dependent membrane remodeling by a transporter protein
eLife 8:e50576.
https://doi.org/10.7554/eLife.50576
Further reading
-
- Chromosomes and Gene Expression
- Structural Biology and Molecular Biophysics
Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.
-
- 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.
