Light-Regulated allosteric switch enables temporal and subcellular control of enzyme activity
Abstract
Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.
Data availability
The raw mass spectrometry data and associated tables have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier: PXD018162. All data generated or analyzed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Funding
National Institutes of Health (R21CA212907)
- Jordan Fauser
National Institutes of Health (CA238720)
- Forest M White
Chicago Biomedical Consortium
- Andrei V Karginov
Army Research Office (W911NF-17-1-0395)
- Denis Tsygankov
National Institutes of Health (R21CA159179)
- Andrei V Karginov
National Institutes of Health (R01GM118582)
- Andrei V Karginov
National Institutes of Health (R21CA223915)
- Jalees Rehman
- Andrei V Karginov
National Institutes of Health (HL007829-22)
- Mark Shaaya
- Jordan Fauser
- Martin Brennan
National Institutes of Health (P01 HL060678)
- Viswanathan Natarajan
- Jalees Rehman
- Andrei V Karginov
National Institutes of Health (CA210180)
- Forest M White
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Luis F Larrondo, Pontificia Universidad Católica de Chile, Chile
Version history
- Received: July 2, 2020
- Accepted: September 22, 2020
- Accepted Manuscript published: September 23, 2020 (version 1)
- Accepted Manuscript updated: September 24, 2020 (version 2)
- Version of Record published: October 21, 2020 (version 3)
- Version of Record updated: November 11, 2020 (version 4)
Copyright
© 2020, Shaaya et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,794
- Page views
-
- 638
- Downloads
-
- 24
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
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)
Further reading
-
- Biochemistry and Chemical Biology
Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named ‘L’ and ‘R,’ where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.
-
- Biochemistry and Chemical Biology
Organizations that fund research are keen to ensure that their grant selection processes are fair and equitable for all applicants. In 2020, the Arnold and Mabel Beckman Foundation introduced blinding to the first stage of the process used to review applications for Beckman Young Investigator (BYI) awards: applicants were instructed to blind the technical proposal in their initial Letter of Intent by omitting their name, gender, gender-identifying pronouns, and institutional information. Here we examine the impact of this change by comparing the data on gender and institutional prestige of the applicants in the first four years of the new policy (BYI award years 2021–2024) with data on the last four years of the old policy (2017–2020). We find that under the new policy, the distribution of applicants invited to submit a full application shifted from those affiliated with institutions regarded as more prestigious to those outside of this group, and that this trend continued through to the final program awards. We did not find evidence of a shift in the distribution of applicants with respect to gender.