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Title: Minimally disruptive optical control of protein tyrosine phosphatase 1B

Abstract

Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)—an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer—and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.

Authors:
 [1];  [2];  [2]; ORCiD logo [1]
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  1. Univ. of Colorado, Boulder, CO (United States)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Science Foundation (NSF); National Institutes of Health (NIH)
OSTI Identifier:
1615301
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Hongdusit, Akarawin, Zwart, Peter H., Sankaran, Banumathi, and Fox, Jerome M. Minimally disruptive optical control of protein tyrosine phosphatase 1B. United States: N. p., 2020. Web. doi:10.1038/s41467-020-14567-8.
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Hongdusit, Akarawin, Zwart, Peter H., Sankaran, Banumathi, & Fox, Jerome M. Minimally disruptive optical control of protein tyrosine phosphatase 1B. United States. https://doi.org/10.1038/s41467-020-14567-8
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Hongdusit, Akarawin, Zwart, Peter H., Sankaran, Banumathi, and Fox, Jerome M. Fri . "Minimally disruptive optical control of protein tyrosine phosphatase 1B". United States. https://doi.org/10.1038/s41467-020-14567-8. https://www.osti.gov/servlets/purl/1615301.
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@article{osti_1615301,
title = {Minimally disruptive optical control of protein tyrosine phosphatase 1B},
author = {Hongdusit, Akarawin and Zwart, Peter H. and Sankaran, Banumathi and Fox, Jerome M.},
abstractNote = {Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)—an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer—and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.},
doi = {10.1038/s41467-020-14567-8},
journal = {Nature Communications},
number = 1,
volume = 11,
place = {United States},
year = {2020},
month = {2}
}
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Journal Article:
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https://doi.org/10.1038/s41467-020-14567-8
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Cited by: 22 works
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Figures / Tables:

Fig. 1 Fig. 1: Minimally disruptive photocontrol of PTP1B. a Left: An alignment of a competitively inhibited structure of PTP1B (orange, pdb entry 2f71) and an apo structure of PTP1B (yellow, pdb entry 3a5j) highlights an allosteric control system. Closure of the WPD loop (black) over an inhibitor orders the α7 helix;more » opening of the loop (light blue) hinders this ordering. Right: An alignment of the LOV2 domain from A. sativa (blue) and an N-terminal segment of the same domain of A. thaliana (white) that is identical between the two proteins (pdb entries 2v0w and 4hhd, respectively). Two terminal α-helices (gray and white) are stable in the dark state, but not the light state. b Design of a photoswitchable chimera. Light-induced unwinding of the A’α helix of LOV2 destabilizes the α7 helix of PTP1B, causing an allosteric conformational change that inhibits catalysis. We attached the C-terminal α7 helix of PTP1B to the N-terminal A’α helix of LOV2 at crossover points in a primary sequence alignment (1–8). These points are highlighted in blue (PTP1B) and red (LOV2) in (a). c Assays on 4-methylumbelliferyl phosphate (4MUP) show the results of chimera optimization. Construct 7 has the largest dynamic range (DR) of the crossover variants; 7.1 has a higher activity than 7, and 7.1(T406A), termed PTP1BPS, has a larger DR than 7.1. The dashed gray and blue lines denote values for 7.1 and 7.1(T406A), respectively. The plotted data depict the mean, SE, and associated estimates of DR for n= 6 independent experiments. d Aligned catalytic domains of PTP1B in three structures: photoswitchable (6ntp), apo (3a5j), and competitively inhibited (2f71, α6 and α7 only). e An analysis of the activity of PTP1BPS on p-nitrophenyl-phosphate (pNPP) indicates that light affects kcat, but not Km (kcat-dark/kcat-light= 2.50 ± 0.04). Error bars denote SE for n= 3 independent reactions. f The DR of PTP1BPS is similar for substrates of different sizes. The plotted data depict the mean, SE, and associated estimates of DR for n≥ 3 independent reactions. g Structures of pNPP, 4MUP, and a peptide (PEP) derived from epidermal growth factor receptor (EGFR). Source data are provided as a Source Data file.« less
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