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Kinetic and structural parameters governing Fic-mediated adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78

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Cell Stress and Chaperones Aims and scope

Abstract

Fic (filamentation induced by cAMP) proteins regulate diverse cell signaling events by post-translationally modifying their protein targets, predominantly by the addition of an AMP (adenosine monophosphate). This modification is called Fic-mediated adenylylation or AMPylation. We previously reported that the human Fic protein, HYPE/FicD, is a novel regulator of the unfolded protein response (UPR) that maintains homeostasis in the endoplasmic reticulum (ER) in response to stress from misfolded proteins. Specifically, HYPE regulates UPR by adenylylating the ER chaperone, BiP/GRP78, which serves as a sentinel for UPR activation. Maintaining ER homeostasis is critical for determining cell fate, thus highlighting the importance of the HYPE-BiP interaction. Here, we study the kinetic and structural parameters that determine the HYPE-BiP interaction. By measuring the binding and kinetic efficiencies of HYPE in its activated (Adenylylation-competent) and wild type (de-AMPylation-competent) forms for BiP in its wild type and ATP-bound conformations, we determine that HYPE displays a nearly identical preference for the wild type and ATP-bound forms of BiP in vitro and preferentially de-AMPylates the wild type form of adenylylated BiP. We also show that AMPylation at BiP’s Thr366 versus Thr518 sites differentially affect its ATPase activity, and that HYPE does not adenylylate UPR accessory proteins like J-protein ERdJ6. Using molecular docking models, we explain how HYPE is able to adenylylate Thr366 and Thr518 sites in vitro. While a physiological role for AMPylation at both the Thr366 and Thr518 sites has been reported, our molecular docking model supports Thr518 as the structurally preferred modification site. This is the first such analysis of the HYPE-BiP interaction and offers critical insights into substrate specificity and target recognition.

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Abbreviations

Fic:

filamentation induced by cAMP

AMP:

adenosine monophosphate

HYPE:

Huntingtin yeast interacting protein E

UPR:

unfolded protein response

ER:

endoplasmic reticulum

GS:

glutamine synthetase

GS-ATase:

glutamine synthetase adenylyltranferase

BiP:

binding immunoglobulin protein

NBD:

nucleotide binding domain

SBD:

substrate binding domain

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Acknowledgements

We thank Dr. Clint Chapple for advice on the design of kinetic experiments. We also thank the Purdue University office of Radiological and Environmental Management (REM) and Dr. Nicholas Carpita for access to the Liquid Scintillation Counter. Wild type HYPEΔ102/pSMT3 was a gift from Dr. Junyu Xiao and ERdJ6/pGEX-4T1 was kindly provided by Dr. David Ron. We also thank Dr. Linda Hendershot for additional J protein constructs and helpful discussions. Finally, we are grateful to members of the Mattoo lab for their helpful discussions.

Funding

This work was funded in part by the National Institute of General Medical Sciences of the National Institute of Health (R01GM10092), an Indiana Clinical and Translational Research Grant (CTSI-106564), and a Purdue Institute for Inflammation, Immunology, and Infectious Disease Core Start Grant (PI4D-209263) to SM; the National Institute of General Medical Sciences of the National Institutes of Health (R01GM123055) and the National Science Foundation (IOS1127027, DMS1614777) to DK; and grants from the Purdue Research Foundation (PRF-209104) and the Cancer Prevention Internship Program to AS.

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Authors and Affiliations

  1. From the Department of Biological Sciences, Purdue University, 915 W. State St., LILY G-227, West Lafayette, IN, 47907, USA

    Anwesha Sanyal, Erica A. Zbornik, Ben G. Watson, Daisuke Kihara & Seema Mattoo

  2. Department of Computer Science, Purdue University, West Lafayette, IN, 47907, USA

    Charles Christoffer & Daisuke Kihara

  3. Bindley Biosciences Center, Purdue University, West Lafayette, IN, 47907, USA

    Jia Ma

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  1. Anwesha Sanyal
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  2. Erica A. Zbornik
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  4. Charles Christoffer
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  5. Jia Ma
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  6. Daisuke Kihara
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  7. Seema Mattoo
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Contributions

S.M. and A.S. conceived and designed the study. A.S., E.A.Z., B.G.W., and C.C. conducted the experiments. J.M. trained and provided advice on the BLI experiments. D.K. supervised the molecular docking studies. S.M. supervised the overall study. S.M. and A.S. wrote the manuscript. All authors revised and agreed to the final version of the manuscript.

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Correspondence to Seema Mattoo.

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Sanyal, A., Zbornik, E.A., Watson, B.G. et al. Kinetic and structural parameters governing Fic-mediated adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78. Cell Stress and Chaperones 26, 639–656 (2021). https://doi.org/10.1007/s12192-021-01208-2

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  • DOI: https://doi.org/10.1007/s12192-021-01208-2

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