Cyclic di-AMP traps proton-coupled K<sup>+</sup> transporters of the KUP family in an inward-occluded conformation.

MF Fuss; JP Wieferig; RA Corey; Y Hellmich; I Tascón; JS Sousa; PJ Stansfeld; J Vonck; Inga Hänelt

doi:10.1038/s41467-023-38944-1

Cyclic di-AMP traps proton-coupled K<sup>+</sup> transporters of the KUP family in an inward-occluded conformation.

MF Fuss, JP Wieferig, RA Corey, Y Hellmich, I Tascón, JS Sousa, PJ Stansfeld, J Vonck, Inga Hänelt^*

^*Corresponding author for this work

School of Physiology, Pharmacology & Neuroscience

Research output: Contribution to journal › Article (Academic Journal) › peer-review

11 Citations (Scopus)

Abstract

Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K+/H+ symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K+ concentrations, further K+ accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation.

Original language	English
Article number	3683
Journal	Nature Communications
Volume	14
Issue number	1
DOIs	https://doi.org/10.1038/s41467-023-38944-1
Publication status	Published - 21 Jun 2023

Bibliographical note

Funding Information:
We thank the Central Electron Microscopy Facility of the Max Planck Institute of Biophysics for cryo-EM infrastructure and technical support, and Juan Castillo-Hernández and Özkan Yildiz for support in cryo-EM data processing. This work was supported by the German Research Foundation via SPP1879 to J.V. and I.H. (VO 1449/1-1 and HA 6322/4-1), and the Heisenberg programme to I.H. (HA 6322/5-1). Research in PJS’s lab is also funded by Wellcome (208361/Z/17/Z) and BBSRC (BB/P01948X/1, BB/R002517/1 and BB/S003339/1). This project made use of time on ARCHER2 and JADE2 granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim ( http://hecbiosim.ac.uk ), supported by EPSRC (grant no. EP/R029407/1). This project also used Athena and Sulis at HPC Midlands+, which were funded by the EPSRC on grants EP/P020232/1 and EP/T022108/1. We thank the University of Warwick Scientific Computing Research Technology Platform for computational access.

Funding Information:
We thank the Central Electron Microscopy Facility of the Max Planck Institute of Biophysics for cryo-EM infrastructure and technical support, and Juan Castillo-Hernández and Özkan Yildiz for support in cryo-EM data processing. This work was supported by the German Research Foundation via SPP1879 to J.V. and I.H. (VO 1449/1-1 and HA 6322/4-1), and the Heisenberg programme to I.H. (HA 6322/5-1). Research in PJS’s lab is also funded by Wellcome (208361/Z/17/Z) and BBSRC (BB/P01948X/1, BB/R002517/1 and BB/S003339/1). This project made use of time on ARCHER2 and JADE2 granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (http://hecbiosim.ac.uk), supported by EPSRC (grant no. EP/R029407/1). This project also used Athena and Sulis at HPC Midlands+, which were funded by the EPSRC on grants EP/P020232/1 and EP/T022108/1. We thank the University of Warwick Scientific Computing Research Technology Platform for computational access.

Publisher Copyright:
© 2023, The Author(s).

Keywords

Ion Transport
membrane protein
structural biology
molecular dynamics (MD) simulations

Access to Document

10.1038/s41467-023-38944-1Licence: CC BY

Handle.net

Persistent link

Cite this

@article{2157e091b53e40b3ad32b58101014472,

title = "Cyclic di-AMP traps proton-coupled K+ transporters of the KUP family in an inward-occluded conformation.",

abstract = "Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K+/H+ symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K+ concentrations, further K+ accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation.",

keywords = "Ion Transport, membrane protein, structural biology, molecular dynamics (MD) simulations",

author = "MF Fuss and JP Wieferig and RA Corey and Y Hellmich and I Tasc{\'o}n and JS Sousa and PJ Stansfeld and J Vonck and Inga H{\"a}nelt",

note = "Funding Information: We thank the Central Electron Microscopy Facility of the Max Planck Institute of Biophysics for cryo-EM infrastructure and technical support, and Juan Castillo-Hern{\'a}ndez and {\"O}zkan Yildiz for support in cryo-EM data processing. This work was supported by the German Research Foundation via SPP1879 to J.V. and I.H. (VO 1449/1-1 and HA 6322/4-1), and the Heisenberg programme to I.H. (HA 6322/5-1). Research in PJS{\textquoteright}s lab is also funded by Wellcome (208361/Z/17/Z) and BBSRC (BB/P01948X/1, BB/R002517/1 and BB/S003339/1). This project made use of time on ARCHER2 and JADE2 granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim ( http://hecbiosim.ac.uk ), supported by EPSRC (grant no. EP/R029407/1). This project also used Athena and Sulis at HPC Midlands+, which were funded by the EPSRC on grants EP/P020232/1 and EP/T022108/1. We thank the University of Warwick Scientific Computing Research Technology Platform for computational access. Funding Information: We thank the Central Electron Microscopy Facility of the Max Planck Institute of Biophysics for cryo-EM infrastructure and technical support, and Juan Castillo-Hern{\'a}ndez and {\"O}zkan Yildiz for support in cryo-EM data processing. This work was supported by the German Research Foundation via SPP1879 to J.V. and I.H. (VO 1449/1-1 and HA 6322/4-1), and the Heisenberg programme to I.H. (HA 6322/5-1). Research in PJS{\textquoteright}s lab is also funded by Wellcome (208361/Z/17/Z) and BBSRC (BB/P01948X/1, BB/R002517/1 and BB/S003339/1). This project made use of time on ARCHER2 and JADE2 granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (http://hecbiosim.ac.uk), supported by EPSRC (grant no. EP/R029407/1). This project also used Athena and Sulis at HPC Midlands+, which were funded by the EPSRC on grants EP/P020232/1 and EP/T022108/1. We thank the University of Warwick Scientific Computing Research Technology Platform for computational access. Publisher Copyright: {\textcopyright} 2023, The Author(s).",

year = "2023",

month = jun,

day = "21",

doi = "10.1038/s41467-023-38944-1",

language = "English",

volume = "14",

journal = "Nature Communications",

issn = "2041-1723",

publisher = "Springer Nature",

number = "1",

}

TY - JOUR

T1 - Cyclic di-AMP traps proton-coupled K+ transporters of the KUP family in an inward-occluded conformation.

AU - Fuss, MF

AU - Wieferig, JP

AU - Corey, RA

AU - Hellmich, Y

AU - Tascón, I

AU - Sousa, JS

AU - Stansfeld, PJ

AU - Vonck, J

AU - Hänelt, Inga

N1 - Funding Information: We thank the Central Electron Microscopy Facility of the Max Planck Institute of Biophysics for cryo-EM infrastructure and technical support, and Juan Castillo-Hernández and Özkan Yildiz for support in cryo-EM data processing. This work was supported by the German Research Foundation via SPP1879 to J.V. and I.H. (VO 1449/1-1 and HA 6322/4-1), and the Heisenberg programme to I.H. (HA 6322/5-1). Research in PJS’s lab is also funded by Wellcome (208361/Z/17/Z) and BBSRC (BB/P01948X/1, BB/R002517/1 and BB/S003339/1). This project made use of time on ARCHER2 and JADE2 granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim ( http://hecbiosim.ac.uk ), supported by EPSRC (grant no. EP/R029407/1). This project also used Athena and Sulis at HPC Midlands+, which were funded by the EPSRC on grants EP/P020232/1 and EP/T022108/1. We thank the University of Warwick Scientific Computing Research Technology Platform for computational access. Funding Information: We thank the Central Electron Microscopy Facility of the Max Planck Institute of Biophysics for cryo-EM infrastructure and technical support, and Juan Castillo-Hernández and Özkan Yildiz for support in cryo-EM data processing. This work was supported by the German Research Foundation via SPP1879 to J.V. and I.H. (VO 1449/1-1 and HA 6322/4-1), and the Heisenberg programme to I.H. (HA 6322/5-1). Research in PJS’s lab is also funded by Wellcome (208361/Z/17/Z) and BBSRC (BB/P01948X/1, BB/R002517/1 and BB/S003339/1). This project made use of time on ARCHER2 and JADE2 granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (http://hecbiosim.ac.uk), supported by EPSRC (grant no. EP/R029407/1). This project also used Athena and Sulis at HPC Midlands+, which were funded by the EPSRC on grants EP/P020232/1 and EP/T022108/1. We thank the University of Warwick Scientific Computing Research Technology Platform for computational access. Publisher Copyright: © 2023, The Author(s).

PY - 2023/6/21

Y1 - 2023/6/21

N2 - Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K+/H+ symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K+ concentrations, further K+ accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation.

AB - Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K+/H+ symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K+ concentrations, further K+ accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation.

KW - Ion Transport

KW - membrane protein

KW - structural biology

KW - molecular dynamics (MD) simulations

UR - http://europepmc.org/abstract/med/37344476

U2 - 10.1038/s41467-023-38944-1

DO - 10.1038/s41467-023-38944-1

M3 - Article (Academic Journal)

C2 - 37344476

SN - 2041-1723

VL - 14

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 3683

ER -

Cyclic di-AMP traps proton-coupled K<sup>+</sup> transporters of the KUP family in an inward-occluded conformation.

Abstract

Bibliographical note

Keywords

Access to Document

Handle.net

Other files and links

Fingerprint

Cite this