Structural basis for Mep2 ammonium transceptor activation by phosphorylation

Bert van den Berg, Anupama Chembath, Damien Jefferies, Arnaud Basle, Syma Khalid, Julian C Rutherford

Research output: Contribution to journalArticle

Abstract

Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

Original languageEnglish
Article number11337
JournalNature Communications
Volume7
DOIs
Publication statusPublished - 18 Apr 2016

Fingerprint

phosphorylation
Phosphorylation
Ammonium Compounds
Chemical activation
activation
transporter
Candida albicans
Crystal structure
saccharomyces
crystal structure
Fungal Proteins
Candida
Yeast
Saccharomyces cerevisiae
Conformations
Nitrogen
X-Rays
proteins
nitrogen
X rays

Bibliographical note

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Keywords

  • Amino Acid Sequence
  • Ammonium Compounds/chemistry
  • Candida albicans/genetics
  • Cation Transport Proteins/chemistry
  • Crystallography, X-Ray
  • Gene Expression Regulation, Fungal
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Nitrogen/metabolism
  • Phosphorylation
  • Protein Isoforms/chemistry
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae/genetics
  • Saccharomyces cerevisiae Proteins/chemistry
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Static Electricity
  • Substrate Specificity
  • Thermodynamics

Cite this

van den Berg, B., Chembath, A., Jefferies, D., Basle, A., Khalid, S., & Rutherford, J. C. (2016). Structural basis for Mep2 ammonium transceptor activation by phosphorylation. Nature Communications, 7, [11337]. https://doi.org/10.1038/ncomms11337
van den Berg, Bert ; Chembath, Anupama ; Jefferies, Damien ; Basle, Arnaud ; Khalid, Syma ; Rutherford, Julian C. / Structural basis for Mep2 ammonium transceptor activation by phosphorylation. In: Nature Communications. 2016 ; Vol. 7.
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Structural basis for Mep2 ammonium transceptor activation by phosphorylation. / van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C.

In: Nature Communications, Vol. 7, 11337, 18.04.2016.

Research output: Contribution to journalArticle

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AU - van den Berg, Bert

AU - Chembath, Anupama

AU - Jefferies, Damien

AU - Basle, Arnaud

AU - Khalid, Syma

AU - Rutherford, Julian C

N1 - This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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N2 - Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

AB - Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

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KW - Kinetics

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Nitrogen/metabolism

KW - Phosphorylation

KW - Protein Isoforms/chemistry

KW - Protein Structure, Secondary

KW - Protein Structure, Tertiary

KW - Saccharomyces cerevisiae/genetics

KW - Saccharomyces cerevisiae Proteins/chemistry

KW - Sequence Alignment

KW - Sequence Homology, Amino Acid

KW - Static Electricity

KW - Substrate Specificity

KW - Thermodynamics

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DO - 10.1038/ncomms11337

M3 - Article

VL - 7

JO - Nature Communications

JF - Nature Communications

SN - 2041-1723

M1 - 11337

ER -