The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae

Jan Abendroth; Daniel D. Mitchell; Konstantin V. Korotkov; Tanya L. Johnson; Allison Kreger; Maria Sandkvist; Wim G.J. Hol

doi:10.1016/j.jsb.2009.03.009

The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae

Jan Abendroth, Daniel D. Mitchell, Konstantin V. Korotkov, Tanya L. Johnson, Allison Kreger, Maria Sandkvist, Wim G.J. Hol

Research output: Contribution to journal › Article › peer-review

47 Scopus citations

Abstract

The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix α6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology.

Original language	English
Pages (from-to)	303-315
Number of pages	13
Journal	Journal of Structural Biology
Volume	166
Issue number	3
DOIs	https://doi.org/10.1016/j.jsb.2009.03.009
State	Published - Jun 2009

Bibliographical note

Funding Information:
We acknowledge Stewart Turley for expert help with data collection and Michael Bagdasarian at Michigan State University for anti-EpsF antiserum. We thank the staff of beamline 9.2 at the Stanford Synchrotron Radiation Lightsource for support during data collection. This research was supported by NIH grant AI34501 to W.G.J.H. from the National Institute of Allergy and Infectious Diseases (NIAID) and by the Howard Hughes Medical Institute (HHMI) and by grant AI49294 from NIAID to M.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of the HHMI, NIAID or the National Institutes of Health.

Funding

We acknowledge Stewart Turley for expert help with data collection and Michael Bagdasarian at Michigan State University for anti-EpsF antiserum. We thank the staff of beamline 9.2 at the Stanford Synchrotron Radiation Lightsource for support during data collection. This research was supported by NIH grant AI34501 to W.G.J.H. from the National Institute of Allergy and Infectious Diseases (NIAID) and by the Howard Hughes Medical Institute (HHMI) and by grant AI49294 from NIAID to M.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of the HHMI, NIAID or the National Institutes of Health.

Funders	Funder number
National Institutes of Health (NIH)	AI34501
Howard Hughes Medical Institute
National Institute of Allergy and Infectious Diseases	R56AI049294

Keywords

General secretory pathway
GspF
PilG
Type 4 pilin biogenesis
XcpS

ASJC Scopus subject areas

Structural Biology

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10.1016/j.jsb.2009.03.009

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title = "The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae",

abstract = "The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix α6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology.",

keywords = "General secretory pathway, GspF, PilG, Type 4 pilin biogenesis, XcpS",

author = "Jan Abendroth and Mitchell, {Daniel D.} and Korotkov, {Konstantin V.} and Johnson, {Tanya L.} and Allison Kreger and Maria Sandkvist and Hol, {Wim G.J.}",

note = "Funding Information: We acknowledge Stewart Turley for expert help with data collection and Michael Bagdasarian at Michigan State University for anti-EpsF antiserum. We thank the staff of beamline 9.2 at the Stanford Synchrotron Radiation Lightsource for support during data collection. This research was supported by NIH grant AI34501 to W.G.J.H. from the National Institute of Allergy and Infectious Diseases (NIAID) and by the Howard Hughes Medical Institute (HHMI) and by grant AI49294 from NIAID to M.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of the HHMI, NIAID or the National Institutes of Health. ",

year = "2009",

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language = "English",

volume = "166",

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AU - Abendroth, Jan

AU - Mitchell, Daniel D.

AU - Korotkov, Konstantin V.

AU - Johnson, Tanya L.

AU - Kreger, Allison

AU - Sandkvist, Maria

AU - Hol, Wim G.J.

N1 - Funding Information: We acknowledge Stewart Turley for expert help with data collection and Michael Bagdasarian at Michigan State University for anti-EpsF antiserum. We thank the staff of beamline 9.2 at the Stanford Synchrotron Radiation Lightsource for support during data collection. This research was supported by NIH grant AI34501 to W.G.J.H. from the National Institute of Allergy and Infectious Diseases (NIAID) and by the Howard Hughes Medical Institute (HHMI) and by grant AI49294 from NIAID to M.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of the HHMI, NIAID or the National Institutes of Health.

PY - 2009/6

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N2 - The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix α6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology.

AB - The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix α6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology.

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JO - Journal of Structural Biology

JF - Journal of Structural Biology

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ER -

The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

Access to Document

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