Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems

Anne T. Tuukkanen; Diana Freire; Sum Chan; Mark A. Arbing; Robert W. Reed; Timothy J. Evans; Grasilda Zenkeviciutė; Jennifer Kim; Sara Kahng; Michael R. Sawaya; Catherine T. Chaton; Matthias Wilmanns; David Eisenberg; Annabel H.A. Parret; Konstantin V. Korotkov

doi:10.1016/j.jmb.2018.11.003

Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems

Anne T. Tuukkanen, Diana Freire, Sum Chan, Mark A. Arbing, Robert W. Reed, Timothy J. Evans, Grasilda Zenkeviciutė, Jennifer Kim, Sara Kahng, Michael R. Sawaya, Catherine T. Chaton, Matthias Wilmanns, David Eisenberg, Annabel H.A. Parret, Konstantin V. Korotkov

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

20 Scopus citations

Abstract

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE–PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG₁ chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG₁ adopts a quasi 2-fold symmetric structure that consists of a central β-sheet and two α-helical bundles. In addition, we describe the structures of EspG₃ chaperones from four different crystal forms. Alternate conformations of the putative PE–PPE binding site are revealed by comparison of the available EspG₃ structures. Analysis of EspG₁, EspG₃, and EspG₅ chaperones using small-angle X-ray scattering reveals that EspG₁ and EspG₃ chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG₃–PE5–PPE4 complex based on the small-angle X-ray scattering analysis.

Original language	English
Pages (from-to)	289-307
Number of pages	19
Journal	Journal of Molecular Biology
Volume	431
Issue number	2
DOIs	https://doi.org/10.1016/j.jmb.2018.11.003
State	Published - Jan 18 2019

Bibliographical note

Publisher Copyright:
© 2018 Elsevier Ltd

Funding

We thank Marcel Behr for providing M. kansasii genomic DNA, Wilbert Bitter for providing M. marinum genomic DNA, and Carlo Carolis for the espG 3mtu construct. We thank the staff of the UCLA-DOE Institute Protein Expression Technology Center, supported by the US Department of Energy, Office of Biological and Environmental Research (BER) program under Award Number DE-FC02-02ER63421, and the UCLA Crystallization Core for assistance in protein purification and crystallization screening. The authors thank staff members of the Northeastern Collaborative Access Team and Southeast Regional Collaborative Access Team at the Advanced Photon Source, Argonne National Laboratory for assistance during data collection. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. We acknowledge the Sample Preparation and Characterization facility of EMBL at PETRA3 (DESY, Hamburg) for technical support. Work performed in the laboratory of D.E. is supported by the Howard Hughes Medical Institute and National Institutes of Health grants 23616-002-06 F3:02 , TBSGC P01AI068135 , and TBSGC P01AI095208 . Research reported in this publication was partially supported by an Institutional Development Award from the National Institute of General Medical Sciences of the National Institutes of Health under grant numbers P20GM103486 and P30GM110787 , and by the National Institute of Allergy and Infectious Diseases grant number R01AI119022 to K.V.K. A.T.T. was supported by the EMBL EIPOD program under Marie Curie COFUND actions and by the Bundesministerium für Bildung and Forschung project BIOSCAT (grant 05K12YE1 ). We thank Marcel Behr for providing M. kansasii genomic DNA, Wilbert Bitter for providing M. marinum genomic DNA, and Carlo Carolis for the espG3mtu construct. We thank the staff of the UCLA-DOE Institute Protein Expression Technology Center, supported by the US Department of Energy, Office of Biological and Environmental Research (BER) program under Award Number DE-FC02-02ER63421, and the UCLA Crystallization Core for assistance in protein purification and crystallization screening. The authors thank staff members of the Northeastern Collaborative Access Team and Southeast Regional Collaborative Access Team at the Advanced Photon Source, Argonne National Laboratory for assistance during data collection. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. We acknowledge the Sample Preparation and Characterization facility of EMBL at PETRA3 (DESY, Hamburg) for technical support. Work performed in the laboratory of D.E. is supported by the Howard Hughes Medical Institute and National Institutes of Health grants 23616-002-06?F3:02, TBSGC P01AI068135, and TBSGC P01AI095208. Research reported in this publication was partially supported by an Institutional Development Award from the National Institute of General Medical Sciences of the National Institutes of Health under grant numbers P20GM103486 and P30GM110787, and by the National Institute of Allergy and Infectious Diseases grant number R01AI119022 to K.V.K. A.T.T. was supported by the EMBL EIPOD program under Marie Curie COFUND actions and by the Bundesministerium f?r Bildung and Forschung project BIOSCAT (grant 05K12YE1).

Funders	Funder number
Office of Basic Energy Sciences
Office of Biological and Environmental Research
Southeast Regional Collaborative Access Team at the Advanced Photon Source
TBSGC
US Department of Energy
National Institutes of Health (NIH)	23616-002-06, 23616-002-06 F3:02
National Institutes of Health (NIH)
Howard Hughes Medical Institute
U.S. Department of Energy EPSCoR
National Institute of General Medical Sciences	P30GM110787, P20GM103486
National Institute of General Medical Sciences
National Institute of Allergy and Infectious Diseases	P01AI095208, P01AI068135, R01AI119022
National Institute of Allergy and Infectious Diseases
Office of Science Programs
DOE Basic Energy Sciences	W-31-109-Eng-38
DOE Basic Energy Sciences
Biological and Environmental Research	DE-FC02-02ER63421
Biological and Environmental Research
Argonne National Laboratory
University of California, Los Angeles
European Molecular Biology Laboratory	05K12YE1
European Molecular Biology Laboratory
Bundesministerium für Bildung und Frauen

Keywords

Mycobacterium tuberculosis
PE–PPE proteins
protein export
small-angle X-ray scattering

ASJC Scopus subject areas

Biophysics
Structural Biology
Molecular Biology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.jmb.2018.11.003

Cite this

Tuukkanen, A. T., Freire, D., Chan, S., Arbing, M. A., Reed, R. W., Evans, T. J., Zenkeviciutė, G., Kim, J., Kahng, S., Sawaya, M. R., Chaton, C. T., Wilmanns, M., Eisenberg, D., Parret, A. H. A., & Korotkov, K. V. (2019). Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems. Journal of Molecular Biology, 431(2), 289-307. https://doi.org/10.1016/j.jmb.2018.11.003

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title = "Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems",

abstract = "Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE–PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central β-sheet and two α-helical bundles. In addition, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE–PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3, and EspG5 chaperones using small-angle X-ray scattering reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3–PE5–PPE4 complex based on the small-angle X-ray scattering analysis.",

keywords = "Mycobacterium tuberculosis, PE–PPE proteins, protein export, small-angle X-ray scattering",

author = "Tuukkanen, \{Anne T.\} and Diana Freire and Sum Chan and Arbing, \{Mark A.\} and Reed, \{Robert W.\} and Evans, \{Timothy J.\} and Grasilda Zenkeviciutė and Jennifer Kim and Sara Kahng and Sawaya, \{Michael R.\} and Chaton, \{Catherine T.\} and Matthias Wilmanns and David Eisenberg and Parret, \{Annabel H.A.\} and Korotkov, \{Konstantin V.\}",

note = "Publisher Copyright: {\textcopyright} 2018 Elsevier Ltd",

year = "2019",

month = jan,

day = "18",

doi = "10.1016/j.jmb.2018.11.003",

language = "English",

volume = "431",

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Tuukkanen, AT, Freire, D, Chan, S, Arbing, MA, Reed, RW, Evans, TJ, Zenkeviciutė, G, Kim, J, Kahng, S, Sawaya, MR, Chaton, CT, Wilmanns, M, Eisenberg, D, Parret, AHA & Korotkov, KV 2019, 'Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems', Journal of Molecular Biology, vol. 431, no. 2, pp. 289-307. https://doi.org/10.1016/j.jmb.2018.11.003

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T1 - Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems

AU - Tuukkanen, Anne T.

AU - Freire, Diana

AU - Chan, Sum

AU - Arbing, Mark A.

AU - Reed, Robert W.

AU - Evans, Timothy J.

AU - Zenkeviciutė, Grasilda

AU - Kim, Jennifer

AU - Kahng, Sara

AU - Sawaya, Michael R.

AU - Chaton, Catherine T.

AU - Wilmanns, Matthias

AU - Eisenberg, David

AU - Parret, Annabel H.A.

AU - Korotkov, Konstantin V.

PY - 2019/1/18

Y1 - 2019/1/18

N2 - Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE–PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central β-sheet and two α-helical bundles. In addition, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE–PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3, and EspG5 chaperones using small-angle X-ray scattering reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3–PE5–PPE4 complex based on the small-angle X-ray scattering analysis.

AB - Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE–PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central β-sheet and two α-helical bundles. In addition, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE–PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3, and EspG5 chaperones using small-angle X-ray scattering reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3–PE5–PPE4 complex based on the small-angle X-ray scattering analysis.

KW - Mycobacterium tuberculosis

KW - PE–PPE proteins

KW - protein export

KW - small-angle X-ray scattering

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DO - 10.1016/j.jmb.2018.11.003

M3 - Article

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AN - SCOPUS:85057352843

SN - 0022-2836

VL - 431

SP - 289

EP - 307

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

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ER -

Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

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