Structures of the Insecticidal Toxin Complex Subunit XptA2 Highlight Roles for Flexible Domains

Cole L. Martin; David W. Chester; Christopher D. Radka; Lurong Pan; Zhengrong Yang; Rachel C. Hart; Elad M. Binshtein; Zhao Wang; Lisa Nagy; Lawrence J. DeLucas; Stephen G. Aller

doi:10.3390/ijms241713221

Structures of the Insecticidal Toxin Complex Subunit XptA2 Highlight Roles for Flexible Domains

Cole L. Martin, David W. Chester, Christopher D. Radka, Lurong Pan, Zhengrong Yang, Rachel C. Hart, Elad M. Binshtein, Zhao Wang, Lisa Nagy, Lawrence J. DeLucas, Stephen G. Aller

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

Abstract

The Toxin Complex (Tc) superfamily consists of toxin translocases that contribute to the targeting, delivery, and cytotoxicity of certain pathogenic Gram-negative bacteria. Membrane receptor targeting is driven by the A-subunit (TcA), which comprises IgG-like receptor binding domains (RBDs) at the surface. To better understand XptA2, an insect specific TcA secreted by the symbiont X. nematophilus from the intestine of entomopathogenic nematodes, we determined structures by X-ray crystallography and cryo-EM. Contrary to a previous report, XptA2 is pentameric. RBD-B exhibits an indentation from crystal packing that indicates loose association with the shell and a hotspot for possible receptor binding or a trigger for conformational dynamics. A two-fragment XptA2 lacking an intact linker achieved the folded pre-pore state like wild type (wt), revealing no requirement of the linker for protein folding. The linker is disordered in all structures, and we propose it plays a role in dynamics downstream of the initial pre-pore state.

Original language	English
Article number	13221
Journal	International Journal of Molecular Sciences
Volume	24
Issue number	17
DOIs	https://doi.org/10.3390/ijms241713221
State	Published - Sep 2023

Bibliographical note

Funding Information:
This research was supported by a UAB competitive COVID-19 relief award and start-up funds to S.G.A.

Funding Information:
CryoEM data was collected at the Baylor College of Medicine CryoEM ATC, which includes equipment purchased under the support of CPRIT Core Facility Award RP190602. EM data collection from the FEI Polara was conducted at the Center for Structural Biology Cryo-EM Facility at Vanderbilt University. The XptA2 2-fragment data was collected in the UAB cryo-EM core, and the DSF data was collected in the UAB X-ray Crystallography Core Facility supported by the Comprehensive Cancer Center Support Grant (NIH grant # 2P30CA013148-49). C.D.R. is supported by the National Institute of Health, United States, grant 1K99AI166116. We thank Joel Sheets for the initial purification of XptA2 for crystallization. This work is based on C.L.M.’s dissertation submitted to fulfill, in part, the requirements for the degree of Doctor of Philosophy at the University of Alabama at Birmingham.

Publisher Copyright:
© 2023 by the authors.

Keywords

Cryo-EM
TcA
X-ray crystallography
toxin translocase

ASJC Scopus subject areas

Catalysis
Molecular Biology
Spectroscopy
Computer Science Applications
Physical and Theoretical Chemistry
Organic Chemistry
Inorganic Chemistry

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10.3390/ijms241713221

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title = "Structures of the Insecticidal Toxin Complex Subunit XptA2 Highlight Roles for Flexible Domains",

abstract = "The Toxin Complex (Tc) superfamily consists of toxin translocases that contribute to the targeting, delivery, and cytotoxicity of certain pathogenic Gram-negative bacteria. Membrane receptor targeting is driven by the A-subunit (TcA), which comprises IgG-like receptor binding domains (RBDs) at the surface. To better understand XptA2, an insect specific TcA secreted by the symbiont X. nematophilus from the intestine of entomopathogenic nematodes, we determined structures by X-ray crystallography and cryo-EM. Contrary to a previous report, XptA2 is pentameric. RBD-B exhibits an indentation from crystal packing that indicates loose association with the shell and a hotspot for possible receptor binding or a trigger for conformational dynamics. A two-fragment XptA2 lacking an intact linker achieved the folded pre-pore state like wild type (wt), revealing no requirement of the linker for protein folding. The linker is disordered in all structures, and we propose it plays a role in dynamics downstream of the initial pre-pore state.",

keywords = "Cryo-EM, TcA, X-ray crystallography, toxin translocase",

author = "Martin, {Cole L.} and Chester, {David W.} and Radka, {Christopher D.} and Lurong Pan and Zhengrong Yang and Hart, {Rachel C.} and Binshtein, {Elad M.} and Zhao Wang and Lisa Nagy and DeLucas, {Lawrence J.} and Aller, {Stephen G.}",

note = "Funding Information: This research was supported by a UAB competitive COVID-19 relief award and start-up funds to S.G.A. Funding Information: CryoEM data was collected at the Baylor College of Medicine CryoEM ATC, which includes equipment purchased under the support of CPRIT Core Facility Award RP190602. EM data collection from the FEI Polara was conducted at the Center for Structural Biology Cryo-EM Facility at Vanderbilt University. The XptA2 2-fragment data was collected in the UAB cryo-EM core, and the DSF data was collected in the UAB X-ray Crystallography Core Facility supported by the Comprehensive Cancer Center Support Grant (NIH grant # 2P30CA013148-49). C.D.R. is supported by the National Institute of Health, United States, grant 1K99AI166116. We thank Joel Sheets for the initial purification of XptA2 for crystallization. This work is based on C.L.M.{\textquoteright}s dissertation submitted to fulfill, in part, the requirements for the degree of Doctor of Philosophy at the University of Alabama at Birmingham. Publisher Copyright: {\textcopyright} 2023 by the authors.",

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AU - Martin, Cole L.

AU - Chester, David W.

AU - Radka, Christopher D.

AU - Pan, Lurong

AU - Yang, Zhengrong

AU - Hart, Rachel C.

AU - Binshtein, Elad M.

AU - Wang, Zhao

AU - Nagy, Lisa

AU - DeLucas, Lawrence J.

AU - Aller, Stephen G.

N1 - Funding Information: This research was supported by a UAB competitive COVID-19 relief award and start-up funds to S.G.A. Funding Information: CryoEM data was collected at the Baylor College of Medicine CryoEM ATC, which includes equipment purchased under the support of CPRIT Core Facility Award RP190602. EM data collection from the FEI Polara was conducted at the Center for Structural Biology Cryo-EM Facility at Vanderbilt University. The XptA2 2-fragment data was collected in the UAB cryo-EM core, and the DSF data was collected in the UAB X-ray Crystallography Core Facility supported by the Comprehensive Cancer Center Support Grant (NIH grant # 2P30CA013148-49). C.D.R. is supported by the National Institute of Health, United States, grant 1K99AI166116. We thank Joel Sheets for the initial purification of XptA2 for crystallization. This work is based on C.L.M.’s dissertation submitted to fulfill, in part, the requirements for the degree of Doctor of Philosophy at the University of Alabama at Birmingham. Publisher Copyright: © 2023 by the authors.

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N2 - The Toxin Complex (Tc) superfamily consists of toxin translocases that contribute to the targeting, delivery, and cytotoxicity of certain pathogenic Gram-negative bacteria. Membrane receptor targeting is driven by the A-subunit (TcA), which comprises IgG-like receptor binding domains (RBDs) at the surface. To better understand XptA2, an insect specific TcA secreted by the symbiont X. nematophilus from the intestine of entomopathogenic nematodes, we determined structures by X-ray crystallography and cryo-EM. Contrary to a previous report, XptA2 is pentameric. RBD-B exhibits an indentation from crystal packing that indicates loose association with the shell and a hotspot for possible receptor binding or a trigger for conformational dynamics. A two-fragment XptA2 lacking an intact linker achieved the folded pre-pore state like wild type (wt), revealing no requirement of the linker for protein folding. The linker is disordered in all structures, and we propose it plays a role in dynamics downstream of the initial pre-pore state.

AB - The Toxin Complex (Tc) superfamily consists of toxin translocases that contribute to the targeting, delivery, and cytotoxicity of certain pathogenic Gram-negative bacteria. Membrane receptor targeting is driven by the A-subunit (TcA), which comprises IgG-like receptor binding domains (RBDs) at the surface. To better understand XptA2, an insect specific TcA secreted by the symbiont X. nematophilus from the intestine of entomopathogenic nematodes, we determined structures by X-ray crystallography and cryo-EM. Contrary to a previous report, XptA2 is pentameric. RBD-B exhibits an indentation from crystal packing that indicates loose association with the shell and a hotspot for possible receptor binding or a trigger for conformational dynamics. A two-fragment XptA2 lacking an intact linker achieved the folded pre-pore state like wild type (wt), revealing no requirement of the linker for protein folding. The linker is disordered in all structures, and we propose it plays a role in dynamics downstream of the initial pre-pore state.

KW - Cryo-EM

KW - TcA

KW - X-ray crystallography

KW - toxin translocase

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SN - 1661-6596

VL - 24

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

IS - 17

M1 - 13221

ER -

Structures of the Insecticidal Toxin Complex Subunit XptA2 Highlight Roles for Flexible Domains

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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