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Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action

The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltran...

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Autores principales: Rosa, Brenda, Marchetti, Marialaura, Paredi, Gianluca, Amenitsch, Heinz, Franko, Nina, Benoni, Roberto, Giabbai, Barbara, De Marino, Maria Giovanna, Mozzarelli, Andrea, Ronda, Luca, Storici, Paola, Campanini, Barbara, Bettati, Stefano
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829319/
https://www.ncbi.nlm.nih.gov/pubmed/31640223
http://dx.doi.org/10.3390/ijms20205219
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author Rosa, Brenda
Marchetti, Marialaura
Paredi, Gianluca
Amenitsch, Heinz
Franko, Nina
Benoni, Roberto
Giabbai, Barbara
De Marino, Maria Giovanna
Mozzarelli, Andrea
Ronda, Luca
Storici, Paola
Campanini, Barbara
Bettati, Stefano
author_facet Rosa, Brenda
Marchetti, Marialaura
Paredi, Gianluca
Amenitsch, Heinz
Franko, Nina
Benoni, Roberto
Giabbai, Barbara
De Marino, Maria Giovanna
Mozzarelli, Andrea
Ronda, Luca
Storici, Paola
Campanini, Barbara
Bettati, Stefano
author_sort Rosa, Brenda
collection PubMed
description The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein–protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS.
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spelling pubmed-68293192019-11-18 Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action Rosa, Brenda Marchetti, Marialaura Paredi, Gianluca Amenitsch, Heinz Franko, Nina Benoni, Roberto Giabbai, Barbara De Marino, Maria Giovanna Mozzarelli, Andrea Ronda, Luca Storici, Paola Campanini, Barbara Bettati, Stefano Int J Mol Sci Article The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein–protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS. MDPI 2019-10-21 /pmc/articles/PMC6829319/ /pubmed/31640223 http://dx.doi.org/10.3390/ijms20205219 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Rosa, Brenda
Marchetti, Marialaura
Paredi, Gianluca
Amenitsch, Heinz
Franko, Nina
Benoni, Roberto
Giabbai, Barbara
De Marino, Maria Giovanna
Mozzarelli, Andrea
Ronda, Luca
Storici, Paola
Campanini, Barbara
Bettati, Stefano
Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action
title Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action
title_full Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action
title_fullStr Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action
title_full_unstemmed Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action
title_short Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action
title_sort combination of saxs and protein painting discloses the three-dimensional organization of the bacterial cysteine synthase complex, a potential target for enhancers of antibiotic action
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829319/
https://www.ncbi.nlm.nih.gov/pubmed/31640223
http://dx.doi.org/10.3390/ijms20205219
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