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

Brenda Rosa, Marialaura Marchetti, Gianluca Paredi, Heinz Amenitsch, Nina Franko, Roberto Benoni, Barbara Giabbai, Maria Giovanna De Marino, Andrea Mozzarelli, Luca Ronda, Paola Storici, Barbara Campanini, Stefano Bettati

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.

Original languageEnglish
Article number5219
JournalInternational Journal of Molecular Sciences
Volume20
Issue number20
DOIs
Publication statusPublished - 1 Oct 2019

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Keywords

  • Cysteine biosynthesis
  • Cysteine synthase complex
  • O-acetylserine sulfhydrylase
  • Protein painting
  • SAXS
  • Serine acetyltransferase

ASJC Scopus subject areas

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

Fields of Expertise

  • Human- & Biotechnology

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