FisMat2017 - Submission - View

Abstract's title: STRUCTURAL CHARACTERIZATION OF CYSTEINE SYNTHASE ENZYMATIC COMPLEX BY PROTEIN PAINTING AND SAXS
Submitting author: Stefano Bettati
Affiliation: University of Parma
Affiliation Address: Department of Medicine and Surgery, Parco Area delle Scienze 23/A, 43124 Parma
Country: Italy
Oral presentation/Poster (Author's request): Oral presentation
Other authors and affiliations: Heinz Amenitsch (Institute of Inorganic Chemistry, Graz University of Technology, Stremayerg 6/IV, 8010 Graz, Austria), Barbara Campanini (Department of Food and Drug Science, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy), Maria Giovanna De Marino (SITEIA.PARMA Interdepartmental Center, University of Parma, Parco Area delle Scienze 181/A, 43124 Parma, Italy), Nicola Demitri (Elettra-Sincrotrone Trieste, S.S. 14 km 163.5 in Area Science Park, Basovizza, Trieste, Italy), Barbara Giabbai (Elettra-Sincrotrone Trieste, S.S. 14 km 163.5 in Area Science Park, Basovizza, Trieste, Italy), Marialaura Marchetti (Biopharmanet-TEC Interdepartmental Center, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy), Andrea Mozzarelli (Department of Food and Drug Science, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy), Gianluca Paredi (SITEIA.PARMA Interdepartmental Center, University of Parma, Parco Area delle Scienze 181/A, 43124 Parma, Italy), Luca Ronda (Department of Medicine and Surgery, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy), Brenda Rosa (Department of Medicine and Surgery, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy), Paola Storici (Elettra-Sincrotrone Trieste, S.S. 14 km 163.5 in Area Science Park, Basovizza, Trieste, Italy)
Abstract

As a precursor of many sulfur-containing reducing molecules and cofactors, cysteine is a key intermediate in many processes related to bacterial pathogenicity and virulence. The last two steps of cysteine biosynthesis in bacteria are catalyzed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK), respectively. CysE and CysK may form a complex, cysteine synthase (CS), containing two CysK homodimers and a CysE hexamer. Despite a relatively vast literature concerning complex stoichiometry and mechanism of formation, the three-dimensional structure of CS has not been determined yet. This key missing information limits the understanding of the exact regulatory role of complex formation, and its validation as a druggable target in designing innovative antibiotics. To gain insight into CysE-CysK protein-protein interaction and the overall geometry of CS complex, we exploited two complementary techniques: Small Angle X-ray Scattering (SAXS) and protein painting(1). SAXS probes protein shape with a spatial resolution of the order of 15 Å. Protein painting is an innovative technique that takes advantage of the interaction between dye molecules and solvent-exposed positively charged amino-acid side chains to disclose, following proteolysis and mass spectrometry analysis of peptides, surface regions that are involved in protein-protein interactions.

The results are consistent with a CS model where CysK engages in binding to CysE the same regions that account for interactions with other partners in distinct complexes (e.g. that with a toxin involved in contact dependent growth inhibition of bacteria). Of the two equivalent monomers of CysK interacting with a CysE trimer, only one is involved in binding, the other exposing to solvent the active site region.

 

(1) Luchini, A., Espina, V. & Liotta, L.A. Protein painting reveals solvent-excluded drug targets hidden within native protein–protein interfaces. Nature Communications 5, 4413 (2014).