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

doi:10.3390/ijms20205219

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.

Highlights

Four of these enzymes have been the target of significant medicinal chemistry efforts, either in enteric bacteria or in Mycobacterium tuberculosis: O-acetylserine sulfhydrylase isozymes A and B (CysK/CysM) [35,36,37,40,41], phosphoadenosine phosphosulphate reductase (CysH) [38], and serine acetyltransferase (CysE) [42]
The ability of protein painting to correctly identify the hot spots for complex formation between CysK and its binding partners was first tested on the CysK/CdiA-CT complex for which a three-dimensional structure is available
Cysteine biosynthesis is a putative target for enhancers of antibiotic therapy since cysteine-depleted bacteria exhibit a decreased fitness [17]

Summary

Introduction

Four of these enzymes have been the target of significant medicinal chemistry efforts, either in enteric bacteria or in Mycobacterium tuberculosis: O-acetylserine sulfhydrylase isozymes A and B (CysK/CysM) [35,36,37,40,41], phosphoadenosine phosphosulphate reductase (CysH) [38], and serine acetyltransferase (CysE) [42]. Complex stoichiometry is in principle consistent with two structural models (Figure 1B), one in which two C-terminal peptides of CysE bind the two opposite active sites of CysK dimer, and another in which only one CysK active site is occupied This latter model is better supported by data that show partial inhibition of CysK activity by CysE, even at saturating CysE concentrations [43,50,68]. The authors, in addition to the remarkable finding of a moonlighting function for CysK, proposed that the toxin forms a complex with CysK using the same structural motif used by CysE (i.e., insertion of the C-terminus in CysK active site). This work has the aim to gain structural information on the CS in solution and to connect this information with functional data to answer three questions: (i) What regions of CysK are involved in the protein–protein interaction? (ii) What is the geometry of CS, and which of the two possible binding modes of CysK is compatible with it? (iii) Does complex formation induce any long-range conformational changes in the protein that could account for an allosteric control of enzyme activity?

Methods

Results

Conclusion