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).