TY - JOUR
T1 - 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
AU - Rosa, Brenda
AU - Marchetti, Marialaura
AU - Paredi, Gianluca
AU - Amenitsch, Heinz
AU - Franko, Nina
AU - Benoni, Roberto
AU - Giabbai, Barbara
AU - De Marino, Maria Giovanna
AU - Mozzarelli, Andrea
AU - Ronda, Luca
AU - Storici, Paola
AU - Campanini, Barbara
AU - Bettati, Stefano
PY - 2019/10/1
Y1 - 2019/10/1
N2 - 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.
AB - 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.
KW - Cysteine biosynthesis
KW - Cysteine synthase complex
KW - O-acetylserine sulfhydrylase
KW - Protein painting
KW - SAXS
KW - Serine acetyltransferase
UR - http://www.scopus.com/inward/record.url?scp=85073757152&partnerID=8YFLogxK
U2 - 10.3390/ijms20205219
DO - 10.3390/ijms20205219
M3 - Article
C2 - 31640223
AN - SCOPUS:85073757152
SN - 1661-6596
VL - 20
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 20
M1 - 5219
ER -