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
Abstract
:1. Introduction
2. Results and Discussion
2.1. Validation of the Protein Painting Assay
2.2. Mapping Protein–Protein Interaction in Cysteine Synthase
2.3. SAXS Analysis of CysK, CysE, and CS
3. Materials and Methods
3.1. Proteins Expression and Purification
3.2. Spectroscopy
3.3. Size Exclusion Chromatography
3.4. Protein Painting with Small Molecule Dyes
3.5. SAXS Measurements
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
AcCoA | acetyl coenzyme A |
Acid Fuchsin | 2-amino-5-[(4-amino-3-sulfophenyl)(4-imino-3-sulfo-2,5-cyclohexadien-1-ylidene)methyl]- 3-methyl-benzenesulfonic acid, sodium salt |
ACTH | adrenocorticotropic hormone |
ANSA | 8-Anilino-1-naphthalenesulfonic acid ammonium salt |
AO50 | sodium 4-(4-(benzyl-et-amino)-ph-azo)-2,5-di-cl-benzenesulfonate |
BSA | bovine serum albumin |
DTT | dithiothreitol |
EDTA | ethylenediaminetetraacetic acid |
Eosin B | 4’,5’-dibromo-3’,6’-dihydroxy-2’,7’-dinitro-spiro[isobenzofuran-1(3H),9’-[9H]xanthen]-3one |
CS | cysteine synthase |
CR | disodium 4-amino-3-[[4-[4-[(1-amino-4-sulfonatonaphthalen-2yl) diazenyl] phenyl] phenyl] diazenyl] naphthalene-1-sulfonate |
CysD | ATP sulfurylase |
CysE | serine acetyltransferase |
CysH | phosphoadenosine phosphosulphate reductase |
CysK | O-acetylserine sulfhydrylase isozyme A |
CysM | O-acetylserine sulfhydrylase isozyme B (O-phosphoserine sulfhydrylase) |
CysN | CysD-associated GTPase |
GAPDH | glyceraldehyde 3-phosphate dehydrogenase |
HCCA | α-cyano-4-hydroxycinnamic acid |
HPLC-SEC | high-pressure liquid chromatography-size exclusion chromatography |
IPGT | isopropyl β-D-1-thiogalactopyranoside |
MALDI TOF/TOF | matrix-assisted laser desorption ionization-time of flight/time of flight |
OAS | O-acetylserine |
NAS | N-acetylserine |
PDDF | pair distance distribution function |
PLP | pyridoxal 5′-phosphate |
PPIs | protein–protein interactions |
RBB | disodium 1-amino-9,10-dioxo-4-[3-(2-sulfonatooxyethylsulfonyl)anilino]anthracene-2-sulfonate |
SAXS | small angle X-ray scattering |
TCEP | tris(2-carboxyethyl)phosphine) |
TFA | trifluoroacetic acid |
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Parameter | CysK | CysE | CS |
---|---|---|---|
Guinier Analysis | |||
Guinier radius Rg (nm) | 2.64 ± 0.05 | 3.90 ± 0.04 | 6.10 ± 0.11 |
I(0) (cm−1) | 6.71 ± 0.02 | 19.08 ± 0.09 | 27.03 ± 0.23 |
q-range (nm−1) | 0.22–0.39 | 0.13–0.31 | 0.13–0.2 |
Quality (%) | 96 | 91 | 99 |
GNOM | |||
Max. dimension Dmax (nm) | 8.5 | 13.0 | 22.0 |
Guinier radius Rg (nm) | 2.623 ± 0.006 | 3.856 ± 0.009 | 6.606 ± 0.003 |
q-range (nm−1) | 0.1–3.5 | 0.1–3.5 | 0.1–3.5 |
Porod volume (kDa) | 72 | 189 | 303 |
CRYSOL | |||
PDB file | 1oas | Hexamer built from 1t3d | |
Guinier radius Rg (nm) | 2.572 | 3.361 | |
Molecular weight (kDa) | 67.57 | 171.6 | |
Max. dimension Dmax (nm) | 8.675 | 11.0 | |
DAMMIF (def. parameter, 10 runs) | |||
q range for fitting (nm−1) | 0.12–3.9 | 0.12–3.9 | 0.12–3.9 |
Symmetry, anisotropy assumption | P1, unknown | P1, unknown | P1, unknown |
χ2 range | 1.803–1.833 | 2.044–2.119 | 2.077–3.731 |
Resolution from SASRES (nm) | 4.6 ± 0.3 | 2.9 ± 0.2 | 9.4 ± 0.2 |
NSD (standard dev.), no clusters | 0.9 (0.12), 1 | 0.59 (0.01), 1 | 1.43(0.06), 2 |
MW estimate for proteins (kDa) | 58.6 | 155 | 300 |
Phase radius of gyration (nm) | 2.62 | 3.86 | 6.61 |
Maximum phase diameter (nm) | 10.500 | 14.800 | 24.300 |
SASBDB [83] codes | SASDGW6 | SASDGV6 | SASDGX6 |
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Rosa, B.; Marchetti, M.; Paredi, G.; Amenitsch, H.; Franko, N.; Benoni, R.; Giabbai, B.; De Marino, M.G.; Mozzarelli, A.; Ronda, L.; et al. 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. Int. J. Mol. Sci. 2019, 20, 5219. https://doi.org/10.3390/ijms20205219
Rosa B, Marchetti M, Paredi G, Amenitsch H, Franko N, Benoni R, Giabbai B, De Marino MG, Mozzarelli A, Ronda L, et al. 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. International Journal of Molecular Sciences. 2019; 20(20):5219. https://doi.org/10.3390/ijms20205219
Chicago/Turabian StyleRosa, Brenda, Marialaura Marchetti, Gianluca Paredi, Heinz Amenitsch, Nina Franko, Roberto Benoni, Barbara Giabbai, Maria Giovanna De Marino, Andrea Mozzarelli, Luca Ronda, and et al. 2019. "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" International Journal of Molecular Sciences 20, no. 20: 5219. https://doi.org/10.3390/ijms20205219