Structural evidence for the covalent modification of FabH by 4,5-dichloro-1,2-dithiol-3-one (HR45)† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ob01396e

Mass spectrometry and modelling shows the antimicrobial inhibitor 4,5-dichloro-1,2-dithiol-3-one (HR45) acts by forming a covalent adduct with the target β-ketoacyl-ACP synthase III (FabH). The 5-chloro substituent directs attack of the essential active site thiol (C112) via a Michael type addition elimination reaction mechanism.

The structure of HR45 was minimised using B3LYP/6-311G*. The LUMO and LUMO+1 orbitals were degenerate in energy. The LUMO+1 is shown below and is consistent with a substrate that would undergo Michael addition at C5. According to frontier molecular orbital theory, the shape of this orbital should approximate the localisation of the additional pair of electrons following nucleophilic attack of the S atom of Cys112 on HR45. The largest orbital coefficient (lobe) associated with a single atom is located over C5, consistent with the site that would favour formation of a new σ-bond (i.e. between C5 of HR45 and the S atom of Cys112).

S6
Model of HR45 small molecule crystal structure docked into saFabH protein crystal structure (PDB:3IL7) The X-ray structure of HR45 was obtained from the CCDC (CCDC Number: 1517056) 1 . The coordinates for a Cysteine-HR45 C5-SG adduct were produced using JLigand 2 ; and the adduct was then manually docked into the crystal structure of saFabH to replace Cys112 (PDBID: 3IL7) 3 and adjusted to minimise steric clashes and to maximize hydrogen bonding potential using coot 4 . The resulting model was then energy minimized using refmac5 5 . Model images were produced using PyMol (Schrödinger LLC).

Mass Spectrometry Data and Methods
Purified tryptic peptide samples were analysed by direct infusion electrospray ionisation using a TriVersa nanomate (Advion) coupled to a Bruker 12T SolariX with a 10cm infinity cell.
Spectra were acquired in positive polarity over 100-2000m/z with a 2megaword data size. 20 spectra were summed, each with a 50 ms accumulation time. Pepsin digests were analysed by C18 LC-MS/MS over a 50 min gradient from 5% to 45% acetonitrile/0.1% formic acid. MS spectra were acquired on an LTQ Orbitrap in positive polarity over a 333-1800 m/z range using a 371.10124 lockmass. MS scans were acquired at 60k resolution followed by 6 data depended ion trap MSMS spectra, or at 30k resolution followed by 4 7500 resolution Orbitrap MSMS spectra. In additional analyses, candidate modified peptides were fragmented in the Orbitrap, as above, but using a data independent inclusion list. MSMS spectra were converted to mgf format and peptides assigned using the Mascot search engine (www.matrixscience.com).   To an aliquot of saFabH (2.5 mL, 0.4 mg/mL) in non-reducing buffer (20 mM Tris-HCl pH 7.6, 300 mM NaCl, 10% glycerol) was added HCl (100 µL, final conc. 0.04 N). Lyophilised pepsin (Promega) was resuspended in ddH2O (1 mg/mL, pH 5.5), and added (25 µL, 1:40 w:w) to the acidified protein aliquot and incubated overnight at 37 °C with shaking at 300 rpm. The reaction was stopped by heating to 95 °C for 10 minutes. The peptic digest was then desalted S12 into MeCN:H2O (2:1, 0.1% formic acid, 200 µL) using a C18 SPE cartridge and used immediately or lyophilised and stored at -20 °C.

Expression of saFabH
The saFabH/pET-HISTEV construct (4 μL) was transformed into an aliquot (50 μL) of BL21(DE3) (New England BioLabs) cells and set on ice for 25 minutes. The cells were then heat shocked at 42 °C for 40 seconds and set back on ice for a further 2 minutes. SOC media (100 μL) was added and the mixture was agitated at 37 °C for 1 hour. The mixture was spread on LB agar (30 μg.mL -1 kanamycin) and incubated overnight at 37 °C. A single transformant was used to inoculate two seed cultures of sterile LB broth (2 x 250 mL, 30 μg.mL -1 kanamycin) and agitated overnight at 37 °C. One of the overnight seed cultures was used to sub-culture sterile LB broth (5 x 500 mL, 30 μg.mL -1 kanamycin) to an OD600 of 0.1. The cultures were agitated at 37 °C until the OD600 reached 0.6, at which point expression was induced by addition of IPTG (final conc. 0.1 mM). Cells were harvested by centrifugation (30 minutes at 5000 rpm) after a further 3 hours at 30 °C and subsequently stored at -20 °C.
Purification of saFabH N-terminal histidine-tagged saFabH was purified at 4 °C by Ni-affinity chromatography followed by size exclusion chromatography. The BL21 (DE3) cell pellet expressing FabH was resuspended in lysis buffer (30 mL, 20 mM Tris-HCl pH 7.6, 300 mM NaCl, 5 mM imidazole) and lysed for 15 minutes with rounds of 30 second of sonication followed by 30 seconds of rest. The cell lysate was clarified by centrifugation (18,000 g, 30 minutes, 4 °C) and the cellfree extract was injected onto a HisTrap 5 mL (GE Healthcare) Ni 2+ -affinity chromatography column pre-equilibrated in lysis buffer. The column was washed with lysis buffer (5 CV) before the histidine-tagged protein was eluted using a gradient (0-100%) of lysis buffer to elution buffer (20 mM Tris-HCl pH 7.6, 300 mM NaCl, 400 mM imidazole) over 20 CV.
Each elution fraction was analysed by SDS-PAGE, and the fractions containing His-tagged saFabH were pooled, and the protein concentration was determined by a Bradford assay (Thermo Fisher). In order to remove the N-terminal histidine tag, a 5 mL aliquot of histidinetagged saFabH was combined with an aliquot of TEV protease (1:5 w/w) and dialysed against size exclusion mobile phase buffer (500 mL, 20 mM Tris-HCl pH 7.6, 300 mM NaCl, 10% S13 glycerol) at 4 °C for 16 h using 8 kDa MWCO dialysis tubing. The reaction mixture was then injected onto a HisTrap 5 mL (GE) Ni 2+ -affinity chromatography column pre-equilibrated in lysis buffer (30 mL, 20 mM Tris-HCl pH 7.6, 300 mM NaCl, 5 mM imidazole). The flow-through was collected and concentrated to a volume of 2 mL using a 30 kDa MWCO spin-filter.
The concentrated non-histidine-tagged saFabH sample was further purified by size exclusion chromatography (HiLoad Superdex 200 16/60, GE Healthcare) with an isocratic elution of mobile phase buffer (500 mL, 20 mM Tris-HCl pH 7.6, 100 mM NaCl, 10% glycerol) at 1 mL.min -1 over 120 minutes. The saFabH eluted at approximately 70 minutes and the most concentrated fractions were pooled and flash cooled in 500 μL aliquots in liquid nitrogen before storage at -80 °C.

Expression of TEV protease
The TEV/GST fusion construct (4 μL) was transformed into an aliquot (50 μL) of BL21 (DE3) Rosetta Gami (Millipore) cells and set on ice for 25 minutes. The cells were then heat shocked at 42 °C for 40 seconds and set back on ice for a further 2 minutes. SOC media (100 μL) was added and the mixture was agitated at 37 °C for 1 hour. The mixture was spread on LB agar (30 μg.mL -1 kanamycin, 25 μg.mL -1 chloramphenicol) and incubated overnight at 37 °C. A single transformant was used to inoculate two seed cultures of sterile LB broth (2 x 250 mL, 30 μg.mL -1 kanamycin, 25 μg.mL -1 chloramphenicol) and agitated overnight at 37 °C. One of the overnight seed cultures was used to sub-culture sterile LB broth (5 x 500 mL, 30 μg.mL -1 kanamycin, 25 μg.mL -1 chloramphenicol) to an OD600 of 0.1. The cultures were agitated at 37 °C until the OD600 reached 0.6, at which point the temperature was reduced to 20°C and expression was induced by addition of IPTG (final conc. 0.4 mM). Cells were harvested after a further 16 h at 20 °C and subsequently stored at -20 °C.
Purification of TEV protease N-terminal histidine-tagged TEV protease was purified at 4 °C by Ni-affinity chromatography followed by size exclusion chromatography. The BL21 (DE3) Rosetta Gami cell pellet expressing FabH was resuspended in lysis buffer (PBS, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, 1 mM benzamidine) and lysed for 15 minutes with rounds of 30 second of sonication followed by 30 seconds of rest. Excess cell matter was removed by centrifugation (18,000 g, S14 30 minutes, 4 °C) and the supernatant was injected onto a HisTrap 5 mL (GE) Ni 2+ -affinity chromatography column pre-equilibrated in lysis buffer. The column was washed with lysis buffer (5 CV) before the histidine-tagged protein was eluted using a gradient (0-100%) of lysis buffer to elution buffer (PBS, 300 mM NaCl, 500 mM imidazole, 1 mM PMSF, 1 mM benzamidine) over 20 CV.
Each elution fraction was analysed by SDS-PAGE, and the fractions containing His-tagged TEV protease were pooled and dialysed against self-cleavage buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 1 mM PMSF) at 4 °C for 2 h using 8 kDa MWCO dialysis tubing in the absence of benzamidine to allow self-cleavage of the GST solubility-tag domain. The reaction was concentrated to 2 mL using a 30 kDa MWCO spin filter and further purified by size exclusion chromatography (HiLoad Superdex 200 16/60) with an isocratic elution of mobile phase buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol) at 1 mL.min -1 over 120 minutes.
TEV protease eluted at approx. 80 minutes and the most concentrated fractions were pooled, and dialysed against storage buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 50% glycerol). TEV protease was flash cooled in 500 μL aliquots in liquid nitrogen and stored at -80 °C.