model of mechanism of action of the TB drug TMC207 European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o DESY, D-22603
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1 Variations of subunit of the Mycobacterium tuberculosis F-ATPsynthase and a novel model of mechanism of action of the TB drug TMC207 Goran Biuković 1, Sandip Basak 1, Malathy Sony Subramanian Manimekalai 1, Sankaranarayanan Rishikesan 1, Manfred Roessle 2, Thomas Dick 3, Srinivasa P. S. Rao 4, Cornelia Hunke 1 and Gerhard Grüber 1 * 1 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o DESY, D Hamburg, Germany 3 National University of Singapore, Yong Loo Lin School of Medicine, Department of Microbiology, 5 Science Drive 2, Singapore Novartis Institute for Tropical, Diseases Pte Ltd, 10 Biopolis Road, Singapore * Corresponding author CORRESPONDENCE: Corresponding author: Prof. Dr. Gerhard Grüber, School of Biological Sciences, Nanyang Technological University, Singapore , Phone: ; Fax: ; E- mail: ggrueber@ntu.edu.sg 1
2 MATERIALS AND METHODS Determination of binding of subunit to subunit by fluorescence correlation spectroscopy Fluorescence correlation spectroscopy (FCS) was performed on an LSM 510 Meta/ConfoCor 3 (Zeiss, Jena, Germany) using recombinant of the Bacillus subtilis PS3 F 1 F O ATP synthase and the subunit of the M. tuberculosis F 1 F O ATP synthase, which was fluorescently labeled by an Atto488 NHS ester (ATTO-TEC, Siegen, Germany). For the labelling procedure a buffer exchange was performed with subunit using a 10 kda Centricon to amine-free phosphate buffer (ph 8.0, 200 mm NaCl). An equimolar excess of reactive dye to protein solution was used for 8 minutes at 6 C. The access of deactivated, unbound fluorescence dye was removed by using a Superdex S 75 gel filtration column with subsequent washing steps in 10 kda Amicon Ultra-4 Centrifugal Filter Devices (Millipore, Billerica, MA, USA). The quality of the fluorescently labeled protein solution was verified by FCS as well as SDS PAGE (10% total acrylamide and 1.7% crosslinked acrylamide) with fluorescence detection ( ex : 488 nm) followed by Coomassie staining (Supplementary Fig. 2A, insert). For the FCS experiment 50 mm Tris/HCl (ph 8.0) buffer containing 200 mm NaCl was used. The temperature was adjusted to 25 C in an incubation chamber (Zeiss). The 488 nm laser line of an 30 mw Ar-Laser (458/477/488/514 nm) was focused into the aqueous solution by a water immersion objective (40 x / 1,2 W Korr UL-VIS-IR, Zeiss). FCS was measured in 15 μl droplets of recombinant produced, purified fluorescently labelled subunit and unlabelled, which were placed on Nunc 8 well chambered cover glass. Before usage, the glass surface was treated with an aqueous solution with 3 % gelatine, in order to prevent unspecific binding (1). The access was removed by washing steps with H 2 O. Following filter sets were used: MBS: HFT 488, EF2: LP 505, EF: None, DBS: Mirror. Out- 2
3 of-focus fluorescence was rejected by a 90 μm pinhole in the detection pathway. Solution of Rhodamine 6G (Rho6G) in water was used as references for the calibration of the confocal instrument. Variable concentrations of were mixed after addition of fluorescently labelled subunit. The droplets were incubated on the glass slip surface for about 3 min, and monitored during this time by FCS. The fluorescence autocorrelation functions were determined by measurements of at least 10 repetitions with 30 sec each. The calculations of the bound fractions and dissociation constants were done by the ConfoCor 3-software 4.2, Excel2003 and OriginPro 8 SR4. A 'Standard AC normalized triplet' model was used to determine the diffusion time of the fluorescently labelled protein. The determined value for the detected protein was fixed during the fitting of the titration experiments. A 'Standard AC 2 diffusion coefficients normalized triplet' model has been used (G (τ) = 1 + G t * G d ) for the subsequent titration experiments which takes two fluorescent components in consideration (Atto488- and Atto complex). This model comprises for the correlation function (G (τ) ) the triplet relaxation time (G t ) and triplet fraction (G d ). The calculations and the figures were done in Windows Excel in combination with OriginPro. To determine the binding constant a sigmoidal non-linear curve fit by a Hill equation was applied. References 1. Hunke C, Chen W-Y, Schäfer H-J, Grüber G Cloning, purification, and nucleotide-binding traits of the catalytic subunit A of the catalytic V 1 V O ATPase from Aedes albopictus. Prot. Expr. Purif. 53:
4 Figure legends: Figure S1. Purification of M. tuberculosis subunit (A) Following purification on Ni 2+ -NTA resin, the proteins were first applied onto anion exchange chromatography and then further purified by Superdex 75 column using buffer (50 mm Tris-HCl, ph 8.5, 200 mm NaCl, 10% glycerol) at a flow rate of 0.5 ml/min. Insert in figures show SDS gels after purification on Superdex 75. The fractions highlighted by an gray bar indicate the monomeric recombinant of Mt, while the fractions of the peaks at 9 ml and 10.5 ml include oligomeric forms of. (B) Circular dichroism (CD) spectroscopy of Mt. (C) SDS-Page of Mtε (lane 2) ε-t19a (lane 3) and ε-r37g (lane 3). Lane 1 shows a marker of individual proteins with molecular masses expressed in kda. (D) intrinsic tryptophan fluorescence titrations of the mutant protein MtεR37G in the absence and presence of TMC207. The identical spectra were observed for the mutant MtεT19A (not shown). Excitation was at 295 nm. Figure S2. FCS analysis of subunit -α 3 β 3 γ formation. (A) Normalized autocorrelation functions of Atto488 labelled Mtε by increasing the quantity of unlabeled α 3 β 3 γ. The inset shows the SDS PAGE of the used proteins. Lane 1 displays Atto488-subunit detected by fluorescence and in lane 2 by Coomassie dye. In lane 3 the unlabeled α 3 β 3 γ complex and a 1:10 diluted α 3 β 3 γ sample in lane 4 stained by Coomassie Brilliant Blue R. Lane 5 illustrates the protein standard. (B) Concentration dependent binding of to α 3 β 3 γ. Best fits yielding the binding constants are represented as fitted line by a non-linear, Hill curve fit. Figure S3. (A) CD spectrum of the C-terminal segment Mt Assignment of cross-peaks in the NOESY spectrum of Mt in the HA-HN (B) and HN-HN (C) region of the spectrum. Peak picking was done in Sparky 3.1 software and cross peaks were identified based on TOCSY and NOESY spectrum. 4
5 Figure S4. (A) Secondary structure prediction using Hα chemical shifts of by PREDITOR software. (B) The NOESY connectivity plot of peptide Figure S5. Nucleotide binding affinity measurements of Mtε (A, C) and the control protein subunit A of the A-ATP synthase (P. horikoshii OT3) (B, D) using ITC. The top panel in the figure shows the injection profile after baseline correction, and the bottom panels show the integration (heat release) for each injection (except for the first one). Continuous lines in the bottom panel reveal the fit of the data to a function based on a one-site binding model. Figure S6. 2D 1 H- 15 N-HSQC spectra of Mtε in the absence (red) and presence (green) of 2 equivalent of mefloquine/hcl. The HSQC spectra were acquired at 303 with 250 μm of protein dissolved in 50 mm sodium phosphate (ph 6.5) buffer containing 200 mm NaCl and 10% glycerol. No significant changes could be observed upon addition of the drug. 5
6 Figure S1A-B: (A) (B) (C) (D) 6
7 Figure S2A-B: (A) (B) 7
8 Figure S3A-C: (A) (B) (C) 8
9 Figure S4A-B: (A) (B) 9
10 Figure S5A-D: (A) Mtε + ATP + Mg (B) Subunit A + ATP + Mg (C) Mtε + ADP + Mg (D) Subunit A + ADP + Mg 10
11 Figure S6: 11
12 Table legend: Table S1. Structural statistics for Mt
13 Table S1. Structural statistics for Mt Total number of residues 18 Total number of NMR restraints 197 Intraresidual ( i j = 0) 84 Short range ( i j = 1) 80 Medium-range (2 i j 5) 33 Long-range ( i j 5) 0 Ramachandran plot statistics (%) Residues in most favoured regions 90.4 Residues in additionally allowed regions 9.6 Residues in generously allowed regions 0 Residues in disallowed region 0 Structural precision for well ordered region r.m.s.d. backbone (C α, C and N) (residues ) r.m.s.d. heavy atoms (residues ) Å Å 13
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