Computational protocol: The Crystal Structure and Small-Angle X-Ray Analysis of CsdL/TcdA Reveal a New tRNA Binding Motif in the MoeB/E1 Superfamily

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Protocol publication

[…] Crystals of TcdA (full length, as a C-terminal hexa-histidine fusion) in complex with ATP or as obtained directly from the cell (AMP) were grown at 5 mg/ml TcdA in presence of 0.05 M potassium phosphate and 10% PEG1000 at basic pH at 20°C. The construct of TcdA and the crystallization conditions used in this study are different to those used in a recent crystallization report []. Two X-ray diffraction data sets were collected from crystals of the ATP complex, one to a maximum resolution of 1.77 Å at a wavelength of 0.97948 Å, and a second data set to 2.35-Å resolution at a long wavelength (1.99976 Å) for sulfur single anomalous diffraction (SAD), at the BL13-XALOC beamline (ALBA, Barcelona, Spain). A data set for the AMP complex was collected to 1.89-Å resolution at the PROXIMA 2A beamline (Synchrotron SOLEIL, Paris, France). All the data sets were integrated with XDS [] and scaled with Aimless [] from the CCP4 suite of programs [] ().The structure of TcdA in complex with ATP was solved at 1.77-Å resolution using MR-SAD by first placing residues 21–199 of MoeB (PDB 1JWA) [] with PHASER [] using the high-resolution data set, and then using the partial model and the anomalous signal of sulfur, phosphorus (from ATP) and potassium measured on the long wavelength data set to phase the full length structure. The complete structure of TcdA could be built into the minimally biased electron density maps calculated from MR-SAD phases; the only exception is a solvent exposed loop spanning residues 217–236 above the ATP binding pocket. The structure of the AMP complex was solved by MR using the ATP complex as search model. Both structures were built, refined and validated with Coot [], phenix.refine [] and MolProbity []. [...] The dissociation constant for the TcdA-tRNALys(UUU) binding interaction was obtained by following the quenching of the intrinsinc fluorescence of tryptophan (2) and phenylalanine (8) residues in TcdA upon tRNALys(UUU) addition. All fluorescence spectroscopy experiments (100 μl) were conducted in 20 mM sodium/potassium phosphate, pH 7.4, 300 mM NaCl, at 25°C using a Varioskan (Thermo Fisher) instrument, in black 96-well plates, setting the excitation and emission wavelengths to 280 nm and 340 nm, respectively. TcdA variants at 5 μM were challenged with increasing concentrations of tRNA (0.1–10 μM) in triplicate. Primary inner-filter effects due to tRNA absorption at the excitation wavelength were corrected for by standard methods (secondary inner filter effects for tRNA at 340 nm are negligible) and collisional induced quenching (as opposed to binding) was ruled out as the mechanism for the quenching of TcdA intrinsic fluoresence []. The reduction in fluorescence signal that accompanied tRNA addition was analyzed by non-linear regression methods in SigmaPlot v12, and the dissociation constant, K D, and the Hill coefficient were calculated according to the following equation: F0−FF−Fc=LnKD+Ln where F0 is the intrinsic fluorescence of TcdA in the absence of ligand, Fc is the minimal residual fluorescence of the TcdA-tRNALys(UUU) complex and L is the tRNALys(UUU) concentration. [...] SAXS experiments were performed at the BM29 BioSAXS beamline at the ESRF (Grenoble, France) []. SAXS data from purified TcdA were collected using a batch setup at three concentrations between 0.5–5 mg/ml, with ten successive time frames and 20 s exposures. For TcdA-tRNALys(UUU), SAXS data (1 s per frame) were collected from two identical experiments using an online size-exclusion chromatography (SEC) setup [] after injecting 100 μl of 8-mg/ml complex on a Superdex 200 Increase column (GE Healthcare). Since the complex was prepared in presence of a molar excess of the tRNALys(UUU) component, this step allowed to obtain scattering data from both the protein-tRNA complex and the excess tRNALys(UUU) on a single experiment. All SAXS measurements were performed at 5°C in 20 mM sodium/potassium phosphate buffer, pH 7.4, 300 mM NaCl, 2 mM β-mercaptoethanol. Data were recorded using a 1 M PILATUS detector (DECTRIS) at a sample-to-detector distance of 2.7 m and a wavelength of 1.5 Å, covering the range of momentum transfer 0.020 < s < 0.5 Å-1. Data from the batch setup (TcdA) or from the two equivalent peaks from the replicated SEC-SAXS measurements [TcdA-tRNALys(UUU) and tRNA] were averaged, buffer subtracted and merged using the procedures outlined in Round et al. []. The radius of gyration (Rg) was evaluated using the Guinier approximation [] and also from the entire scattering curve using Porod’s law [], and the pair-distance distribution function P(r) was calculated using GNOM []. Guinier plots of all SAXS data support monodispersity of the analyzed samples (.). The measured scattering curves were compared with the theoretical scattering curves of the macromolecular models using CRYSOL []. Ab initio shape restoration was performed using 10–20 independent runs of DAMMIF [] followed by DAMAVER [] to create the final ab initio shape. ScÅtter was used to calculate the molecular weights of the macromolecules and for the calculation of the fitting parameter R SAS, the small-angle scattering invariant VC and the parameter QR []. The summary of SAXS statistics is given in . […]

Pipeline specifications

Software tools XDS, CCP4, Coot, PHENIX, MolProbity, SigmaPlot, ATSAS, CRYSOL, DAMMIF
Applications Miscellaneous, Small-angle scattering, Protein structure analysis
Organisms Escherichia coli, Dipturus trachyderma, Saccharomyces cerevisiae
Chemicals Adenosine Triphosphate, Ampicillin, Cysteine, Sodium, Sulfur