Computational protocol: Tetrahydrodipicolinate N-Succinyltransferase and Dihydrodipicolinate Synthase from Pseudomonas aeruginosa: Structure Analysis and Gene Deletion

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[…] Crystallization screening was carried out using the vapor diffusion method and a Phoenix crystallisation robot. DapA crystals were grown in hanging drops containing 2 µl of protein solution (12.5 mg/ml) and 2 µl of reservoir solution (18% of PEG6000, 0.2 M MgCl2, 0.1 M TRIS-HCl, pH 7.6). The crystals used for diffraction analysis were cyo-protected by a short soak in a buffer identical to the mother liquor containing 30% PEG6000. Diffraction quality crystals of DapD were obtained by equilibrating a mixture of 2 µl of protein solution (26 mg/ml) and 2 µl of reservoir solution (19–20% of PEG3350, 0.3–0.4 M succinate, pH 6.2) against the reservoir solution in hanging drops. Single rod-shaped crystals appeared after 24–48 hours. These were flash-frozen in liquid nitrogen without addition of any cryoprotectant. The crystals of the CoA complex were produced by co-crystallization in conditions identical to that of the apo-protein by adding CoA (final concentration 10–15 mM) to the protein solution for 10 minutes prior to crystallization. The crystals were isomophous with those obtained for the apo-enzyme. Incubation of PaDapD with formyl-CoA for 15 minutes resulted in crystals that belong to the monoclinic space group (P21). These crystals diffracted reproducibly to higher resolution. The complexes with L-2-aminopimelate and D-2-aminopimelate were obtained by soaking the monoclinic crystals in crystallization liquor containing 0.4 M of either D- or L-2-aminopimelate instead of succinate.X-ray data to 2.95 Å for the DapD apo-enzyme were collected at the beamline ID14:4 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) at 110 K. The dataset for the binary complex DapD-CoA was collected at beamline ID14:1 (ESRF, Grenoble, France) at 110 K to 2.4 Å resolution. The X-ray data were processed and scaled with the programs MOSFLM and SCALA from the CCP4 suite . These crystals belong to the tetragonal space group P41212 with cell dimensions a = b = 122.4 Å, and c = 199.3 Å. A second dataset for the apo-enzyme was collected from the monoclinic crystal form at beamline I911-3 at MaxLab (Lund, Sweden) to 1.8 Å resolution. The diffraction datasets for the D- and the L-2-aminopimelate complexes were collected at MaxLab (Lund, Sweden) at the beamline I911-2 to 1.9 Å and at I911-3 to 2.5 Å resolution, respectively (). The datasets scaled well in the orthorhombic Laue group P222, but a more detailed analysis indicated crystal twinning. The true space group is P21, with a twinning operator (-h, -k, l). The twinning fractions vary between crystals from 0.35 to 0.43.A 1.6 Å diffraction dataset from a DapA crystal was collected at the beamline ID14:4 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) at 110 K. DapA crystals belong to the space group P1 with cell dimensions a = 43.1 Å, b = 51.7 Å, c = 140.3 Å, and α = 95.7°, β = 90.0°, γ = 113.3°. The statistics of the data set are given in . [...] The structure of PaDapD was determined initially by molecular replacement in space group P41212 using the program MOLREP . A polyalanine model of the trimer of the putative tetrahydropyridine-2-carboxylate N-succinyltransferase from Campylobacter jejuni (PDB code 2RIJ) with the cofactor and the solvent atoms omitted was used as the search model. The best solution had a score of 0.295 and an R-factor of 55.9%. In order to monitor the refinement process, 5% of the X-ray data were removed for the calculation of R-free. Initial cycles of restrained refinement using Refmac5 resulted in a drop of the R-factor by 15%. The correctness of the molecular replacement solution was confirmed by the appearance of electron density for the side chains specific for PaDapD. Tight NCS restraints were applied throughout refinement for the three subunits in the asymmetric unit.The structure of PaDapD in the monoclinic crystal form was determined by MOLREP using the refined coordinates for the PaDapD trimer from the tetragonal crystal form. The P21 crystals contain two trimers in the asymmetric unit. Since these crystals exhibited various fractions of twinning, twin refinement as implemented in Refmac5 was employed. NCS restraints were used throughout the refinement. Isotropic B-factor refinement was used in all cases, except for the PaDapD complex with L-2-aminopimelic acid. In this case TLS refinement was carried out in the final cycles, resulting in a decrease of Rfree by 1.5%.The structure of PaDapA was determined by molecular replacement using the program MOLREP , and the coordinates from Escherichia coli DapA (2OJP) as search model. The best solution contained four polypeptide chains in the P1 unit cell, with a MOLREP score of 0.401 and R-factor of 0.489. The program Arp/warp was used in the initial model building and refinement steps.Manual rebuilding of all models was carried out with the program Coot, based on sigma-A weighted 2Fo-Fc and Fo-Fc electron density maps . Examination and adjustment of the models were interspersed with rounds of refinement using Refmac5 . In the structures derived from the monoclinic crystal form, i.e apo-enzyme, and the binary complexes with L-2 and D-2-aminopimelic acid, respectively, the C-terminal part of the protein is disordered, and residues 330–344 were not modeled. Water molecules were added based on peak heights, shape of the electron density, temperature factor and capability to form hydrogen bonds with surrounding protein residues and/or other water molecules. Well defined difference electron density allowed modeling of CoA and succinate for the CoA-succinate complex of PaDapD. The final model of this complex contains three chains of DapD, amino acid residues 1–245 and 251–343, three CoA and succinate molecules and 147 water molecules. No electron density was observed for the loop comprising residues 246–250. The electron densities for the inhibitor/substrate molecules in the binary complexes are well defined in all six subunits of the asymmetric units, with the exception of subunit B of the DapD-L-2-AP complex. The difference electron density for a ligand in the active site of this subunit is not well defined, indicating low occupancy, and we therefore did not model any bound substrate.At the final stage anisotropic B-factors were used in the refinement of the DapA structure as implemented in Refmac5. The final model of PaDapA contains four polypeptide chains (chain A: residues 1–291, and chains B, C and D: residues 2–291), four chloride ions and 875 water molecules. Details of the refinement and protein models are given in . The crystallographic data have been deposited with the Protein Data Bank, accession codes 3R5A, 3R5B, 3R5C, 3R5D for PaDapD and 3QZE for the PaDapA.Protein structures were analyzed and validated through the MOLPROBITY server . Structural comparisons were carried out using the DALI algorithm molecular contacts and interacting surfaces were analyzed with the PISA server . Figures were made using the program Pymol (http://www.pymol.org). […]

Pipeline specifications

Software tools CCP4, Molrep, REFMAC5, ARP/wARP, Coot, MolProbity, PyMOL
Databases DAPD
Application Protein structure analysis
Organisms Pseudomonas aeruginosa, Mus musculus, Homo sapiens, Pseudomonas aeruginosa PAO1
Diseases Lung Diseases
Chemicals Diaminopimelic Acid, Succinic Acid