M

M., Smith B. targeted amino acidity mutagenesis indicate the fact that backbone amide of Glu224 and the medial side string of Arg233 type an oxyanion gap in sortase B that stabilizes high energy tetrahedral catalytic intermediates. Amazingly, an extremely conserved threonine residue inside the destined sorting sign substrate facilitates structure from the oxyanion gap by stabilizing the positioning of the energetic site arginine residue via hydrogen bonding. Molecular dynamics simulations and major sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily. are significantly attenuated in virulence, whereas transpeptidation measurements suggest that Arg233 and the backbone amide of Glu224 form an oxyanion hole that stabilizes high energy tetrahedral catalytic intermediates. Interestingly, a highly conserved threonine residue CD213a2 within the sorting signal actively participates in constructing the oxyanion hole by hydrogen bonding to the active site arginine residue. MD simulations of SrtA, as well as primary sequence conservation, suggest that all sortases will use a similar substrate-stabilized mechanism to anchor proteins to the cell wall or to assemble pili. EXPERIMENTAL PROCEDURES Production, Crystallization, and Structure Determination of SrtB-NPQT* Complex DNA encoding SrtB (residues 31C244) was amplified by PCR from genomic DNA, cloned into a pE-SUMO vector (LifeSensors) and transformed into Rosetta (DE3) pLysS cells (Novagen). Protein expression was induced by addition of 1 1 mm isopropyl -d-1-thiogalactopyranoside and allowed to continue for 16 h at 16 C. Protein was purified by affinity purification using HisPur cobalt resin (Thermo) per the manufacturer’s instructions. The His6-SUMO tag was then cleaved by incubating the protein overnight at 4 C with recombinant ULP1 protease and removed by reapplying the protein mixture to the HisPur cobalt resin. Cbz-NPQT* (where T* is (2transpeptidation reactions were performed based on the method developed by Kruger (43). 100 m SrtB (wild-type or mutant) was incubated with 2 mm GGGGG and 200 m peptide substrate in 100 l of assay buffer (300 mm Tris-HCl and 150 mm NaCl) at 37 C for 24 h. The reactions were quenched by adding 50 l of 1 1 m HCl and injected onto a Waters XSelect HSS C18 reversed phase HPLC column. Peptides were eluted by applying a gradient from 3 to 23% acetonitrile (in 0.1% trifluoroacetic acid) over 25 min at a flow rate of 1 1 ml/min. Elution of the peptides was monitored by absorbance at 215 nm. Peak fractions were collected, and their identities were confirmed by MALDI-TOF mass spectrometry. Computational Modeling and Molecular Dynamics Molecular dynamics simulations were performed with NAMD (44), using the AMBER99SB-ILDN force field (45), a 2-fs time step, and the SHAKE algorithm to constrain all hydrogen containing bonds (46). Nonbonded interactions were truncated at 10 ?, with the use of a smoothing function beginning at 9 ?, and long range electrostatics were handled with the particle mesh Ewald method using a maximum grid spacing of 1 1 ? and a cubic B spline (47). Parameters for the Cys-Thr linkage were generated with GAFF (48, 49), with the charges derived from a RESP fit (48). Constant temperature was maintained through the use of Langevin dynamics with a damping coefficient of 2 ps?1, whereas the barostat was controlled through a Nos-Hoover method with a target pressure of 1 Pazopanib (GW-786034) 1 atm, a piston period of 100 fs, and a damping time of 50 fs (50, 51). Models of the thioacyl intermediate were originally constructed from the SrtB-NPQT* structure by replacing the disulfide bond with a thioester in PyMOL (52). The models were solvated in a periodic water box with a solvent distance of 10 ? and parameterized in tLeap (53). Models were then energy-minimized and equilibrated in NAMD (44) by slowly removing restraints from the initial atom positions over 1 ns with 2-fs steps. For simulations of SrtA, the NMR structure 2KID was utilized (24). Potential of mean force calculations were performed using two-dimensional replica-exchange umbrella sampling calculations (54). For the first dimension (the coordinate in Fig. 6, axis in Fig. 6,.L. of computational modeling, molecular dynamics simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu224 and the side chain of Arg233 form an oxyanion hole in sortase B that stabilizes high energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. Molecular dynamics simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily. are significantly attenuated in virulence, whereas transpeptidation measurements suggest that Arg233 and the backbone amide of Glu224 form an oxyanion hole that stabilizes high energy tetrahedral catalytic intermediates. Interestingly, a highly conserved threonine residue within the sorting signal actively participates in constructing the oxyanion hole by hydrogen bonding to the active site arginine residue. MD simulations of Pazopanib (GW-786034) SrtA, as well as primary sequence conservation, suggest that all sortases will use a similar substrate-stabilized mechanism to anchor proteins to the cell wall or to assemble pili. EXPERIMENTAL PROCEDURES Production, Crystallization, and Structure Determination of SrtB-NPQT* Complex DNA encoding SrtB (residues 31C244) was amplified by PCR from genomic DNA, cloned into a pE-SUMO vector (LifeSensors) and transformed into Rosetta (DE3) pLysS cells (Novagen). Protein expression was induced by addition of 1 1 mm isopropyl -d-1-thiogalactopyranoside and allowed to continue for 16 h at 16 C. Protein was purified by affinity purification using HisPur cobalt resin (Thermo) per the manufacturer’s instructions. The His6-SUMO tag was then cleaved by incubating the Pazopanib (GW-786034) protein overnight at 4 C with recombinant ULP1 protease and removed by reapplying the protein mixture to the HisPur cobalt resin. Cbz-NPQT* (where T* is (2transpeptidation reactions were performed based on the method developed by Kruger (43). 100 m SrtB (wild-type or mutant) was incubated with 2 mm GGGGG and 200 m peptide substrate in 100 l of assay buffer (300 mm Tris-HCl and 150 mm NaCl) at 37 C for 24 h. The reactions were quenched by adding 50 l of 1 1 m HCl and injected onto a Waters XSelect HSS C18 reversed phase HPLC column. Peptides were eluted by applying a gradient from 3 to 23% acetonitrile (in 0.1% trifluoroacetic acid) over 25 min at a flow rate of 1 1 ml/min. Elution of the peptides was monitored by absorbance at 215 nm. Peak fractions were collected, and their identities were confirmed by MALDI-TOF mass spectrometry. Computational Modeling and Molecular Dynamics Molecular dynamics simulations were performed with NAMD (44), using the AMBER99SB-ILDN force field (45), a 2-fs time step, and the SHAKE algorithm to constrain all hydrogen containing bonds (46). Nonbonded interactions were truncated at 10 ?, with the use of a smoothing function beginning at 9 ?, and long range electrostatics were handled with the particle mesh Ewald method using a maximum grid spacing of 1 1 ? and a cubic B spline (47). Parameters for the Cys-Thr linkage were generated with GAFF (48, 49), with the charges derived from a RESP fit (48). Constant temperature was maintained through the use of Langevin dynamics with a damping coefficient of 2 ps?1, whereas the barostat was controlled through a Nos-Hoover method with a target pressure of 1 1 atm, a piston period of 100 fs, and a damping time of 50 fs (50, 51). Models of the thioacyl intermediate were originally constructed from the SrtB-NPQT* structure by replacing the disulfide bond with a thioester in PyMOL (52). The models were solvated in a periodic water box with a solvent distance of 10 ? and parameterized in tLeap (53). Models were then energy-minimized and equilibrated in NAMD (44) by slowly removing restraints from the initial atom positions over 1 ns with 2-fs steps. For simulations of SrtA, the NMR structure 2KID was utilized (24). Potential of mean force calculations were performed using two-dimensional replica-exchange umbrella sampling calculations (54). For the.