Computational protocol: Evidence That the C-Terminal Domain of a TypeB PutA Protein Contributes to Aldehyde Dehydrogenase Activity andSubstrate Channeling

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Protocol publication

[…] All steady-state assays were performed at 23 °C. Two PRODH assays were used. First, the PRODH kinetic constants for wild-type RcPutA were determined using proline and CoQ1 as the substrates as described previously. Second, the PRODH activities of wild-type RcPutA, RcPutA variants, and BjPutA variants were measured using dichlorophenolindophenol (DCPIP) as the terminal electron acceptor and phenazine methosulfate as the mediator (proline/DCPIP oxidoreductase assay) as previously described.Km and kcat for proline were determined using PutA (0.085–0.09 μM) and proline (0–300 mM) while fixing the DCPIP concentration at 75 μM. The assays described above were conducted in 20 mM Tris buffer (pH 8.0, 10% glycerol). P5CDH activity was measured in 50 mM potassium phosphate (pH 7.5, 600 mM NaCl) as previously described using PutA enzyme (0.17–0.19 μM) and DL-P5C (0–5.5 mM) with the NAD+ concentration fixed at 200 μM. The progress of the reaction was monitored by following NADH formation at 340 nm (ε340 = 6.2 mM–1 cm–1). Data were collected using a path length of 0.15 cm on a Hi-Tech Scientific SF-61DX2 stopped-flow instrument. Assays were performed in triplicate, and values for Km and kcat were estimated by fitting initial velocities to the Michaelis–Menten equation (SigmaPlot version 12.0).The PRODH–P5CDH coupled activity, in which proline is converted to glutamate, was measured as described previously. Briefly, PutA enzyme (0.17–0.19 μM) was mixed with 200 μM CoQ1, 40 mM proline, and 200 μM NAD+ in 50 mM potassium phosphate (pH 7.5, 600 mM NaCl). The progress of the reaction was followed by NAD+ reduction at 340 nm (ε340 = 6200 M–1 cm–1). The transient time was estimated by fitting the linear portion of the product concentration progress curve to a line and extrapolating to the x-axis. [...] Homology models of RcPutA domains were used in SAXS rigid body modeling. The models were obtained from the following servers using default settings: the SWISS-MODEL Workspace server, MODELER via the HHPred server of MPI Bioinformatics Toolkit, the Phyre2 server, and the I-TASSER server.All four servers identified BjPutA (PDB entry 3HAZ) as the best template for modeling the catalytic core (RcPutA residues 1–972). The level of sequence identity between BjPutA and RcPutA in this region is 52% (Figure S1 of the ). The four models are very similar; the pairwise rmsds for Cα atoms span the range of 0.5–0.7 Å. A representative model of the catalytic core is shown in Figure S2A of the .The four servers were also used to calculate models of the CTDUF (residues 994–1097). All four servers identified BjPutA residues 622–756 as the template (27% identical). This region of BjPutA corresponds to the oligomerization hairpin and Rossmann dinucleotide-binding domain (Figure A). The pairwise rmsds of the four models span the range of 0.7–2.3 Å. A representative model of the CTDUF is shown in Figure S2B of the .A model of the conserved C-terminal motif of RcPutA (residues 1108–1119) was built using MODELER. The other servers did not produce models because of the short sequence length. The template structure consisted of BjPutA residues 978–989. This region of BjPutA corresponds to a β-strand followed by a turn of α-helix (Figure A). The sequence identity of the modeled region is 5 of 12 residues (Figure S1 of the ). The model of the RcPutA C-terminal motif is shown in Figure S2B of the . [...] Prior to SAXS data collection, purified RcPutA was subjected to size exclusion chromatography using a Superdex 200 column. The column buffer consisted of 50 mM Tris, 5% glycerol, 0.5 mM THP, and 50 mM NaCl (pH 7.8). The fractions were pooled, concentrated to ∼8.5 mg/mL, and dialyzed at 4 °C for 24 h against 50 mM Tris, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM THP, and 5% glycerol (pH 7.8). The dialysate was reserved for use as the SAXS reference.SAXS experiments were performed at SIBYLS beamline 12.3.1 of the Advanced Light Source through the mail-in program., For each sample, scattering intensities were measured at three nominal protein concentrations. Data were collected for each protein concentration at exposure times of 0.5, 1.0, 3.0, and 6.0 s. The scattering curves collected from the protein samples were corrected for background scattering using intensity data collected from the reference buffer.The SAXS data were analyzed as follows. Composite scattering curves were generated with PRIMUS by scaling and merging the background-corrected low-q region data from the 0.5 or 1.0 s exposure with the high-q region data from the 3.0 s exposure. PRIMUS was also used to perform Guinier analysis. GNOM was used to calculate pair distribution functions. GASBOR was used to calculate shape reconstructions. Fifty independent models were generated with GASBOR using a maximal particle dimension (Dmax) of 107 Å and no symmetry constraints. DAMAVER was used to average and filter the GASBOR models. Situs module pdb2vol was used to convert the averaged, filtered models into volumetric maps. SUPCOMB was used to superimpose dummy atom models. CRYSOL was used to calculate theoretical SAXS curves from atomic models. The molecular mass in solution was determined from SAXS data using the volume of correlation invariant as implemented previously and Porod–Debye analysis. [...] CORAL (COmplexes with RAndom Loops) of the ATSAS package was used to determine the structural relationship between the catalytic core and the CTDUF of RcPutA. The default settings of CORAL were used for all calculations. Three sets of rigid body calculations, denoted as CORAL set 1, CORAL set 2, and CORAL decoy set, were performed as follows.For CORAL set 1, the catalytic core model (residues 1–972) was held fixed, residues 973–993 were modeled as a string of dummy residues, and the CTDUF model (residues 994–1097) was treated as a movable, rigid body. Each of the four models of the catalytic core was combined with each of the four models of the CTDUF for a total of 16 pairs. For every pair, 10 independent simulated annealing optimization calculations were performed, each starting from a different random number seed. Thus, a total of 160 CORAL poses were generated. The starting configuration for these calculations is shown in Figure S2C of the .A second set of calculations (CORAL set 2) was performed in which the model of the conserved C-terminal motif (residues 1108–1119) was combined with the CTDUF models using structural similarity to the oligomerization flap of BjPutA. The resulting composite model was considered to be a single, movable rigid body in these calculations. Four such composite models were made by adding the conserved C-terminal motif model to each of the four CTDUF models. One of these composite models is shown in Figure S2B of the . These four composite models were paired with the four catalytic core models, and 30 CORAL calculations were performed for each pair to generate a total of 480 poses. Two different starting configurations were used for these calculations to ensure that the initial arrangement of domains did not bias the results (Figure S2C,D of the ).The CORAL decoy set was generated to validate the results of rigid body modeling. These calculations were performed using decoy structures in place of the CTDUF model. Four decoy structures were used: profilin IB (PDB entry 1ACF, 125 residues), ketosteroid isomerase (PDB entry 3SED, 126 residues), a VH domain (PDB entry 1T2J, 116 residues), and human bromodomain (PDB entry 3HMF, 118 residues). These structures are shown in Figure S3 of the . Each decoy domain was paired with each of the four models of the RcPutA catalytic core, and 20 CORAL calculations were performed for each pair for a total of 320 decoy poses. […]

Pipeline specifications

Software tools SigmaPlot, SWISS-MODEL, HHPred, MPI Bioinformatics Toolkit, Phyre, I-TASSER, ATSAS, GASBOR, CRYSOL, CORAL
Applications Miscellaneous, Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Rhodobacter capsulatus
Chemicals Acetaldehyde, Glutamic Acid