Computational protocol: Mechanistic and Evolutionary Insights from the Reciprocal Promiscuity of Two Pyridoxal Phosphate-dependent Enzymes*

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Protocol publication

[…] Immediately prior to crystallization attempts with ALR(Y274F), a 20-ml spin concentrator with nominal molecular mass cutoff of 50 kDa (GE Healthcare) was used to exchange the protein into a buffer consisting of 20 mm Tris·HCl (pH 8.0) and 100 μm PLP. Protein was crystallized by vapor diffusion using the hanging drop method at a temperature of 291 K and with a reservoir solution (500 μl) consisting of 1.6 m ammonium sulfate, 100 mm HEPES (pH 7.0). A 2:1 ratio of protein to crystallization buffer yielded yellow-colored hexagonal plate crystals that appeared overnight and grew to approximate dimensions of 0.4 × 0.4 × 0.1 mm over 7 days. After a week, crystals were supplemented with 10 mm PLP for 3 days to ensure complete cofactor incorporation.Prior to data collection, an inhibited ALR(Y274F) structure was produced by soaking pre-formed crystals with 10 mm l-Ala-P. Inhibitor soaking was for 30 min, before a brief soak in crystallization buffer supplemented with 30% glycerol, and then flash-cooling in liquid nitrogen. Data were collected to 2.25 Å for the inhibitor-treated crystal and to 1.86 Å for an untreated crystal on a Rigaku MicroMax-007 HR rotating anode instrument equipped with a MAResearch Mar345dtb detector and Oxford Cryosystem Cobra cooling system. Data were processed using XDS () and scaled using SCALA (). The structures were solved by molecular replacement using the wild type E. coli ALR structure (PDB 2RJG) and the program PHASER () and were modeled using COOT (). The structures were refined using REFMAC5 () to R-factors of 17.5% (uninhibited) and 16.7% (inhibited), and free R values of 20.4 and 20.2%, respectively. The apparent space group of these crystals was P622, but subsequently the true space group was determined as P6, with twin refinement in REFMAC5 improving the refinement statistics markedly. The final models were assessed by Ramachandran plot and geometrical analysis using both the MOLPROBITY server () and the PDB validation server. PLP and l-Ala-P-PLP aldimine molecules were modeled into discrete and unambiguous electron density, and sulfate and water molecules were added in the final rounds of refinement. The non-inhibited structure contains four protein molecules (two dimers), four PLPs, five sulfates, three glycerols, and 467 water molecules. The inhibited structure contains four protein molecules, three l-Ala-P-PLP aldimines, eight sulfates, and 236 water molecules. Active site electron density is shown in the supplemental Fig. S1, and data collection and refinement statistics are in supplemental Table S2.The CBL(P113S) protein was treated similarly to ALR(Y274F), apart from the following specific details. Buffer exchange prior to crystallization used 5 mm HEPES (pH 7.5) supplemented with 10 μm PLP. Protein was finally concentrated to 7.5 mg·ml−1 and crystallized by vapor diffusion using the hanging drop method and with a reservoir solution consisting of 10% PEG400, 100 mm HEPES (pH 7.3), and 150 mm CaCl2. Various ratios of protein to reservoir solution were mixed (1 + 1 μl, 2 + 2 μl, 3 + 1.5 μl, and 1.5 + 3 μl) and inverted over the reservoir solution. In all conditions, yellow-colored coffin-shaped crystals grew overnight, with the largest reaching a final size of ∼0.2 × 0.2 × 0.4 mm within 2 days. A large and perfect looking crystal was coated in a 70:30 mixture of paratone N and mineral oil before flash-cooling in liquid nitrogen; it was diffracted to a resolution of 1.74 Å. The structure was solved by molecular replacement using the wild type CBL structure (PDB 1CL1). The P113S mutation was incorporated, and a HEPES molecule plus 379 water molecules were included in the final model. The PLP cofactor was clearly observed in the structure at full occupancy.An inhibited CBL(P113S) structure was produced by soaking a pre-formed crystal overnight in a crystallization solution supplemented with 10 mm l-Ala-P. Data were collected in-house and processed, and the structure was solved and refined as outlined above. The l-Ala-P-PLP aldimine was clearly observed in discrete and unambiguous electron density and was included in final refinement along with 514 water molecules. Active site electron density is shown in supplemental Fig. S1, and data collection and refinement statistics for the uninhibited and inhibited CBL(P113S) structures are in supplemental Table S2. […]

Pipeline specifications

Software tools XDS, CCP4, Coot, REFMAC5, MolProbity
Applications Small-angle scattering, Protein structure analysis
Organisms Escherichia coli
Chemicals Alanine, Amino Acids, Cystathionine, Pyridoxal Phosphate