Computational protocol: Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins

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Protocol publication

[…] The web interface of NCBI Protein BLAST () was used to search for sequences similar to the MTG of S. mobaraensis. The amino acid sequence of S. mobaraensis protein–glutamine γ-glutamyltransferase (Uniprot accession number P81453) was entered as a query. Manual screening of the results for E values < 10−10 and polypeptide sequences shorter than that of S. mobaraensis MTG yielded hypothetical gene product KALB_7456 from bacterial strain K. albida DSM 43870 (GenBankTM accession number AHI00814.1, Uniprot accession number W5WHY8). Sequence alignment of the S. mobaraensis and the K. albida sequences with Clustal Omega version 1.2.1 () yielded a value of 32% in the percent identity matrix and identified conservation of the catalytically active residues of MTG (Cys-140, Asp-331, and His-350; P81453 numbering). The ProP 1.0 server from the Technical University of Denmark () was used to predict the propeptide and signal sequences of the hypothetical K. albida microbial transglutaminase. VAAPTPR↓AP was the only predicted propeptide cleavage site with a score (0.513) above the threshold. [...] KalbTG in PBS was crystallized at 22 °C using the sitting drop (200-nl) vapor diffusion method by 1:1 mixing of 8 mg/ml protein with an unbuffered reservoir consisting of 0.2 m ammonium tartrate, 20% PEG 3350. Crystals were cryoprotected in reservoir solution containing 20% ethylene glycol before flash-cooling in liquid nitrogen. Data were collected at 100 K at SLS beamline PX-II using a Pilatus 6M detector and integrated and scaled in space group P3 with XDS (). The l = 3n reflections have I/σ of >9, rendering the presence of a screw axis unlikely. Self-Patterson and twinning analyses did not reveal suspicious data pathologies. The cell volume is consistent with two or three KalbTG molecules in the asymmetric unit, with Matthews parameters of 3.5 Å3/Da and 2.3 Å3/Da, respectively. Whereas the κ = 180° section of the self-rotation function did not indicate a 2-fold NCS axis, a peak in the κ = 164° section at ω = 0°, φ = 0° indicated that at least two molecules in the asymmetric unit are related by a 164° rotation, which turned out to be correct after molecular replacement. Data collection statistics are summarized in supplemental Table 1.The structure of KalbTG (226 residues) was determined by molecular replacement using the S. mobaraensis transglutaminase (354 residues, PDB entry 3IU0) as the search model. The first attempts using the complete S. mobaraensis TG were unsuccessful, probably because the enzymes are of very different sizes. The two transglutaminases share 28.2% sequence identity and 38.9% sequence similarity over the entire length of KalbTG. A variant of S. mobaraensis TG devoid of loop regions and trimmed to the hydrophobic core resulted in a potential solution with PHASER () when searching for two molecules in the asymmetric unit in space group P3 with a log-likelihood gain of 213. Trigonal space groups P31 and P32 did not yield solutions, consistent with the high intensities of the l = 3n reflections. The molecular replacement model was refined in BUSTER () to an initial Rfree of 46%. Some secondary structure elements were visible in the electron density maps and were included in the model, which was then submitted to 10 cycles of automatic model building and refinement in CBUCCANEER and REFMAC5 (). The resulting model included the entire KalbTG catalytic domain and had an Rfree of 30%. The structure was completed in COOT () and refined with PHENIX () to an Rfree value of 23% at 1.9 Å resolution with excellent stereochemistry. There are two molecules in the asymmetric unit that are virtually identical (root mean square deviation 0.26 Å over all atoms) and exhibit excellent electron density (supplemental Fig. 3) and stereochemistry (supplemental Table 2). Model refinement statistics are collected in supplemental Table 2. The first 19 N-terminal amino acids (MGGGSTTAQAAAVAAPTPR) and the C-terminal artificial GGGS-His8 tag are disordered in the structure. […]

Pipeline specifications

Software tools XDS, REFMAC5, Coot, PHENIX
Applications Small-angle scattering, Protein structure analysis
Organisms Escherichia coli
Chemicals Ammonium Compounds