The recombinant proteins MsKGD, MsKGDΔ360, MsGarA, MsDlaT, MsLpd, MtGabD1, and MtGarA and the two FHA domains alone (MsGarAΔ44 used in cocrystallization with KGDΔ360 and MtGarAΔ43 used for kinase assays) were overproduced in E. coli and purified as previously described (21, 26). MsGarA and its mutant versions were also produced in microfermentor units, following established protocols (54). MtPknB1–279 (referred to as PknBCD) and its mutant version carrying the substitution L33E (PknBCD,L33E) were expressed for 15 hours at 22°C with 100 μM isopropyl-β-d-thiogalactopyranoside (IPTG). Both proteins were purified using the same protocol. E. coli cells were harvested by centrifugation, resuspended in lysis buffer [50 mM NaH2PO4 (pH 8), 500 mM NaCl, 5% glycerol, 25 mM imidazole, and 1 mM dithiothreitol (DTT)] supplemented with cOmplete protease inhibitor cocktail (Roche) and sonicated. After centrifugation, the supernatant was loaded onto a 5-ml HisTrap HP column (GE Healthcare), and the His-tagged protein was purified by applying a linear imidazole gradient (25 to 500 mM). Next, the His6 tag was removed by incubating for 18 hours at 18°C in the presence of His6-tagged Tobacco Etch Virus (TEV) protease (55) at a 1:30 ratio (w/w) in buffer [25 mM Hepes (pH 8), 150 mM NaCl, 5% glycerol, and 1 mM DTT] followed by separation on a Ni-nitrilotriacetic acid (NTA) agarose column (Qiagen). Then, the protein was further purified by size exclusion chromatography on a Superdex 200 column (GE Healthcare) equilibrated in 25 mM Hepes (pH 8), 150 mM NaCl, 5% glycerol, and 1 mM DTT. Fractions corresponding to the PknB peaks, as confirmed by SDS-polyacrylamide gel electrophoresis, were later pooled and concentrated up to 40 mg/ml, flash-frozen in liquid nitrogen, and stored at −80°C. All proteins were quantified using the molar absorption coefficient predicted from the amino acid sequence by the ProtParam tool (http://web.expasy.org/protparam/).

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