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Supplementary Information Turbidity measurement of alkaline-phosphatase-induced precipitation. Etk samples with 10 μM and 20 μM concentration (in 50 mM Tris, pH 8.5) were incubated with 0.1 U and 0.4 U of calf intestine alkaline phosphatase (Roche), in a transparent 96-well plate in a total volume of 50 μL (50 mM Tris, pH 8.5). The degree of precipitation (turbidity) was measured in terms of O.D.360nm over a two-hour period in a plate reader (Bio-Tek, Fisher). Other remarks After determining the Etk structure, we have spent over two years of ongoing effort to elucidate the structure of phosphorylated Etk in which P-Y574 would be in the open or active conformation. Unfortunately, neither co-crystallization with ATP/Mg2+ nor soaking existing apo-crystals with high concentrations of ATP cocktails resulted in structures showing Y574 in its phosphorylated state. However, when Etk samples were treated with alkaline phosphatase, as they were dephosphorylated in the crystal structure, the protein precipitated immediately and irreversibly, and could not be used for crystallization trials (Supplementary Figs. 3 and 5). Therefore, the solubilization of Etk requires the aid of phosphorylation at the highly hydrophobic C-terminal tail, and subsequently a steady dephosphorylation may gradually decrease protein solubility and facilitate the formation of a stable EtK protein crystal. We have made extensive efforts in crystallizing Y574E mutant which, if showing a direct E574-R614 connection, would provide 1 further support of our finding. Given the difficulties in the crystallization we have not succeeded yet. No kinetic property has been reported on Wzc/Etk so far, while a few external substrates have been used to demonstrate the presence and extent of PTK activity (Ilan et al., 1999;Grangeasse et al., 2003;Mijakovic et al., 2003). To date, kinase activity of Wzc is commonly quantified by SDS-PAGE gel autoradiography, with the (specifically mutated) PTK itself as the substrate (Soulat et al., 2007). While the PTK serves as both the kinase and the substrate (hence the autokinase activity is measured), it is important to control the initial phosphorylation level of the samples. Supplementary Figure 3 shows a similar starting phosphorylation level for all wild type Etk and mutant samples used in this study. The fully dephosphorylated protein samples precipitated rapidly (probably due to the aggregation of hydrophobic C-terminal Tyr cluster) and were not used. In addition, we have also designed and synthesized a peptide that mimics the C-terminus of Etk as an external substrate. However, no satisfactory phosphorylation levels have been obtained because of the low solubility of the peptide, which contains a large number of tyrosine residues. 2 Supporting Tables Table 1. Crystallographic statistics Dataset Space group Unit cell dimensions (Å) Wavelength (Å) Resolution Range (Å) Unique reflections I/sigma a Completeness (%)a Rmergea,b SAD C2 a = 114.85, b = 51.75, c = 120.73 α = 90, β = 114.50, γ = 90 0.9788 30-2.6 22672 28.1 (4.9) 86.9 (56.2) 0.090 (0.222) Phasing statistics Overall figure of merit 0.67 Refinement Resolution Range (Å) No. of reflections in the working set Rcryst. / Rfreec r.m.s.d. bond length (Å) r.m.s.d. bond angles (º) Average B factor No. of protein atoms No. of solvent atoms Native C2 a = 115.51, b = 51.73, c = 120.52 α = 90, β = 114.57, γ = 90 0.9997 50-2.5 19864 20.0 (2.7) 93.8 (61.8) 0.078 (0.329) Native + ADP C2 a = 115.41, b = 50.90, c = 120.48 α = 90, β = 114.37, γ = 90 0.9177 50-3.0 13128 15.9 (4.5) 91.3 (67.1) 0.098 (0.241) 50-2.5 15757 0.196 / 0.253 0.007 1.2 67.64 3945 114 50-3.0 13045 0.223 / 0.291 0.007 1.3 66.13 3689 99 a Numbers in parentheses refer to statistics for the highest shell. Rmerge = Σ|Iobs - <I>| / ΣIobs, where Iobs is the intensity measurement and <I> is the mean intensity for multiply recorded reflections. c Rcryst and Rfree = Σ|Fobs - Fcalc| / Σ|Fobs| for reflections in the working and test sets, respectively. b 3 Supporting Figure Legends Figure 1. NanoESI/TOF mass spectrum of the Etk R614A mutant. (A) The protein solution was prepared in formic acid/water/methanol (v/v/v; 1:1:2, and the m/z spectrum shown was acquired on the QStar XL QqTOF mass spectrometer. (B) A deconvoluted mass spectrum is shown in the inset. (C) Deconvoluted mass spectrum for the Y574F mutant treated with ATP, showing a maximum of seven extra phosphates. The identity of the extra +258 Da peaks is unclear. Figure 2. Partial MALDI/TOF mass spectra of the digested EtK proteins by endoprotease Lys-C (A) EtK wild-type monomer; (B) EtK high molecular fraction; (C) R614A mutant. Data were recorded by the QStar XL MALDI QqTOF mass spectrometer using 2,5dihydroxybenzoic acid as the matrix. The m/z range is shown the profile of the phosphorylated Y-cluster peptide ions at the C-terminus of EtK (residues 706-726), and the number of phosphorylated groups was labeled on each peak. Figure 3. SDS-PAGE gel of the purified protein fractions by size-exclusion chromatography. (1) Etk wild-type high molecular weight fraction; (2) Etk wild-type monomer fraction; (3) Y574A mutant; (4) Y574G mutant; (5) Y574E mutant; (6) R614A (6). The second column (B) represents samples from the first column (A) incubated with alkaline phosphatase. The third column (C) shows samples from the first column (A) treated with ATP and Mg2+. 4 Figure 4. Circular dichroism spectra of Etk kinase domain. Dashed line: freshly purified Etk. Gray line: the same fraction after ATP and Mg2+ treatment. Figure 5. Etk precipitation induced by alkaline phosphatase (AP) dephosphorylation, measured by O.D.360 to monitor turbidity over time. 5 Figure 1 Figure 2 6 Figure 3 Figure 4 7 Figure 5 8 Supporting Information REFERENCES 1. Grangeasse C, Obadia B, Mijakovic I, Deutscher J, Cozzone AJ, and Doublet P (2003) Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd, a UDP-glucose dehydrogenase. J Biol Chem, 278, 39323-39329. 2. Ilan O, Bloch Y, Frankel G, Ullrich H, Geider K, and Rosenshine I (1999) Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide. EMBO J, 18, 3241-3248. 3. Mijakovic I, Poncet S, Boel G, Maze A, Gillet S, Jamet E, Decottignies P, Grangeasse C, Doublet P, Le MP, and Deutscher J (2003) Transmembrane modulator-dependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases. EMBO J, 22, 4709-4718. 4. Soulat D, Jault JM, Geourjon C, Gouet P, Cozzone AJ, and Grangeasse C (2007) Tyrosine-kinase Wzc from Escherichia coli possesses an ATPase activity regulated by autophosphorylation. FEMS Microbiol Lett, .. 9