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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
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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.
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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
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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+.
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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.
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Figure 1
Figure 2
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Figure 3
Figure 4
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Figure 5
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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, ..
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