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Supplementary Information
Expression and purification of IpaA560-633 and vinculin1-265
The fragment encoding IpaA560-633 was cloned in pGEX4T2 (GE Healthcare) and expression
of GST-IpaA560-633 was induced in Escherichia coli DH5 with isopropyl--D-thiogalactoside
(IPTG) during growth in Luria-Bertani medium (LB) at 310 K. Bacteria were resuspended in
Phosphate Buffered Saline (PBS300) (10 mM Na2HPO4, 1.8 mM KH2PO4, 300 mM NaCl, 2.7
mM KCl, pH = 7.5) and sonicated. The soluble fraction was applied to a GSTrap column (GE
Healthcare) and eluted with PBS300 containing 10 mM reduced glutathione. GST-IpaA560-633
was incubated with thrombin for 16 hours at 293K and simultaneously dialyzed against
PBS300. The mixture was re-applied to a GSTrap column, mounted in series with a
benzamidine column, and IpaA560-633 (carrying additional Gly and Ser residues at its Nterminus) was collected in the flowthrough. IpaA560-633 was applied to a Superdex75 HR
10/30 column using PBS300 as buffer and concentrated to 12.8 mg/ml. IpaA560-633Y567W and
IpaA560-633Y613W were constructed with the Quickchange method (Stratagene). Variant
proteins were purified as described above. Trypsin digestion and MS and MS/MS analysis
(not shown) confirmed the incorporation of Trp in both variants.
The plasmid encoding MBP-vinculin1-265 (MBP-VD1) (Bourdet-Sicard et al., 1999) was
introduced in E. coli Rosetta2 (DE3) strain (Novagen) and expression was induced by IPTG
during growth in LB at 310 K. Bacteria were resuspended in buffer A (20 mM Tris/HCl, pH
7.5, 200 mM NaCl, 1 mM EDTA and 10 mM -mercaptoethanol (ME)) and disrupted using
a French Press. The soluble fraction was applied to an amylose column (New England
Biolabs) and eluted in buffer A containing 10 mM maltose. MBP-VD1 was diluted 5 times in
buffer B (20 mM Tris/HCl, pH 8.0, 5 mM ), applied to a ResourceQ column and eluted
with a linear gradient from 40 to 500 mM NaCl in buffer B. MBP-VD1 was incubated with
Factor Xa for 16 hours at 293K and the mixture was diluted 5 times in buffer B, applied to a
ResourceQ column and eluted with a linear gradient from 40 to 500 mM NaCl in buffer B.
The peak containing VD1 was concentrated to 3.6 mg/ml. VD11-250 was constructed by
replacing the Ser251 codon (TCC) by a stop codon (TAA). MBP-VD11-250 was expressed and
purified as described above, including a last gel filtration step on Superdex75 HR 10/30, using
20 mM Tris/Acetate, pH 7.6, 150 mM NaCl, 5 mM  as buffer.
The IpaA560-633/VD1 complex used for crystallization was prepared by mixing VD1 with an
excess of IpaA560-633 (> 2:1 molar ratio). The mixture was left on ice for 15 minutes and
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applied to a Superdex75 HR 10/30 column, using 20 mM Tris/Acetate, pH 7.6, 150 mM
NaCl, 1 mM EDTA, 5 mM as buffer. The peak containing 1:1 complexes was
concentrated to 8.7 mg/ml.
Expression and purification of IpaAC and gel filtration analysis of IpaAC/VD11-250
mixtures
The fragment encoding residues 394-633 of IpaA (IpaAC) was cloned in pQE30 (Qiagen) and
expression of His-tagged IpaAC was induced in E. coli XL1Blue with IPTG during growth in
2xTY medium at 310 K. Bacteria were resuspended in 20 mM Tris pH 8, 300 mM NaCl, 5 %
glycerol and 20 mM imidazole and opened using a French press. The pellet was collected and
washed in 50 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA and 0.5 % Triton X-100 and
subsequently solubilized in 100 mM Tris pH 8, 50 mM glycine, 10 mM MgSO4, 6 M
guanidine HCl and 10 mM imidazole. This solution was applied to a HisTrap column (GE
Healthcare) loaded with Ni2+, and IpaAC was eluted with 100 mM Tris pH 8, 50 mM glycine,
6 M guanidine HCl and 200 mM imidazole. The eluate was diluted into 100 mM Tris pH 8,
200 mM NaCl, 400 mM L-arginine and 10 % glycerol. IpaAC was concentrated and buffer
was exchanged to 20 mM Tris/Acetate pH 7.6, 150 mM NaCl, 10 % glycerol and 1 mM
EDTA. Analytical gel filtration with IpaAC/VD11-250 mixtures was performed using a
Superdex200 PC 3.2/30 column. IpaAC/VD11-250 molar ratios of 1/1, 1/2, 1/3, 1/4, 1/5 and 1/6
were used in analysis. Experimental procedures were the same as for analysis of IpaA 560633/VD1
mixtures, except that the VD1 concentration was 25 in all cases and samples
were incubated for 1 hour at room temperature before injection.
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Figure S1: Gel filtration profiles of IpaAC/VD1 mixtures at different molar ratios, as
indicated. Fractions were collected and analyzed by SDS-PAGE. IpaAC (27 kDa) runs at a
larger apparent molecular weight in gel filtration. At 1/1, 1/2 and 1/3 molar ratios, a peak for
free VD1 is absent and all VD1 binds IpaAC in complexes of increasing size which do not
resolve into separate peaks. When VD1 is present in excess (F, G and H), a tail can be
discerned trailing the peak of IpaAC/(VD1)3, which could indicate an exchange process
between free VD1 and complexed VD1 during the run.
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