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Supporting Information to

Study of the structural and dynamic effects in the FimH adhesin

upon -D-heptyl mannose binding

Sophie Vanwetswinkel, Alexander N. Volkov, Yann G. J. Sterckx, Abel Garcia-Pino, Lieven Buts,

Wim F. Vranken, Julie Bouckaert, René Roy, Lode Wyns and Nico van Nuland

Table of contents:

Table S1 CING analysis of the apo FimHL NMR structure S2

Table S2 RMSD analysis of the FimHL structures S3

Table S3 X-ray crystallography data collection and refinement statistics S4

Figure S1 2D [1H,15N]-HSQC spectrum of the apo wt FimHL S5

Figure S2 Local RMSD analysis of the FimHL crystal structures S6

Figure S3 NOE coverage of the apo FimHL solution structure S7

Figure S4 NMR relaxation profiles of the apo and HM-bound wt FimHL S8

Figure S5 NMR relaxation profiles of the apo wt FimHL at different concentrations S9

Figure S6 SAXS data analysis of the apo and HM-bound FimHL in HEPES buffer S10

Figure S7 SAXS curve fitting for the apo and HM-bound FimHL in HEPES buffer S11

Figure S8 Chemical shift perturbation analysis of the Y48A FimHL S12

Figure S9 ITC titration of the HM into Y48A FimHL S13

Figure S10 NMR relaxation profiles of the apo and HM-bound Y48A FimHL S14

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Distance restraints total 4622 + 9*

Intra-residual 713

Sequential 1143

Medium Range 599

Long range 2167 + 9*

Dihedral restraints 226

Phi angles 123

Psi angles 123

NOE violations > 0.5 Å 0

Dihedral violations > 5° 3

RMSD from average (Å) Residues 1 - 158

Backbone N, CA, CO, O 0.35 ± 0.06

Heavy atoms 0.68 ± 0.05

Ramachandran plot:

Most favored regions (%) 76.9

Additional allowed regions (%) 22.3

Generously allowed regions (%) 0.8

Disallowed regions (%) 0.0

CING ROG score (%) 28 / 25 / 46

Table S1: CING analysis of the 25 lowest energy NMR structures obtained after structure

calculation and water refinement using the RECOORD protocol. The asterisk (*) refers to the

nine manually introduced ambiguous restraints for the Phe1 aromatic protons.

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PDB ID RMSD (Å) PDB ID RMSD (Å)

1tr7 1.52 ± 0.09 4av4 1.39 ± 0.08

1uwf 1.38 ± 0.08 4av5 1.49 ± 0.12

2vco 1.42 ± 0.11 4avh 1.47 ± 0.09

3mcy 1.42 ± 0.08 4avi 1.41 ± 0.09

4auu 1.43 ± 0.10 4avj 1.45 ± 0.09

4auy 1.44 ± 0.09 4avk 1.40 ± 0.09

4av0 1.43 ± 0.09

Table S2: Comparison between the solution structure of the apo FimHL obtained in this work

and PDB-deposited crystal structures of the FimHL complexes. Pairwise backbone RMSDs

were first computed in Pymol for each of the 25 NMR models and then averaged to the all

ensemble; errors are standard deviations.

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PDB id 4LOV

Wavelength (Å) 0.9791

Resolution range (Å) 38.43 - 1.60 (1.67 - 1.60) Space group P212121

Unit cell

a = 39.9b = 41.8 c = 97.5

= = = 90.0 Total reflections 103267

Unique reflections 24894 (1538)

Completeness (%) 92.39 (91.7)

Mean I/sigma(I) 9.40 (2.00)

Wilson B-factor 10.77

R-sym (%) 9.0 (64.6)

R-factor 0.1554 (0.2283) R-free 0.1922 (0.2885) Total number of atoms 1556

macromolecular atoms 1209

ligand atoms 28

water 319

Protein residues 158

RMS(bonds) 0.010

RMS(angles) 1.36

Ramachandran preferred (%) 97.0

Ramachandran allowed (%) 3.0

Ramachandran outliers (%) 0

Average B-factor 8.00

Table S3: X-ray crystallography data collection and refinement statistics.

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Figure S1: 800MHz [1H,15N]-HSQC spectrum of the apo wt FimHL annotated with complete

backbone amide assignments. Assigned Asn and Gln side-chain amides (*) as well as Arg N H (e)

and Trp N 1H 1 (#) resonances are also indicated.

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Figure S2: Variability of FimHL crystal structures. Local atom RMSDs were calculated for a set of 17

FimHL crystal structures [(PDB ID and chain codes: 1UWF (A), 2VCO (A, B), 4AUU (B), 4AV0 (A,

B), 4AV4 (A), 4AV5 (C, D), 4AVH (A, B), 4AVI (A, B), 4AVJ (A, B), 4AVK (A, B)] in Yasara

(http://www.yasara.com) for the non-hydrogen backbone atoms (CA, CO, O and N). Only atoms

present in all chains were retained for the local RMSD comparison, and the RMSD value was set to

0.0 for missing residues. For each residue, including the relevant atoms of the 2 preceding and 2

following residues, the all-against-all superposition matrix of the atom coordinates of the same

pentapeptide fragment in all models was determined. From this, the pentapeptide fragment with the

lowest RMSD from all others was selected, and the RMSD values of the other fragments

superimposed on this fragment calculated. (A) For each residue, the average RMSD is given as black

dot with the maximum and minimum value depicted as error bar. (B) Stereo representation of the 25

NMR ensemble models coloured by the average RMSD per residue calculated in A, from blue

(RMSD=0.077Å) to red (RMSD 0.800Å); residues for which the local RMSD could not be computed

due to scarce data are shown in grey.

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Figure S3: Experimental coverage of the apo FimHL solution structure. (A) Number of all (light grey)

and long-range (dark grey) NOE-derived distance restraints per residue. (B) Stereo representation of

the 25 NMR ensemble members coloured by the total number of restraints [light grey in (A)] from

blue for 16 to red for 100.

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Figure S4: Backbone-amide NMR relaxation profiles of the apo (black) and HM-bound (red)

1.08 mM wt FimHL in 20 mM sodium phosphate 100 mM NaCl pH 6.0 at 25° C and 600 MHz.

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Figure S5: Backbone-amide NMR relaxation profiles of the apo wt FimHL at 1.08 mM (black) and

1.58 mM (blue) in 20 mM sodium phosphate 100 mM NaCl pH 6.0 at 25° C and 600 MHz.

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Figure S6: SAXS curve analysis for the apo and HM-bound FimHL in Hepes buffer (pH 7.4). Buffer-

subtracted scattering curves of (A) apo and (C) holo FimHL measured at four different concentrations:

2.0, 4.0, 6.0, and 8.2 mg/ml. The curves are shown in different shades of blue and green for apo and

holo FimHL, respectively. In both cases, the curves are displaced vertically relative to each other and

are plotted on an arbitrary intensity scale for clarity. The grey traces depict the experimental error.

The insets show the Guinier regions of the respective scattering curves. (B) The merged scattering

curve for apo FimHL (black dots; grey traces depict the experimental error) is very well described by

an apo FimHL monomer. The red and blue lines represent the CRYSOL fit for the apo FimHL NMR

structure and the fit for the best ab initio model, respectively. An overlay of the apo FimHL NMR

structure (blue ribbon) and the ab initio model (grey beads) is shown on the right. (D) The

percentages of holo FimHL monomer and dimer as a function of concentration obtained by a linear

combination of scattering from the dimer and monomer by OLIGOMER. ( 2 in black). Given for

comparison, the 2 in red correspond to the fits with 100% of monomer. The fits of the different

OLIGOMER runs are shown as red traces in (C).

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Figure S7: SAXS curve fitting using different shape dimers for the holo FimHL in Hepes buffer (pH 7.4). (A) p(r) functions of apo and holo FimH support the occurrence of a concentration-dependent dimerization in the presence of HM. (B) Different AUTODOCK solutions for the holo FimHL dimer are shown in purple, yellow, and red, respectively. The crystallographic dimer is shown in green and the HM is depicted in a sphere representation. (C) Fits of OLIGOMER runs for the different dimers shown in (B) to the experimental data of the highest holo FimHL concentration (black dots; grey traces represent the experimental error). (D) Same as (C) for head-to-tail, tail-to-tail, and head-to-head dimers.

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Figure S8: Combined backbone amide chemical shift perturbations ( avg) induced upon (A and C)

Y48A mutation and (B and D) HM binding to the Y48A FimHL. (A and B) avg plotted for each

observed backbone amide and (C and D) mapped onto a surface representation of the wt FimHL-HM

crystal structure (PDB 4lov, this work). Residues are coloured by the avg as defined in the ramp.

Proline and unassigned residues are shown in light grey; HM in dark grey sticks.

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Figure S9: ITC titration of HM into the Y48A FimHL. The top and bottom panels show, respectively,

the raw data after the baseline correction and the integrated data corrected for the heat of dilution of

the ligand. The solid line in the bottom panel is the best fit of the data to an n identical and

independent site binding model. See table 1 for the measured thermodynamic parameters.

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Figure S10: Backbone-amide NMR relaxation profiles of the apo (black) and HM-bound (red)

1.08 mM Y48A FimHL in 20 mM sodium phosphate 100 mM NaCl pH 6.0 at 25° C and 600 MHz.