SUPPORTING INFORMATION
Membrane Association of the Diphtheria Toxin
Translocation Domain Studied by Coarse-Grained
Simulations and Experiment
Jose C. Flores-Canales,a Mauricio Vargas-Uribe,b Alexey S. Ladokhin,b and Maria Kurnikova*,a
a
Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213.
b
Department of Biochemistry and Molecular Biology, the University of Kansas Medical Center,
Kansas City, KS 66160.
* To whom correspondence should be addressed:
E-mail: kurnikova@cmu.edu Phone: 412-268-9772 Fax: 412-268-1061
Fig. S1. Angle formed by the axis of helix TH8 (Cα atoms of residues 323 – 343) and the normal
axis to the membrane plane along the simulation time. The normal axis direction is towards the
membrane core, i.e. TH8 is parallel to the membrane plane if the angle is close to 90°. Panels A,
B, and C show the angles obtained for the neutral pH T-domain structure and lipid bilayers
composed of POPC, POPC:POPG 3:1, and POPC:POPG 1:3, respectively. Panels D, E and F
display data obtained for the low pH unfolded T-domain structure and lipid bilayers composed of
POPC, POPC:POPG 3:1, and POPC:POPG 1:3, respectively. Each line color represents an
independent CG-MD production simulation initiated with the same protein orientation and a
different seed number. Accumulated CG-MD production simulation time is 60 µs.
Fig. S2. Angle formed by the axis of helix TH9 (Cα atoms of residues 359 – 375) and the normal
axis to the membrane plane along the simulation time. The normal axis direction is towards the
membrane core, i.e. TH9 is parallel to the membrane plane if the angle is close to 90°. Panels A,
B and C display the angles obtained with the neutral pH T-domain structure and lipid bilayers
composed of POPC, POPC:POPG 3:1, and POPC:POPG 1:3, respectively. Panels D, E and F
show data obtained with the low pH unfolded T-domain structure and lipid bilayers composed of
POPC, POPC:POPG 3:1, and POPC:POPG 1:3, respectively. Each line color represents an
independent CG-MD production simulation initiated with the same protein orientation and a
different seed number. Accumulated CG-MD production simulation time is 60 µs.
Fig. S3. Plots of the center of mass (COM) distance (Z) between T-domain and anionic lipid
bilayers of different composition along the bilayer axis obtained from the last 500 ns of
unrestrained CG-MD simulations. In this distance (Z) range the protein forms contacts with the
bilayer-water interface. Panels A and B display the distance between the neutral pH structure and
bilayers composed of POPC:POPG 3:1 and POPC:POPG 1:3, respectively. Panels C and D show
the distance of the low pH unfolded T-domain structure and lipid bilayers composed of
POPC:POPG 3:1 and POPC:POPG 1:3, respectively. Each line color represents an independent
CG-MD production simulation initiated with the same protein orientation and a different seed
number.
Fig. S4. Normalized number of protein-membrane interface contacts as a function of residue
number. Data is obtained from the last 200 ns of two independent unrestrained CG-MD
simulations of the low pH T-domain structure and a pre-formed membrane containing
POPC:POPG 1:3. Blue bars correspond to the membrane bound state B1, while red bars
correspond to the membrane bound state B2. (A) Protein and membrane interface contacts. (B)
Protein and acyl chains contacts. All protein and lipid atoms separated by a distance lower than 5
Å are considered to be in contact.
Fig. S5. Normalized number of protein-membrane interface contacts as a function of residue
number. Data is obtained from the last 200 ns of two independent unrestrained CG-MD
simulations of the neutral pH T-domain structure and a pre-formed membrane composed of
POPC:POPG 1:3. Red and blue bars correspond to two representative trajectories, shown in
black and red lines in Figs. S1C, S2C. All protein and head-group phosphate beads separated by
a distance lower than 5 Å are considered to be in contact.
Fig. S6. Normalized number of protein-membrane interface contacts as a function of residue
number. Data is obtained from the last 200 ns segments from: (A) Two independent unrestrained
CG-MD simulations of the neutral pH T-domain structure. (B) One simulation trajectory of the
low pH T-domain structure. The pre-formed membrane contains POPC:POPG 3:1. All protein
and head-group phosphate beads separated by a distance lower than 5 Å are considered to be in
contact.
Fig. S7. Normalized number densities of coarse-grained groups as a function of their distance
from the center of bilayer along the normal axis to the bilayer. The maximum of each number
density profile is scaled to one. Data is obtained from the last 500 ns of representative
unrestrained CG-MD simulations of T-domain structures with lipid bilayers containing
POPC:POPG 3:1. (A) Membrane bound conformation of low pH T-domain structure. (B)
Membrane bound conformation of neutral pH T-domain structure. The center of the bilayer
coincides with Z = 0. Density profiles of headgroup atoms of POPC (choline) and POPG
(glycerol) are shown in black and yellow lines, respectively. Density profiles of phosphate and
acyl chains from all lipids are shown in red and green lines, respectively. Density profiles of
water and sodium counterions (ions) are shown in blue and dark yellow, respectively. Profiles of
cationic residues (Lys, Arg, and N-terminal), histidines and hydrophobic residues are shown in
cyan, grey and magenta lines, respectively.
Fig. S8. Angle formed by the axes of helices (TH8 and TH9) and the normal axis to the
membrane plane versus simulation time. The normal axis direction is towards the membrane
core, with helix being perpendicular to the membrane plane if the angle is about 0° or 180°. We
present results from US CG-MD simulations in which the low pH T-domain structure begins to
associate to the membrane interface, which spans the range of 35 Å to 45 Å along the reaction
coordinate, as shown by free energy curves in Figure 5. (A) and (D) display the angles formed by
helices TH8 and TH9 obtained from the PMF curve of the membrane bound state B1,
respectively. (B) and (E) display the angles formed by helices TH8 and TH9 obtained from the
PMF curve of the membrane bound state B2, respectively. (C) and (F) show the angles formed
by helices TH8 and TH9 obtained from the PMF curve obtained from a different protein
orientation, see text for details. Protein conformations where helices TH8 and TH9 are
approximately perpendicular to the membrane plane are shown in cyan lines. These correspond
to umbrella simulations spanning the range of 40 Å to 45 Å along the reaction coordinate.
Protein conformations where helices TH8 and TH9 are approximately parallel to the membrane
plane are shown in red. These correspond to US window simulations spanning the range of 35 Å
to 38 Å along the reaction coordinate. The intermediate states are shown in gold lines,
corresponding to umbrella simulations restrained at center of mass distance of 39 Å. All plots
show data obtained from umbrella sampling simulations of the low pH T-domain structure and a
pre-formed membrane of mixture POPC:POPG 1:3.
Fig. S9. Normalized number of protein-membrane interface contacts as a function of residue
number. Data is obtained from the last 600 ns of three US window simulations of the low pH Tdomain structure and a pre-formed membrane composed of POPC:POPG 1:3, shown in Figure 5.
(A) Blue and red bars correspond to the transition conformations of PMF profiles of B1 and B2,
respectively. Data is obtained from US window simulations restrained at COM distance of Z =
39 Å. (B) Blue bars correspond to the transition conformation of an US window simulation
restrained at COM distance of Z = 39 Å and with an initial non-preferable protein orientation. All
protein and head-group, phosphate beads separated by a distance lower than 5 Å are considered
to be in contact.
Fig. S10. Normalized number of protein-membrane interface contacts as a function of residue
number. Data is obtained from the last 600 ns of three US window simulations of the low pH Tdomain structure and a pre-formed membrane containing POPC:POPG 1:3, the center of mass
distance is restrained at 42 Å. (A) Blue and red bars correspond to the intermediate state of PMF
profiles of B1 and B2, respectively. (B) Blue bars correspond to the intermediate state obtained
from an US window simulation with an initial non-preferable protein orientation at COM
distance of Z = 42 Å. All protein and head-group, phosphate atoms separated by a distance lower
than 5 Å are considered to be in contact.