— 1 —
Supplementary Information
Supplementary results and discussion
Structure-based functional analyses of GkPOT
We analyzed the peptide-chain-length preference of GkPOT, using the
liposome-based assay (Fig. 2A). The results revealed that GkPOT exhibits a similar
preference for the peptide length as the other POT family transporters, including L.
lactis DtpT (1) and PepTSt (2). We also analyzed the pH dependence of the H+-driven
uptake activity of GkPOT (Fig. S16A). Maximal transport of the (Ala)2 peptide was
observed at pH 7, consistent with the values obtained for L. lactis DtpT (1) and PepTSt
(2).
To investigate the importance of the residues in the peptide binding site, we
performed the liposome-based assay, using the 3H-(Ala)2 peptide as a substrate. The
present crystal structure suggested that the C-terminal side chain group of the substrate
dipeptide is bound to the hydrophobic pocket formed by Tyr78, Trp306, and Trp440
(Fig. S8E), which is conserved among the POT family members (Fig. S3A). This
hydrophobic pocket has sufficient space to accommodate the side chains of large
hydrophobic amino acids, such as Phe. This model is consistent not only with the
— 2 —
previous reports of PepTSo (3), PepTSt (2), and PepT1 (4, 5), but also with our functional
analysis. The Ala mutations of Tyr78 or Trp306, which may disrupt this pocket,
exhibited decreased transporter activities (Fig. S17). In contrast, the Phe mutation of
Tyr78, which may conserve the size and hydrophobicity of the pocket, did not affect the
transporter activity (Fig. S17). This result also suggested that the OH group of Tyr78 is
not critical for the transporter activity, although it hydrogen bonds with the alafosfalin
phosphonate group (Fig. 3).
Molecular dynamics simulations
In the S-E310p simulation, in which Glu310 was protonated, no large structural
changes were observed. The most noticeable (but small) changes occurred in the loops
between helices H4 and H5, and H9 and H10 (Fig. S10), which are involved in crystal
packing interactions in the crystal structure. In contrast, in the S-E310 simulation, in
which Glu310 was deprotonated, large structural changes were observed. The region
between these two Pro residues (137-173) exhibited higher RMS fluctuations than in the
S-E310p simulation (Fig. S10). In contrast, no significant difference between the RMS
fluctuations of the S-E310 and S-E310p simulations was observed in the C-terminal
bundle (Fig. S10). The structural change observed in the S-E310 simulation seems to be
— 3 —
reasonable as the initial motion toward the outward-open state, since the H4-H5 hairpin
is actually accommodated in the cleft formed by helices H8 and H10 in the crystal
structure of FucP in the outward-open state (6) (Fig. S11C). Furthermore, the resulting
structure of the partially-occluded state is similar to the crystal structure of EmrD in the
occluded state (7) (Fig. S11D).
Next, we performed the simulation starting from the Glu310-protonated GkPOT
in complex with the (Ala)2 dipeptide (S-E310p-AA), based on the alafosfalin complex
structure. The peptide was stably bound to the substrate binding pocket. The hydrogen
bonds between the peptide carboxylate group and the Arg43 and Glu310 side chains
were maintained, while the peptide amide group was slightly shifted toward the Glu413
side chain (Fig. S14A, B). However, no large structural changes were observed in the
100-ns simulation. Then, based on the 100-ns structure of the S-E310p-AA simulation
trajectory, we modeled a (Phe)2 dipeptide in the substrate binding pocket, and performed
the 200-ns simulation (S-E310p-FF). In contrast to the S-E310p-AA simulation, we
observed a large structural change toward the occluded form in the S-E310p-FF
simulation (Fig. 4B, E). These results are consistent with the previous biochemical
results, showing that POT member transporters exhibit higher affinity for hydrophobic
dipeptides, such as (Phe)2, than (Ala)2 (1, 2). In the case of the simulation with the
— 4 —
(Ala)2 peptide, a much longer time may be required for the structural transition, as
compared to that with the (Phe)2 peptide. The structural change observed in the
S-E310p-FF simulation corresponds to the reverse transition of the physiological
transport cycle in vivo (Fig. S1D, E). All of the conformational transitions in the
transport cycle of MFS are reversible, as observed in LacY (8), and thus this structural
transition from E to D in Fig. S1 actually occurs in vitro.
Dynamics of GkPOT investigated by ATR-FTIR measurements
According to the MD simulation, the transition from the inward-open to the
occluded state is accompanied by a structural perturbation of the TM helices. Therefore,
we studied this structural alteration upon dipeptide binding, by attenuated total
reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. The difference
spectrum of GkPOT between the presence and absence of 2 mM (Ala)2 dipeptide at pH
5 coincides with the baseline (blue line in Fig. S15A), indicating the lack of dipeptide
binding to GkPOT. In contrast, clear difference spectra were obtained for 2 mM (Ala)2
dipeptide at pH 7 (red line in Fig. S15A), where the positive and negative signals
originate from the dipeptide-GkPOT complex and the free GkPOT, respectively. These
results are also consistent with the pH dependence of the GkPOT activity (Fig. S16A).
— 5 —
The negative peaks at 1660/1651 cm−1 and 1549 cm−1 are the characteristic amide-I and
-II vibrations of an -helix, respectively, indicating that the -helices are structurally
perturbed upon the binding of the (Ala)2 dipeptide. While many positive peaks appear
for amide-I (1700−1670 cm−1 and 1640−1600 cm−1), the single positive band of
amide-II at 1533 cm−1 shows that dipeptide binding accompanies the weakening of the
hydrogen bond of an -helix that monitors the peptide N-H group. The corresponding
amide-I vibrations probably appear at 1690, 1680 and 1670 cm−1, representing the
weakened hydrogen bond of the peptide C=O group, and the remaining bands at
1640-1600 cm−1 may originate from the bound dipeptide.
At a higher concentration of the (Ala)2 dipeptide, the intensity of the difference
FTIR spectra increased, but the spectral shape remained unchanged (Fig. S15B). The
dissociation constant (Kd) was determined to be 3.5 mM (Hill coefficient = 1.1) from the
analysis in Fig. S15C. Therefore, we concluded that the binding of the (Ala)2 dipeptide
causes the structural perturbation of an -helix, where hydrogen bonds are weakened.
The amplitudes of the negative peaks at 1660 and 1651 cm−1 were 0.00038 and 0.00041
for 2 mM (Ala)2 dipeptide (Fig. S15A), suggesting that the full binding would give
values of 0.00109 and 0.00117, respectively. Each band corresponds to about 1 % of the
entire amide-I band. Among the 496 amino acid residues in GkPOT, about 10 groups of
— 6 —
peptide backbones in -helices are likely to participate in the structural transitions
among the inward-open, occluded, and outward-open conformations (Fig. S1C, D, E).
— 7 —
Supplementary Methods
Cloning, Expression and Purification of GkPOT
The GkPOT gene (GK2020) was cloned from Geobacillus kaustophilus
genomic DNA into a plasmid derived from the expression vector pCGFP-BC (9),
which includes a C-terminal Green Fluorescent Protein (GFP), a His8-tag and a
tobacco etch virus (TEV) protease cleavage site. The GkPOT protein was
overexpressed in Escherichia coli C41(DE3)acrB strain cells (provided by K. Ito),
grown in LB medium containing ampicillin (50 µg ml−1). When the culture reached
an absorbance at 600 nm (A600) of ~0.5, the cells were induced with 0.5 mM
isopropyl -thiogalactopyranoside (IPTG) for 18 h at 293 K. The
selenomethionine-substituted GkPOT (SeMet-GkPOT) protein was overexpressed in
the methionine-auxotrophic mutant of the C41(DE3)acrB strain, cultured in Core
medium (Wako) containing 50 µg ml−1 L-selenomethionine (Nacalai Tesque).
The native and SeMet GkPOT proteins were purified according to the
following procedure at 277 K. The cells were pelleted by centrifugation at 4,000 g,
and were disrupted by a Microfluidizer (Microfluidics). After centrifugation (25,000
g), the supernatant was ultra-centrifuged (200,000 g), and the membrane fraction was
— 8 —
collected. The GkPOT protein was solubilized from the membrane fraction with
n-dodecyl-β-D-maltopyranoside (DDM), and was purified by the following three
chromatography steps. The insoluble material was removed by ultracentrifugation
(Beckman Type 70 Ti rotor, 150,000 g, 30 min), and the supernatant was mixed with
Ni-NTA resin (QIAGEN). The GFP-His8-tag of GkPOT was cleaved by TEV protease
at 4 °C overnight, and the protein was re-chromatographed on a Ni-NTA column. The
GFP-His8-tag-cleaved GkPOT was further purified by gel filtration (Superdex 200
10/300 GL, GE Healthcare) in 20 mM HEPES, pH 7.0, containing 150 mM NaCl,
0.03% (w/v) DDM and 3 mM -ME.
Crystallization of GkPOT
For crystallization, the purified protein was concentrated to approximately 10
mg ml–1, using an Amicon Ultra 50K filter (Millipore). GkPOT was mixed with
liquefied monoolein (Sigma) in a 2:3 protein to lipid ratio (w/w), using the twin-syringe
mixing method. Aliquots (200 nl) of the protein-LCP mixture were spotted on a glass
plate and overlaid with 1 µl of precipitant solution. Free-form GkPOT crystals were
grown at 20 °C, in reservoir solutions containing 36–41% PEG400, 100 mM
ADA-NaOH, pH 6.5, 160 mM Li2SO4, and 4 mM SrCl2. Sulfate-bound GkPOT crystals
— 9 —
were grown at 20 °C, in reservoir solutions containing 36-41% PEG400, 100 mM
ADA-NaOH, pH 6.5, 300 mM Li2SO4, and 4 mM SrCl2. GkPOT-E310Q·alafosfalin
complex crystals were grown at 20 °C, in reservoir solutions containing 26–36%
PEG400, 100 mM Tris-HCl, pH 8.0, 300 mM (NH4)2SO4, and 40 mM alafosfalin. The
crystals were flash-cooled using the reservoir solution as a cryoprotectant, and were
stored in liquid nitrogen.
Data collection and structure determination of GkPOT
All diffraction data sets were collected at the station BL32XU at SPring-8
(Hyogo, Japan). Datasets were processed with the HKL2000 suite (HKL Research)
and the CCP4 suite (10). The data processing statistics are summarized in Table SI.
The structure was determined by the SAD method, using the SeMet GkPOT crystal.
The heavy atom sites were identified with the program SHELXD (11). Heavy-atom
refinement and phase calculations were performed with the program SHARP (12)
using the reflections up to 3.2 Å, followed by solvent flattening with the program
SOLOMON (13). We could identify 12 of the 14 TM helices in the resulting electron
density map. The phase calculation statistics are summarized in Table I. The initial
model was built into the map, using the program COOT (14). The model was
— 10 —
subsequently improved through alternating cycles of manual building with COOT and
refinement with the program PHENIX (15). The structural refinement statistics are
summarized in Table SII. Molecular graphics were illustrated with CueMol
(http://www.cuemol.org/).
Liposome-based 3H-di-alanine uptake assay
H+-driven uptake and H+-independent counterflow transport assays were
performed as described previously (2). Proton driven uptake and competition assays
(Fig. 2A, B) were performed using 10 g of the wild type and variant GkPOT proteins.
For the H+-independent counterflow assay (Fig. 2C), 10 μg of wild type and variant
GkPOT were used per assay, with uptake stopped after 5 minutes. For the pH
dependence assay (Fig. S16), GkPOT proteoliposomes, containing 10 g of protein and
preloaded with 50 mM KPi at various pH values, were diluted 1:50 into 50 mM NaPi at
the same pH, to a total volume of 150 μl. The uptake of 15 μM 3H di-alanine was
measured after 1 min at 25°C. The uptake was stopped after 2 minutes by rapid dilution
into 0.1 M LiCl. Proteoliposomes were collected on 0.22 μm nitrocellulose filters and
washed under vacuum with 0.1 M LiCl, prior to scintillation counting. The 3H signals
were converted to molar concentrations of peptide, using standard curves for di-alanine.
— 11 —
Molecular dynamics simulation
We performed the 5 MD simulation runs listed in Table SIII, based on the
sulfate-bound form of the crystal structure. All lipid molecules and sulfate ions observed
in the crystal structures were removed, while all of the water molecules observed in the
crystal structure were kept. Helix HB was removed from the system, since it is not
conserved and thus does not seem to be involved in the transport mechanism. For the
S-E310p-AA and S-E310p-FF simulations, we respectively docked the (Ala)2 and
(Phe)2 dipeptides to the sulfate-bound form of the crystal structure, as described in the
Supplementary Discussion, and then used them as the initial structures. The missing
side chains and hydrogen atoms were built with the program VMD (16). The
protonation states of the His residues were determined using the program PROPKA (17).
His64 and His398 were protonated at their positions, His21, His109, His186, and
His390 were protonated at their positions, and His332 was protonated at both the
and positions. As for the Asp and Glu side chains, all except Glu310 were indicated as
being ionized. The protonation states of Glu310 used in the simulations are listed in
Table SIII. The prepared structures were then embedded in a fully hydrated POPC
bilayer. In the crystal structure, several sulfate ions were observed around the perimeter
— 12 —
of the TM segments, and these were used as indicators for the locations of the phosphate
moiety in the lipid head group. The lipid molecules overlapping the protein were
removed, resulting in 242 POPC molecules included overall. The lipid-protein complex
was then hydrated to form the 100 Å ×100 Å ×100 Å simulation box. Sodium and
chloride ions were then added, to neutralize the system with a salt concentration of 150
mM. The molecular topologies and parameters from the CHARMM27 force-field
parameters, with φ, ψ cross-term map correction (CMAP) (18), were used.
Molecular dynamics simulations were performed with the program NAMD 2.8
(19). The systems were first energy minimized for 1,000 steps with fixed positions of
the non-hydrogen protein atoms, and then for another 1,000 steps with 10 kcal/mol
restraints for the non-hydrogen protein atoms. For the equilibration, the systems were
subjected to MD simulations in the NPT ensemble with harmonic restraints on the
non-hydrogen protein atoms, and the X-Y plane harmonic restraint was employed for
the phosphorus atoms in the lipid head groups. Finally, 100 ns MD simulations without
any restraints were performed in the NPAT ensemble for the production runs. In these
simulations, constant pressure (1 atm) and temperature (300K) were maintained using
Langevin dynamics and a Langevin piston, respectively. The particle mesh Ewald
(PME) method was employed for the calculation of the electrostatic interactions (20).
— 13 —
The equation of motion was integrated with a time step of 2 fs.
ATR-FTIR measurements
For ATR-FTIR measurements, GkPOT was reconstituted into POPE/POPG
(molecular ratio protein:lipid = 1:30). The sample was placed on the surface of a
diamond ATR crystal (nine internal total reflections). After it was dried in a gentle
stream of N2, the sample was rehydrated with solvent containing 50 mM HEPES (pH
7.0) or phosphate (pH 5.0) buffer with 200 mM NaCl, at a flow rate of 0.7 mL/min.
ATR-FTIR spectra were first recorded at 2 cm–1 resolution, using an FTIR spectrometer
(Agilent, CA, USA) equipped with a liquid nitrogen-cooled MCT detector (an average
of 1,150 interferograms) (21). After recording the FTIR spectrum in the second solvent
additionally containing the (Ala)2 dipeptide, the difference FTIR spectrum was
calculated by subtracting the data obtained for the first and second solvents. The cycling
procedure was repeated 7–17 times, and the difference spectra were calculated as the
averages of the presence-minus-absence spectra of the (Ala)2 dipeptide. The spectral
contributions of the unbound dipeptide, the protein/lipid shrinkage and the water/buffer
components were corrected.
Su
pp
lem
enta
ry T
ab
les
Tab
le S
I D
ata
colle
ctio
n an
d ph
asin
g st
atis
tics.
W
T-fr
ee
WT-
sulfa
te
E310
Q-a
lafo
s E3
10Q
-fre
e E3
10Q
-sul
fate
Se
Met
Dat
a co
llect
ion
X-r
ay so
urce
SP
ring-
8 B
L32X
U
Spac
e gr
oup
P2 1
Cel
l dim
ensi
ons
a, b
, c (Å
) 50
.76,
94.
92, 5
7.25
52
.51,
93.
73, 5
7.27
53
.52,
94.
08, 5
7.85
54.1
4, 9
5.23
, 57.
5952
.63,
93.
53, 5
7.50
50.9
3, 9
4.93
, 57.
29
()
90
, 111
.10,
90
90, 1
12.3
3, 9
0 90
, 112
.65,
90
90, 1
11.2
0, 9
0 90
, 112
.38,
90
90, 1
10.9
4, 9
0
Wav
elen
gth
1.00
000
0.97
944
1.00
000
1.00
000
1.00
000
0.97
910
Res
olut
ion
(Å)
50.0
-1.9
0
(1.9
3-1.
90)
50.0
-2.0
0
(2.0
3-2.
00)
50.0
-2.4
0
(2.4
4-2.
40)
50.0
-2.3
0
(2.3
4-2.
30)
50.0
-2.1
0
(2.1
4-2.
10)
50.0
–3.2
0
(3.2
6–3.
20)
Rm
erge
(%)
5.3
(20.
1)
10.4
(35.
2)
12.1
(36.
6)
10.6
(34.
3)
8.4
(29.
5)
13.0
(20.
0)
I/(I
) 26
.48
(3.3
7)
13.2
9 (1
.92)
8.
74 (2
.00)
11
.96
(2.8
8)
16.7
2 (2
.56)
24
.21
(6.2
5)
Com
plet
enes
s (%
) 88
.2 (7
2.2)
96
.0 (8
8.7)
88
.2 (7
7.9)
92
.4 (8
6.1)
92
.8 (8
5.3)
94
.7 (9
1.8)
Red
unda
ncy
2.8
(1.9
) 4.
1 (2
.5)
2.5
(1.8
) 3.
7 (2
.3)
2.7
(2.0
) 11
.3 (4
.6)
*Hig
hest
reso
lutio
n sh
ell i
s sho
wn
in p
aren
thes
es.
Tab
le S
II
Stru
ctur
e re
finem
ent s
tatis
tics.
W
T-fr
ee
WT-
sulfa
te
E310
Q-a
lafo
s E3
10Q
-fre
e E3
10Q
-sul
fate
Ref
inem
ent
Res
olut
ion
(Å)
50-1
.9 (1
.95-
1.9)
50
-2.0
(2.0
6-2.
0)
50-2
.4 (2
.52-
2.4)
50
-2.3
(2.4
-2.3
) 50
-2.1
(2.1
8-2.
1)
No.
refle
ctio
ns
35,2
67
33,0
30
18,2
15
22,4
60
27,9
20
Rw
ork/R
free
0.
1808
/0.2
157
(0.2
261/
0.26
47)
0.19
62/0
.232
5
(0.
2899
/0.3
242)
0.23
84/0
.287
7
(0.3
697/
0.39
84)
0.22
13/0
.251
6
(0.2
991/
0.32
58)
0.21
38/0
.248
5
(0.3
350/
0.34
67)
No.
ato
ms
Prot
ein
3,73
1 3,
708
3,62
8 3,
680
3,68
8
Ions
/Lig
and
20
45
42
20
35
Lipi
d 10
2 16
2 0
16
100
Wat
er
141
131
49
83
89
Aver
age
B-f
acto
rs (Å
2 )
Prot
ein
31.2
9 32
.60
43.0
1 32
.11
41.6
0
Ions
/Lig
and
54.1
9 60
.4
68.3
5 73
.37
65.8
4
Lipi
d 54
.67
55.2
1 —
48
.23
62.0
5
Wat
er
35.5
5 35
.94
37.0
4 27
.48
40.8
6
Coo
rdin
ates
err
or (Å
) 0.
45
0.55
0.
88
0.63
0.
68
R.m
.s. d
evia
tions
Bon
d le
ngth
s (Å
)
0.00
7 0.
006
0.00
2 0.
002
0.00
3
Bon
d an
gles
(º)
1.05
3 1.
019
0.68
8 0.
660
0.73
3
*Hig
hest
reso
lutio
n sh
ell i
s sho
wn
in p
aren
thes
es.
Tab
le S
III
Mol
ecul
ar d
ynam
ics s
imul
atio
ns p
erfo
rmed
in th
is st
udy.
Syst
em n
ame
Initi
al st
ruct
ure
E310
*Pe
ptid
e Le
ngth
(ns)
St
ruct
ural
cha
nge
S-E3
10p
sulfa
te fo
rm
P —
20
0 N
. D.
S-E3
10
sulfa
te fo
rm
D
—
200
× 2
Parti
ally
occ
lude
d
S-E3
10p-
AA
su
lfate
form
P
(Ala
) 2
100
N. D
.
S-E3
10p-
FF
sulfa
te fo
rm
P (P
he) 2
20
0 Pa
rtial
ly o
cclu
ded
*P: p
roto
nate
d, D
: dep
roto
nate
d
A
H D
B Csubstrate
G F E
H+
Out
In
Out
In
Out
In
Out
In
Out
In
Out
In
Inward-open, apo
Outward-open, apo
Occluded, apo Occluded,substrate/H+-bound
Inward-open,H+-bound
Outward-open,H+-bound
Inward-open,substrate/H+-bound
Outward-open,substrate/H+-bound
Fig. S1 Rocker-switch mechanism of the H+-coupled MFS symporters.
Panels (A)–(C), (D) and (H), and (E)–(G) represent the outward-open, occluded, and inward-open states, respectively. All steps
are reversible, and the transitions between the outward-open and inward-open states are allowed when either both substrate/H+
or none are bound. Cyan and pink rectangles indicate N- and C-terminal bundles. Green and red circles represent the substrate
and H+, respectively.
Supplementary Figures and Legends
90°
90°
HAHA
HAHA
HAHA
HBHB
HBHB
HBHB
HAHA
HBHB
PepTSo PepTSo
PepTSt PepTStC D
BA
Fig. S2 Structural comparison with the crystal structures of bacterial POTs from (A), (B) Shewanella oneidensis (PepTSo, PDB
ID: 2XUT) (3) and (C), (D) Streptococcus thermophilus (PepTSt, PDB ID: 4APS) (2).
The amino acid sequences of PepTSo and PepTSt share 26% and 46% sequence identities with GkPOT, respectively (Fig.
S3A). As expected from the sequence similarity, the structure of GkPOT is much more similar to PepTSt than PepTSo: the RMS
deviations from the PepTSo and PepTSt structures are 2.2 Å over the 311 Cα atoms and 1.7 Å over the 407 Cα atoms,
respectively. All three structures possess similar overall arrangements of the TM segments. Especially, the helices constituting
their peptide binding sites share similar structures. In contrast, several differences between GkPOT and PepTSo were observed
in the structures of the inter-helix loops and the additional helices, as well as in the precise conformations of the TM helices.
The most prominent difference is the conformation of the additional helix HA. Helix HA of PepTSo is located near helix HB
(Panels A and B), whereas that of GkPOT is apart from helix HB, and its N terminus is located near the N-terminal bundle. In
both the GkPOT and PepTSo structures, helix HA and its surrounding region have high B-factors, suggesting their flexibility.
Moreover, in several POTs from other species and other MFS family transporters, helices HA and HB are replaced with a long
loop. Therefore, the flexibility of these helices may simply allow the structural conversion between the inward- and
outward-facing conformations, and not be directly involved in the transport mechanism.
1 10 20 30 40 50 60
M A S I D K Q Q I A A S V P Q R G F F G H P K G L F T L F F T E F W E R F S Y Y G M R A I L V Y Y M Y Y E V S K G G L G L D E H L A L 671 GkPOTM T Q Q N S H G N Q I Q D I P Q T G F F G H P R G L G V L F F V E F W E R F S Y Y G M R A L L I F Y M Y F A V T D N G L G I D K T T A M 681 SauPOTM Q N L N K T E K T F F G Q P R G L L T L F Q T E F W E R F S Y Y G M R A I L V Y Y L Y A L T T A D N A G L G L P K A Q A M 621 Ll_DtpT M E D K G K T F F G Q P L G L S T L F M T E M W E R F S Y Y G M R A I L L Y Y M W F L I S T G D L H I T R A T A A 571 PepT_St X P L S I F F I V V N E F C E R F S Y Y G M R A I L I L Y F T N F I S W D D N L S T 421 Hs_PepT1 X P L S I A F I V V N E F C E R F S Y Y G M K A V L I L Y F L Y F L H W N E D T S T 421 Hs_PepT2 M T T P V D A P K W P R Q I P Y I I A S E A C E R F S F Y G M R N I L T P F L M T A L L L S I P E E L R G A V A K 571 PepT_So
70 80 90 100 110 120
A I M S I Y G A L V Y M S G I I G G W L A D R V F G T S R A V F Y G G L L I M A G H I A L A I P G G V A A 12068 GkPOTS I M S V Y G S L I Y M T S I P G G W I A D R I T G T R G A T L L G A V F I I I G H I C L S L P F A L I G 12169 SauPOTA I V S I Y G A L V Y L S T I V G G W V A D R L L G A S R T I F L G G I L I T L G H I A L A T P F G L S S 11563 Ll_DtpTS I M A I Y A S M V Y L S G T I G G F V A D R I I G A R P A V F W G G V L I M L G H I V L A L P F G A S A 11058 PepT_StA I Y H T F V A L C Y L T P I L G A L I A D S W L G K F K T I V S L S I V Y T I G Q A V T S V S S I N D L T D H N H D G T P D S L P V H V V L 11343 Hs_PepT1S I Y H A F S S L C Y F T P I L G A A I A D S W L G K F K T I I Y L S L V Y V L G H V I K S L G A L P I L G G Q V V H T V L 10443 Hs_PepT2D V F H S F V I G V Y F F P L L G G W I A D R F F G K Y N T I L W L S L I Y C V G H A F L A I F E H S V Q G F Y 11358 PepT_So
130 140 150 160 170 180
L F V S M A L I V L G T G L L K P N V S S I V G D M Y K P G D D R R D A G F S I F Y M G I N L G A F L A P L V V G T A G M K Y N 184121 GkPOTL F T S M F F I I I G S G L M K P N I S N I V G R L Y P E N D K R M D A G F V I F Y M S V N M G A L L S P I I L Q H F V N V K N 185122 SauPOTL F V A L F L I I L G T G M L K P N I S N M V G H L Y S K D D S R R D T G F N I F V V G I N M G S L I A P L I V G T V G Q G V N 179116 Ll_DtpTL F G S I I L I I I G T G F L K P N V S T L V G T L Y D E H D R R R D A G F S I F V F G I N L G A F I A P L I V G A A Q E A A G 174111 PepT_StS L I G L A L I A L G T G G I K P C V S A F G G D Q F E E G Q E K Q R N R F F S I F Y L A I N A G S L L S T I I T P M L R V Q Q C G I H S K Q 184114 Hs_PepT1S L I G L S L I A L G T G G I K P C V A A F G G D Q F E E K H A E E R T R Y F S V F Y L S I N A G S L I S T F I T P M L R G D V Q C F G E 173105 Hs_PepT2 T G L F L I A L G S G G I K P L V S S F M G D Q F D Q S N K S L A Q K A F D M F Y F T I N F G S F F A S L S M P L L L K N 174114 PepT_So
190 200 210 220 230 240 250
F H L G F G L A A V G M F L G L V V F V A T R K K N L G L A G T Y V P N P L T P A E K K K A A A I M A V G A V V I A V L L A I L I P N G 252185 GkPOT F H G G F L I A A V G M A L G L V W Y V L F N R K N L G S V G M K P T N P L T P A E K K K Y G L I I G S V V L A I V L I I V I G A L T N 253186 SauPOT Y H L G F S L A A I G M I F A L F A Y W Y G R L R H F P E I G R E P S N P M D S K A R R N F L I T L T I V V I V A I I G F F L L Y Q A S P 248180 Ll_DtpT Y H V A F S L A A I G M F I G L L V Y Y F G G K K T L D P H Y L R P T D P L A P E E V K P L L V K V S L A V A G F I A I I V V M N L V G 242175 PepT_StA C Y P L A F G V P A A L M A V A L I V F V L G S G M Y K K F K P Q G N I M G K V A K C I G F A I K N R F R H R S K A 243185 Hs_PepT1D C Y A L A F G V P G L L M V I A L V V F A M G S K I Y N K P P P E G N I V A Q V F K C I W F A I S N R F K N R S G D 232174 Hs_PepT2F G A A V A F G I P G V L M F V A T V F F W L G R K R Y I H M P P E P K D P H G F L P V I R S A L L T K V E G K G N I G L V L A L I G G V S 244175 PepT_So
260 270 280 290 300
W F T V E T F I S L V G I L G I I I P I I Y F V V M Y R S P K T T A E E R S R V I A Y I P L F V A S A M F W A I Q 309253 GkPOTS L S F N L V S N T V L V L G I A L P I I Y F T L I I R S K D V T D T E R S R V K A F I P L F I L G M V F W A I Q 310254 SauPOTA N F I N N F I N V L S I I G I V V P I I Y F V M M F T S K K V E S D E R R K L T A Y I P L F L S A I V F W A I E 305249 Ll_DtpTW N S L P A Y I N L L T I V A I A I P V F Y F A W M I S S V K V T S T E H L R V V S Y I P L F I A A V L F W A I E 299243 PepT_St F P K R E H W L D W A K E K Y D E R L I S Q I K M V T R V M F L Y I P L P M F W A L F 286244 Hs_PepT1 I P K R Q H W L D W A A E K Y P K Q L I M D V K A L T R V L F L Y I P L P M F W A L L 275233 Hs_PepT2A A Y A L V N I P T L G I V A G L C C A M V L V M G F V G A G A S L Q L E R A R K S H P D A A V D G V R S V L R I L V L F A L V T P F W S L F 315245 PepT_So
310 320 330 340 350 360 370
E Q G S T I L A N Y A D K R T Q L D V A G I H L S P A W F Q S L N P L F I I I L A P V F A W M W V K L G K R Q P T I P Q K F A L 373310 GkPOTE Q G S N V L N I Y G I E H S D M K L N L F G W K T N F G E A I F Q S I N P L F I L L L A P I I S L L W Q K L G T K Q P S L P V K F A I 378311 SauPOTE Q S S T I I A V W G E S R S N L D P T W F G I T F H I D P S W Y Q L L N P L F I V L L S P I F V R L W N K L G E R Q P S T I V K F G L 373306 Ll_DtpTE Q G S V V L A T F A A E R V D S S W F P V S W F Q S L N P L F I M L Y T P F F A W L W T A W K K N Q P S S P T K F A V 359300 PepT_StD Q Q G S R W T L Q A T T M S G K I G A L E I Q P D Q M Q T V N A I L I V I M V P I F D A V L Y P L I A K C G F N F T S L K K M A V 352287 Hs_PepT1D Q Q G S R W T L Q A I R M N R N L G F F V L Q P D Q M Q V L N P L L V L I F I P L F D F V I Y R L V S K C G I N F S S L R K M A V 341276 Hs_PepT2D Q K A S T W I L Q A N D M V K P Q W F E P A M M Q A L N P L L V M L L I P F N N F V L Y P A I E R M G V K L T A L R K M G A 378316 PepT_So
380 390 400 410 420 430 440
G L L F A G L S F I V I L V P G H L S G G G L V H P I W L V L S Y F I V V L G E L C L S P V G L S A T T K L A P A A F S A Q T M S L W F L S 443374 GkPOTG T F L A G A S Y I L I G I V G Y A S G S S N F S V N W V I L S Y I I C V I G E L C L S P T G N S A A V K L A P K A F N A Q M M S I W Y L T 448379 SauPOTG L M L T G I S Y L I M T L P G L L N G T S G R A S A L W L V L M F A V Q M A G E L L V S P V G L S V S T K L A P V A F Q S Q M M A M W F L A 444374 Ll_DtpTG L M F A G L S F L L M A I P G A L Y G T S G K V S P L W L V G S W A L V I L G E M L I S P V G L S V T T K L A P K A F N S Q M M S M W F L S 430360 PepT_StG M V L A S M A F V V A A I V Q V E I D X T V N M A L Q I P Q Y F L L T C G E V V F S V T G L E F S Y S Q A P S N M K S V L Q A G W L L T 421353 Hs_PepT1G M I L A C L A F A V A A A V E I K I N X K M S I A W Q L P Q Y A L V T A G E V M F S V T G L E F S Y S Q A P S S M K S V L Q A A W L L T 410342 Hs_PepT2G I A I T G L S W I V V G T I Q L M M D G G S A L S I F W Q I L P Y A L L T F G E V L V S A T G L E F A Y S Q A P K A M K G T I M S F W T L S 449379 PepT_So
450 460 470 480 490
N A A A Q A I N A Q L V R F Y T P E N E T A Y F G T I G G A A L V L G L I L L A I A P R I G R L M K G I R 496444 GkPOTN A S A Q A I N G T L V K L I E P L G Q T N Y F I F L G V V A I I V T T I V L A F S P L I I K A M K G I R 501449 SauPOTD S T S Q A I N A Q I T P L F K A A T E V H F F A I T G I I G I I V G I I L L I V K K P I L K L M G D V R 497445 Ll_DtpTS S V G S A L N A Q L V T L Y N A K S E V A Y F S Y F G L G S V V L G I V L V F L S K R I Q G L M Q G V E 483431 PepT_StV A V G N I I V L I V A G A G Q F S K Q W A E Y I L F A A L L L V V C V I F A I M A R F Y T Y I N P A E I 474422 Hs_PepT1I A V G N I I V L V V A Q F S G L V Q W A E F I L F S C L L L V I C L I F S I M G Y Y Y V P V K T E D M 462411 Hs_PepT2V T V G N L W V L L A N V S V K S P T V T E Q I V Q T G M S V T A F Q M F F F A G F A I L A A I V F A L Y A R S Y Q M Q D H Y R Q 514450 PepT_So
A
R43R36E32
Y78
K136 N166
W306
N342
E413
E35
Peptide binding site
N bundleC bundle
N bundleC bundle
Intracellular
Extracellular
B
90°
Fig. S3 Conserved residues of the POT family transporters.
(A) Amino-acid sequence alignment of POT family transporters from Geobacillus kaustophilus (GkPOT), Staphylococcus
aureus (SauPOT), Lactococcus lactis (Ll_DtpT), Streptococcus thermophilus (PepT_St), Homo sapiens (Hs_PepT1 and
Hs_PepT2), and Shewanella oneidensis (PepT_So). The conserved amino acids involved in the transport mechanism and
discussed in the main text are enclosed by rectangles. (B) Conserved residues mapped on the molecular surface of GkPOT. The
surface is colored in increasing shades of red reflecting increasing residue conservation, calculated using the program rate4site
(22) based on the sequence alignment in panel (A).
A
E413E413
E310E310
2.7 Å
N342N342
SO42-SO42-
Y40Y40
Y78Y78
R43R43
B
Central cleftCytoplasm Central cleft
Periplasm
HydrophobicLayer
ExtracellularHydrogen-bondNetwork
IntracellularHydrogen-bondNetwork
Fig. S4 The electron density maps of the GkPOT crystal structures.
(A) Stereo view of the unbiased 2m|Fo|–D|Fc| electron density map of GkPOT in the substrate-free form around the central cleft,
contoured at 1.0 σ. The GkPOT structure is depicted by stick models.
(B) The unbiased m|Fo|–D|Fc| omit electron density maps of the SO42–-bound structure, contoured at 3.5 σ. In the calculation of
the maps, the models of Glu310 and the SO42– ion were omitted.
Fig. S5 The hydrophilic and hydrophobic interactions around the extracellular gate.
The magenta and violet lines indicate the extracellular and intracellular hydrogen-bonding interaction networks, respectively.
Hydrophobic residues involved in the extracellular gate formation are depicted by sticks.
Arg43H4
H2
H11
H10
H8
H7
Tyr78
SO42–
Tyr78
SO42–
Arg43
A B
HAHB
H4
N bundle
N bundle
C bundle
C bundle
H2
H11
H10
H8
H7
A B
R43R43
R36R36 E32E32
E35E35
K136K136
W440W440
W306W306
E413E413
E310QE310Q
N342N342
F441F441
Y40Y40
Y78Y78
A169A169P343P343
R43R43
R36R36 E32E32
E35E35
K136K136W440W440
W306W306
E413E413E310QE310Q
N342N342
F441F441
Y40Y40
Y78Y78
A169A169P343P343
SO42-SO42-
Fig. S6 Structural comparison between the free and sulfate-bound forms of GkPOT.
(A) Cytoplasmic view of the GkPOT structure in the sulfate-bound form (in gray), compared to the structure in the free form
(with the same coloring as in Fig. 1). (B) Close-up view of the central cleft. The sulfate ion and the side chains of Arg43 and
Tyr78 are depicted by ball-and-stick models. As shown in this figure, the only differences between the free and sulfate-bound
forms (and the alafosfalin-bound form, as well) are the side chain conformations of Arg43 and Tyr78. In the sulfate-bound
form, the guanidinium group of the Arg43 side chain is shifted toward the bound sulfate ion. The side chain of Tyr78 is flipped
into the peptide-binding site, and recognizes the sulfate ion (and the phosphonate group of alafosfalin in the alafosfalin-bound
form). This inward flipping of the Tyr78 side chain into the peptide binding site drastically changes the shape of the
peptide-binding site, which may enable the recognition of the C-terminal side chain of the substrate dipeptide (Fig. S8D, E).
Fig. S7 The structures of the substrate binding site of the GkPOT E310Q variant, in the (A) free and (B) sulfate-bound forms.
In panels (A) and (B), the crystal structures of the wild-type protein are also shown, with a transparent, gray color. The
resulting structures of the variant are almost the same as those of the wild-type protein, with Cα RMSDs of 0.48 Å and 0.24 Å,
respectively. The glutamine side chain can mimic the neutral charge of the protonated glutamate side chain. The results
indicated that the replacement of Glu310 with Gln does not perturb the surrounding structures, and suggested that the charge
around the Glu310 side chain in the wild type protein is also neutral, and thus it is protonated.
PHO
O
CH3
NH
CH3
O
NH3+
-O
A
D E
N342
N342Q309
E310Q
E413E413
N166
N166
Y40
Y78
Y78
W440
W440
F441
W306
W306
R36
K136
90°
B C
N342
N342Q309
E310Q
E413E413
N166
N166
Y40
Y78
Y78
W440
W440
F441
W306
W306
R36
K136
E310QE310Q
Y40Y40
R36R43 R43
H1
AlafosfalinAlafosfalin
H5H8
H7
Y78Y78
N342N342
Fig. S8 Crystal structure of the GkPOT-E310Q variant in complex with alafosfalin.
(A) Chemical structure of alafosfalin, [(1R)-1-[[(2S)-2-amino-1-oxopropyl]amino]ethyl]phosphonic acid. (B), (C) The electron
density maps of alafosfalin bound to the GkPOT-E310Q variant. (B) The unbiased 2m|Fo|–D|Fc| density map contoured at 1.1 σ
and (C) the unbiased m|Fo|–D|Fc| omit electron density map contoured at 3.5 σ are shown. In panel (B), the density map around
alafosfalin and its surrounding side chains is shown. In panel (C), the model of alafosfalin was omitted in the calculation of the
omit map. (D), (E) Interaction between alafosfalin and the peptide binding pocket of GkPOT. In panels (D) and (E), cross
sections of the molecular surface cut by the plane in the membrane and the plane perpendicular to the membrane are shown,
respectively. The surfaces of the additional helices, N, and C bundles are colored yellow, cyan, and pink, respectively. The
alafosfalin molecule is shown by a space-filling model. The protein side chains involved in the interaction are depicted by stick
models. The cavities found around the alafosfalin are indicated by dashed white circles.
R43R43
R36R36E32E32
E35E35K136K136
W440W440
W306W306
E413E413
E310E310
N342N342
F441F441
Y40Y40
Y78Y78
A169A169P343P343
S-E310pS-E310
S-E310pS-E310
0 50 100 150 200 250 250 300 350 400 450 500Residue number Residue number
0
1
2
3
4
5
RM
SF
(Å)
0
1
2
3
4
5
RM
SF
(Å)
H1 H2 H3 H4 H5 H6 HB H7 H8 H9 H10 H11 H12
Fig. S9 The (Ala)3 peptide docking model of GkPOT, based on the GkPOT-alafosfalin complex structure.
The present complex structure with alafosfalin revealed extra space on the C-terminal side of the alafosfalin (Fig. S8D), which
can accommodate the third residue of the tripeptide. Based on this observation, we created a docking model with the (Ala)3
tripeptide. In this model, the protonated Glu310 and Arg43 side chains recognize the carbonyl group of the tripeptide. The
C-terminal carboxylate group could be recognized by the Lys136 and/or Arg36 side chains. The side chain of Tyr39 could also
be involved in the tripeptide recognition, which is consistent with the observation that the Phe mutation of the corresponding
Tyr29 of PepTSt only affected the tripeptide recognition (2).
Fig. S10 Plots of the time-averaged RMS fluctuations of each residue from the initial crystal structures, during the S-E310p
and S-E310 simulations.
The left and right panels are the plots of the N- and C-bundles, respectively. The locations of the TM segments are indicated by
the shaded boxes.
H4 & H5
H4
Pro137
Pro173
H5
C bundle
S-E310 (84.52 ns)
H4 & H5 C bundle
FucP
H4 & H5 C bundle
EmrD
A B
C D
Fig. S11 Structural change observed in the MD simulations.
(A) The structure of the H4-H5 hairpin. The crystal structure and the most-closed snapshot of the S-E310 simulation (84.52 ns)
are colored green and cyan, respectively. The helix-breaking Pro residues, discussed in the main text, are depicted by stick
models. (B) The structures of the H4-H5 hairpin and the C-terminal bundle in the most-closed snapshot of the S-E310
simulation (84.52 ns). The snapshot structure is colored green, while the initial crystal structure is cyan (H4-H5) and pink
(C-terminal bundle). (C), (D) The structures of the H4-H5 hairpin and the C-terminal bundle of FucP (PDB ID: 3O7P) and
EmrD (PDB ID: 2GFP). The crystal structures of FucP and EmrD are colored blue, while the crystal structure of GkPOT is
cyan (H4-H5) and pink (C-terminal bundle).
S-E310p-FFS-E310p
S-E310S-E310p
A B
Time (ns)0
Dom
ain
rota
tion
(°)
12
10
8
6
4
2
020 40 60 80 100 120 140 160 180 200
Time (ns)0
Dom
ain
rota
tion
(°)
12
10
8
6
4
2
020 40 60 80 100 120 140 160 180 200
C bundleN bundle
Fig. S11 The rotation angle of the N-terminal bundle against the C-terminal bundle as a function of time, during the (A)
S-E310 and (B) S-E310p-FF simulations. In both panels, the rotational angle of the S-E310p simulation is plotted for
comparison.
Fig. S11 Time series of the distance changes during the MD simulations.
(A), (B) Time series of the distance between the Cα atoms of Arg36 (N bundle) and Asn444 (C bundle), which are located at
the two extremities of the peptide binding site (Fig. 3A). (C) Time series of the distance between the cytosolic-side helices of
the N- and C-terminal bundles (residues 141-156 and 420-440, respectively) in the second run of the S-E310 simulations. The
plots of S-E310p and S-E310 are also shown, for comparison. (D) The distance between Arg43 and Glu310 during the S-E310p
and S-E310 simulations. The shortest distance between the side-chain guanidinium nitrogen atoms and the carboxylate oxygen
atoms is plotted.
H4
5-H
1011
Dis
tanc
e (Å
)
Time (ns)0
24
22
20
18
16
14
1220 40 60 80 100 120 140 160 180 200
R36
-N44
4 C
Dis
tanc
e (Å
)
21
20
19
18
17
16
15
Time (ns)0 50 100 150 200
A BR
36-N
444
C D
ista
nce
(Å)
21
20
19
18
17
16
15
Time (ns)0 50 100 150 200
S-E310p-FFS-E310p
S-E310S-E310p
S-E310S-E310p
C
Arg
43-G
lu31
0 di
stan
ce (Å
)
0
2
4
6
8
10
Time (ns)0 20 40 60 80 100 120 140 160 180 200
S-E310S-E310-2
S-E310p
D
A S-E310p-AA initial structure B S-E310p-AA 100 ns
C S-E310p-FF initial structure D S-E310p-FF 200 ns
R43R43
W440W440
W306W306
E413E413E310E310
N342N342
F441F441
Y40Y40
Y78Y78
A169A169P343P343
R43R43
W440W440
W306W306
E413E413E310E310
N342N342
F441F441
Y40Y40
Y78Y78
A169A169P343P343
R43R43
W440W440
W306W306
E413E413E310E310
N342N342
F441F441
Y40Y40
Y78Y78
A169A169P343P343
R43R43
W440W440
W306W306
E413E413E310E310
N342N342
F441F441
Y40Y40
Y78Y78
A169A169P343P343
Fig. S14 MD simulations of the peptide-bound structures.
(A), (B) The structures of the substrate binding site of (A) the initial structure and (B) the final (100 ns) snapshot of the
S-E310p-AA simulation. (C), (D) The structures of the substrate binding site of (C) the initial structure and (D) the final (200
ns) snapshot of the S-E310p-FF simulation.
0.0005
0.0000
-0.0005
Abs
.
1700 1600 1500
Wavenumber (cm-1)
16511660
16701680
1549
1533
1609
1624
1690
Abs
.
1700 1600 1500
Wavenumber (cm-1)
16511660
16701680
1549
15331609
16241690
0.002
0.001
0.000
Abs
. at 1
533
and
1549
cm
-1
0.12 3 4 5 6 7 8
12 3 4 5 6 7 8
102 3 4 5 6 7 8
100
Concentration of AlaAla (mM)
A C
B
Fig. S15 Dynamics of GkPOT investigated by ATR-FTIR measurements.
(A) Difference ATR-FTIR spectra between the presence (positive signal) and absence (negative signal) of 2 mM (Ala)2
dipeptide, at pH 7.0 (red line) and pH 5.0 (blue line), in the 1712–1470 cm−1 region. (B) Difference ATR-FTIR spectra between
the presence (positive signal) and absence (negative signal) of 1 mM (top), 2 mM (second from top), 4 mM (second from
bottom) and 10 mM (bottom) (Ala)2 dipeptide, at pH 7.0 in the 1712–1490 cm−1 region. One division of the y-axis corresponds
to 0.001 absorbance unit. (C) Binding affinity of the (Ala)2 dipeptide to GkPOT at pH 7.0, estimated from the difference
absorbance of the amide-II band (the value at 1533 cm−1 minus that at 1549 cm−1). The Hill equation was used for the curve
fitting.
A H+-driven uptake, pH profile, WT B H+-driven uptake, pH profile, R43Q
3 H A
laAl
a [n
mol
es m
g-1 G
kPO
T ]
3 H A
laAl
a [n
mol
es m
g-1 G
kPO
T ]
pH 6
pH 6.
5pH
7
pH 7.
5pH
80
20
40
60
80
pH 6
pH 6.
5pH
7
pH 7.
5pH
80
5
10
15
* * *WT
no pr
oteinCCCP
Y78A
Y78F
W30
6A0
50
40
30
20
10
3 H (A
la) 2
[nm
oles
mg–1
GkP
OT]
Fig. S16 The pH profiles of the H+-driven 3H-labeled (Ala)2 peptide uptake activity of (A) wild type GkPOT and (B) R43Q
mutant.
Fig. S17 Effect of substitutions within the putative peptide substrate binding site on H+-driven uptake.
‘CCCP’ refers to the addition of the proton ionophore, carbonyl cyanide m-chlorophenyl hydrazine, to the external buffer. Error
bars indicate the standard deviations from triplicate experiments. Asterisks indicate that activity was not detectable.
— 30 —
References
1. Fang G, Konings WN, & Poolman B (2000) Kinetics and substrate specificity of
membrane-reconstituted peptide transporter DtpT of Lactococcus lactis. J.
Bacteriol. 182(9):2530-2535.
2. Solcan N, et al. (2012) Alternating access mechanism in the POT family of
oligopeptide transporters. EMBO J 31:3411 - 3421.
3. Newstead S, et al. (2011) Crystal structure of a prokaryotic homologue of the
mammalian oligopeptide-proton symporters, PepT1 and PepT2. EMBO J.
30(2):417-426.
4. Bolger MB, et al. (1998) Structure, function, and molecular modeling approaches
to the study of the intestinal dipeptide transporter PepT1. J. Pharm. Sci.
87(11):1286-1291.
5. Bailey PD, et al. (2000) How to make drugs orally active: A substrate template for
peptide transporter PepT1. Angew. Chem.-Int. Edit. 39(3):505-508.
6. Dang SY, et al. (2010) Structure of a fucose transporter in an outward-open
conformation. Nature 467(7316):734-738.
7. Yin Y, He X, Szewczyk P, Nguyen T, & Chang G (2006) Structure of the multidrug
transporter EmrD from Escherichia coli. Science 312(5774):741-744.
— 31 —
8. Guan L & Kaback HR (2006) Lessons from lactose permease. Annual Review of
Biophysics and Biomolecular Structure, Annual Review of Biophysics, (Annual
Reviews, Palo Alto), Vol 35, pp 67-91.
9. Kawate T & Gouaux E (2006) Fluorescence-detection size-exclusion
chromatography for precrystallization screening of integral membrane proteins.
Structure 14(4):673-681.
10. Collaborative Computational Project N (1994) The CCP4 Suite: Programs for
Protein Crystallography. Acta Crystallogr. D50:760-763.
11. Schneider TR & Sheldrick GM (2002) Substructure solution with SHELXD. Acta
Crystallogr. D58(Pt 10 Pt 2):1772-1779.
12. de La Fortelle E & Bricogne G (1997) Maximumlikelihood heavy-atom parameter
refinement for multiple isomorphous replacement and multiwavelength anomalous
diffraction methods. Methods Enzymol. 276:472-494.
13. Abrahams JP & Leslie AGW (1996) Methods used in the structure determination of
bovine mitochondrial F-1 ATPase. Acta Crystallogr. D52:30-42.
14. Emsley P & Cowtan K (2004) Coot: model-building tools for molecular graphics.
Acta Crystallogr. D60:2126-2132.
15. Adams PD, et al. (2002) PHENIX: building new software for automated
— 32 —
crystallographic structure determination. Acta Crystallogr. D58:1948-1954.
16. Humphrey W, Dalke A, & Schulten K (1996) VMD: Visual molecular dynamics. J.
Mol. Graph. 14(1):33-38.
17. Dolinsky TJ, et al. (2007) PDB2PQR: expanding and upgrading automated
preparation of biomolecular structures for molecular simulations. Nucleic Acids Res.
35:W522-W525.
18. Mackerell AD, Feig M, & Brooks CL (2004) Extending the treatment of backbone
energetics in protein force fields: Limitations of gas-phase quantum mechanics in
reproducing protein conformational distributions in molecular dynamics
simulations. J. Comput. Chem. 25(11):1400-1415.
19. Phillips JC, et al. (2005) Scalable molecular dynamics with NAMD. J. Comput.
Chem. 26(16):1781-1802.
20. Darden T, York D, & Pedersen L (1993) Particle Mesh Ewald - An N.Log(N)
Method For Ewald Sums In Large Systems. J. Chem. Phys. 98(12):10089-10092.
21. Kitade Y, Furutani Y, Kamo N, & Kandori H (2009) Proton Release Group of
pharaonis Phoborhodopsin Revealed by ATR-FTIR Spectroscopy. Biochemistry
48(7):1595-1603.
22. Pupko T, Bell RE, Mayrose I, Glaser F, & Ben-Tal N (2002) Rate4Site: an