— 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 —

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