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Table1. Calcium content of purified AlgQ2

Ca (µM）

(Ca mol)/(protein mol)

wtAlgQ2

11

1.1

mtAlgQ2

1.7

0.17

510

511

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Table 2. Data collection and refinement statistics

Data collection

Wavelength (Å)

Resolution range (Å)

Space group

Unit-cell parameters (Å, °)

a, b, c

α, β, γ

Total observations

Unique reflections

Completeness (%)

I/σ(I)

Rmerge

Rmean

CC1/2

Wilson B (Å2)

Refinement

Resolution range (Å)

Rwork/Rfree

Protein molecules/ASU

No. atoms

Protein

Saccharide

Calcium ion

Water molecule

RMSD

Bond lengths (Å)

Bond angles (deg.)

Ramachandran plot

Most favored (%)

Allowed (%)

Outlier (%)

PDB ID

512

1.00000

50.0–2.20 (2.24–2.20) a

P1

45.87, 60.82, 87.23

80.37, 89.81, 88.17

124992 (5993)

47003 (2305)

97.8 (97.9)

63.6 (5.5)

0.145 (0.362)

0.178 (0.452)

0.821

29.6

50.0-2.20 (2.26-2.20)

0.210 (0.268)/0.271 (0.336)

7974

72

200

0.012

1.517

97.3

2.3

0.4

6JHX

The highest resolution shell is shown in parentheses.

513

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Table 3. Apparent Tm (°C)

No ligand

(Topen)

wtAlgQ2

45.63

mtAlgQ2

<25

CaCl2

(Topen)

45.63

<25

Δ4M

(Tclosed)

63.18

55.72

514

515

25

CaCl2+Δ4M

(Tclosed)

63.18

55.72

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Figure legends

517

Figure 1. X-ray crystal structures of ABC transporter and AlgQ2. (a) (Left) The complex

518

structure of AlgQ2 and AlgM1M2SS (PDB ID: 4TQU). Magenta, AlgQ2; green, AlgM1;

519

cyan, AlgM2; orange, AlgS. Calcium ion in AlgQ2 is indicated by a yellow ball. (Right)

520

Magnified image at the interface between AlgQ2 and AlgM2. (b) EF-hand-like calcium-

521

binding site of AlgQ2. (c) calcium (yellow ball) is far from the substrate (red/white balls)-

522

binding site of AlgQ2.

523

Figure 2. X-ray crystal structure of mtAlgQ2. (a) Overall structure of mtAlgQ2. Gray

524

and red-colored sticks indicate unsaturated alginate trisaccharide. (b) (Left) Structure of

525

mutated EF-hand-like motif. Red, EF-hand-like motif helix; green, loop. (Right) The

526

figure shows the 2Fo-Fc map contoured at 1.2 σ around the EF-hand-like motif. (c)

527

Superimposing of wtAlgQ2 and mtAlgQ2 for all Cα atoms. Blue, mtAlgQ2; orange,

528

wtAlgQ2. Calcium ion is indicated by a yellow ball.

529

Figure 3. Thermal shift assay by DSF. Top, fluorescent profile. Bottom, negative

530

derivative curve plot of the fluorescent profile. Red, wtAlgQ2 without Δ4M; blue,

531

mtAlgQ2 without Δ4M; black, wtAlgQ2 with Δ4M; green, mtAlgQ2 with Δ4M.

532

Figure 4. Native PAGE profile of AlgQ2. (a) wtAlgQ2 or mtAlgQ2 with or without 50-

533

µM Δ4M. (b) Binding ability of wtAlgQ2 (upper) and mtAlgQ2 (lower) to Δ4M in

534

proportion to increasing substrate concentration. (c) Profile of formation of AlgQ2 and

535

Δ4M complex. Circle; wtAlgQ2, square; mtAlgQ2.

536

Figure 5. ATPase and transport activity of AlgM1M2SS. (a) ATPase activity in the

537

presence (+) or absence (−) of each component in the reaction mixture. ATPase activity

538

was represented as phosphate (nmol) produced by 1-mg AlgM1M2SS per 1 min. (b)

539

Transport activity. Transport activity used PAΔ4M as a substrate in the presence (+) or

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absence (−) of each component in the reaction mixture. Activity of AlgM1M2SS in the

541

presence of wtAlgQ2 was taken as 100%. Negative values indicate no transport activity

542

calculated by subtraction of the mean values of the observed transport activity in

543

liposomes without AlgM1M2SS. (c) ATPase activity using PAΔ4M as a substrate in the

544

presence (+) or absence (−) of each component in the reaction mixture. Assays were

545

performed three times, and the error bars represent SE.

546

Figure 6. Mechanistic model of ABC transporter for alginate import. The dynamics of

547

ABC transporter machinery model. Pink, wtAlgQ2; purple, mtAlgQ2; light yellow,

548

calcium; gray, alginate; green, AlgM1; blue, AlgM2; dark yellow, AlgS.

549

550

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552

Graphical abstract. The calcium bound to EF-hand-like motif stabilizes the substrate-

553

unbound AlgQ2, while calcium-free AlgQ2 adopts a closed conformation in complex

554

with substrate.

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556

Figure 1.

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Figure 2.

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560

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Figure 3.

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Figure 4.

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567

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569

Figure 5.

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571

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Figure 6.

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577

Figure S1. PAΔ4M-binding ability of wtAlgQ2 and mtAlgQ2 with increasing PAΔ4M

578

concentration. Top, wtAlgQ2; bottom, mtAlgQ2. The difference in the band profile from

579

Figure 4b was thought to be due to positive-charge donated by PA.

580

581

35

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