University of Groningen Insights into the transport mechanism of energy-coupling factor transporters Stanek, Weronika Karolina

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University of Groningen

Insights into the transport mechanism of energy-coupling factor transporters

Stanek, Weronika Karolina

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SUPPLEMENT

AR

Y

T

ABLES

Supplementary

Table 1 Substrate binding affinity determined for

differ

ent S-components

Protein Substrate Method Sample Value Author FolT Lactobacillus casei [ 3H]folate

Radiolabeled substrate binding

to cells Whole cells at 4°C kd 36 nM, binding 0.35 nmol/10 10 cells 1 ThiT Lactobacillus casei [thiazole-2-14C]thiamine

to cells

Whole cells with glucose

and iodoacetate kd < 10 nM 2 ECF-FolT Lactobacillus casei [3’,5’,9(n)-3H]folate

to cells

Whole cells, not ener

gized 0.45 nmol/10 10 cells 3 ECF-ThiT Lactobacillus casei [thiazole-2-14C]thiamine

to cells

Whole cells, not ener

gized 0.70 nmol/10 10 cells 3 ECF-FolT Lactobacillus casei [3’,5’,7,9-3H]folate

to cells at 0°C

Whole cells and membranes

- kd 0.26 nM cells - kd 0.35 nM membranes k1 4.9x 10 7 liter/mol/min; k-1 0.028/min 4 ECF-ThiT Lactobacillus casei [ 35S]thiamine

to cells

Whole cells, deener

gized kd 0.03 nM 5 ECF BioY Lactobacillus casei [ 3H]biotin

to cells

Whole cells, deener

gized kd 0.15 nM 5 RibU Lactococcus lactis - Riboflavin - Riboflavin - Riboflavin - Roseoflavin - FMN - FAD - Riboflavin - ITC - ITC - Fluorescence titration - Fluorescence Trp titration - Fluorescence titration - Fluorescence titration - Flow dialysis - Purified - membrane vesicles - Purified - Purified - Purified - Purified - Purified - kd 1.8 ± 0.7 nM - kd 5.0 ± 1.0 nM - kd 0.6 ± 0.2 nM - kd 2.5 ± 1.2 nM - kd 36 ± 6 nM - no binding - kd 1.7 ± 3.2 nM 6 FolT Lactobacillus casei Folate Fluorescence quenching of tryptophan Purified kd 9 nM 7 ThiT Lactobacillus casei Thiamine Fluorescence quenching of tryptophan Purified kd 0.5 nM 7

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ThiT Lactococcus lactis - Thiamine - TMP - TPP - Pyrithiamin Fluorescence quenching of tryptophan Purified - kd 122 ± 13 pM - kd 1.01 nM - kd 1.6 nM - kd 180 ± 70 pM 8 BioY Lactococcus lactis D-biotin Fluorescence quenching of tryptophan Purified kd 0.3 nM 9 BioY Rhodobacter capsulatus D-biotin Fluorescence quenching of tryptophan Purified

Very high affinity

, not possible to determine 9 ThiT Lactococcus lactis Thiamine Fluorescence quenching of tryptophan Purified kon 3.4 x 10 8 M -1 s -1 koff very slow (0.004 s -1) (kd 1 1 pM) 10 FolT Enterococcus faecalis Folate ITC Purified kd 29.8 ± 4.7 nM 11 FolT1 Lactobacillus delbrueckii Folate Fluorescence quenching of tryptophan Purified kd 1.0 ± 0.24 nM 12 FolT2 Lactobacillus delbrueckii Folate Fluorescence quenching of tryptophan Purified kd 20 ± 2.19 nM 12 YkoE Bacillus subtilis Thiamine Fluorescence quenching of tryptophan Purified kd 4.5 nM 13 RibU Listeria monocytogenes Riboflavin FSEC

Purified, released from

whole complex kd 526 ± 74 nM 14 ECF(H>A) RibU Listeria monocytogenes AT P

SEC with samples preincubated with varying

ATP

concentrations

Purified full complex with double H-loop

mutation kd 101 ± 19 µM 14 CbrT Lactobacillus delbrueckii - Cyanocobalamine - Cobinamide - Hydroxycobalamine - Methylcobalamine ITC Membrane vesicles kd 9.2 ± 4.5 nM k 36 ± 15 nMd kd 9.6 ± 6.9 nM kd 4.5 ± 0.3 nM 15 PanT Lactobacillus delbrueckii Pantothenate ITC Membrane vesicles kd 21.4 ± 22.9 nM This study FolT2 Lactobacillus delbrueckii Folate ITC Membrane vesicles kd 14.4 ± 9.3 nM This study

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Supplementary

Table 2 Structurally characterized pr

oteins in ECF

transporters family

, classified by the

type.

Transporter Organism Nucleotide state Orientation PDB code Resolution [Å] Refer ences Type I ECF transporters Tt NikM Thermoanaer obacter tengcongensis

-Nickel bound (The four

nitrogen atoms form an

square-planar geometry)

4M58 (truncated structures with Ni

2+

4M5B and Co

2+

4M5C)

3,2 (1,83 for the truncated) 2,5 (Co bound)

16 CbiO Thermoanaer obacter tengcongensis No nucleotide -4MKI 2,3 17 YkoE Bacillus subtilis -Thiamine bound 5EDL 1,5 (LCP) 13 CbiMQO Rhodobacter capsulatus

-Inward-facing (no CbiN)

5X3X 2,79 18 CbiMQO Rhodobacter capsulatus

-Inward-facing (no CbiN)

5X41

3,47 (LCP)

18

CbiO E166Q dimer

Rhodobacter capsulatus AMP-PCP -5X40 1,45 18 Type II ECF transporters ECF-FolT Lactobacillus br evis Nucleotide free Inward-facing, no substrate bound 4HUQ 3 19 ECF-HmpT (pyridoxin transporter) Lactobacillus br evis Nucleotide free Inward-facing, no substrate bound 4HZU 3,5 20 ECF-PanT Lactobacillus br evis Nucleotide free Inward-facing, no substrate bound 4RFS 3,25 21 ECF-FolT2 Lactobacillus delbrueckii Nucleotide free Inward-facing, no substrate bound 5JSZ 3 12 ECF-FolT2 Lactobacillus delbrueckii AMP-PNP Inward-facing, no substrate bound 5D3M 3,3 12

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ECF-CbrT Lactobacillus delbrueckii -Inward-facing, no substrate bound 6FNP 3,4 15 RibU Staphylococcus aur eus -Riboflavin bound 3P5N 3,6 22 RibUΔCt Thermotoga maritima -Riboflavin bound 5KBW 5KC0 5KC4 2,61 (LCP) 3,2 3,4 14 BioY Lactoccocus lactis -D-biotin bound 4DVE 2,1 9 ThiT Lactococcus lactis

-Thiamin bound (not

V-shape conformation) 3RLB 2,0 23 ThiT Lactococcus lactis

-Small molecule modulators:

• LMG1 16 • BA T-25 • Pyrithamine • AV -38 • LMG139 • LMG135 • 4MES • 4MHW • 4MUU • 4N4D • 4POP • 4POV 2,0 2,5 2,1 2,4 2,2 2,2 To be published 24 FolT1 Lactobacillus delbrueckii -Folate bound 5D0Y 3,01 12 FolT Enter ococcus faecalis -Folate bound 4Z7F 3,19 11 EcfAA ’ Thermotoga maritima ADP -4HLU 2,7 25 EcfAA‘ Thermotoga maritima AMP-PNP -4ZIR 3,0 26

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Supplementary

Table 3

ATPase activity characterization for

differ

ent ECF

transporters

Protein Substrate Method Sample Value Author BioMNY Rhodobacter capsulatus Changing ATP concentrations

Free phosphate colorimetric determination

27 Purified Vmax 390 nmol P i /min/mg 28

ECF-RibU Streptococcus thermophilus

5

mM A

TP

29 Purified Vmax 0.8 ATP molecules/s/ECF-RibU (47.9 mol/min/mol) 25 BioMNY Rhodobacter capsulatus 2 mM A TP

30 - nanodiscs - purified - Vmax 0.73 ± 0.01 µmol P i /min/ mg (70 nmol/min/nmol), Km 0.14 ± 0.04µm; k 1.27 scat -1 - Vmax 0.53 ± 0.04 µmol/min/mg 31 ECF-RibU L ysteria monocytogenes Changing ATP concentrations

29 - prot eol iposom es - purified - Vmax 488 ± 67 nmol/min/mg (56.12 nmol/min/nmol), - Vmax 1 150 ± 28 nmol/min/mg Km 165 ± 16 µM 26 CbiMNQO Rhodobacter capsulatus Changing ATP concentrations Free phosphate colorimetric determination Purified kcat 29.7 min -1 Km 153.8 m 18 CbiMQO Rhodobacter capsulatus Changing ATP concentrations Free phosphate colorimetric determination Purified kcat 33.9 min -1 Km 150.7 m 18 CbiQO Rhodobacter capsulatus Changing ATP concentrations Free phosphate colorimetric determination Purified kcat 2.5 min -1 Km 277.3 m 18

ECF-FolT2 Lactobacillus delbrueckii

Changing ATP concentrations, 100 nM folate Radiolabeled substrate uptake assay Proteoliposomes Km 5.54 mM, n H 1.75 This study

ECF-PanT Lactobacillus delbrueckii

Changing ATP concentrations, 100 nM pantothenate Radiolabeled substrate uptake assay Proteoliposomes Km 5.61 mM, n H 1.74 This study

ECF-PanT Lactobacillus delbrueckii

Changing

ATP

concentrations,

500 nM pantothenate

Coupled enzyme assay

32 Proteoliposomes Km 16.38 mM, Vmax 200.23 pmol/min/pmol, n1.83H This study

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Supplementary

Table 4 Kinetic parameters of transport determined in ECF

transporters

Protein Substrate Sample Value Author ECF-FolT Lactobacillus casei - [G-3H]Folate - [ 14CH 3 ]Amethopterin - [3’,5’-3H]5-methyl tetrahydrofolate Whole cells - Km 0.35 µM, Vmax 0.44 nmol/min/1 0 10 cells - Km 0.21 µM, Vmax 0.44 nmol/min/1 0 10 cells - Km 0.90 µM, Vmax 0.56 nmol/min/1 0 10 cells 33 ThiT Lactobacillus casei [thiazole-2-14C]thaimine

Whole cells, ener

gized Km < 10 nM 2 ECF-FolT Lactobacillus casei [3’,5’,9(n)-3H]folate

Whole cells, ener

gized Vmax 0.32 nmol/min/10 10 cells 3 ECF-ThiT Lactobacillus casei [thiazole-2-4C]thiamine

Whole cells, ener

gized Vmax 0.35 nmol/min/10 10 cells 3 BioY Rhodobacterim capsulatus D-[8,9-3H]biotin

Whole cells, ener

gized Km 250 nM, Vmax 60 pmol/mg/min 34 BioMNY Rhodobacterim capsulatus D-[8,9-3H]biotin

Whole cells, ener

gized Km 5 nM, Vmax 6 pmol/mg/min 34 BioY Chlamydia trachomatis D-[8,9-3H(N)] biotin

Whole cells (nothing about ener

gizing) Km 3.35 nM, Vmax 55.1 pmol/mg/min 35

ECF-CbrT Lactobacillus delbrueckii

Changing 57Co-cyanocobalamine _{concentrations} Proteoliposomes Km 2.1 ± 0.4 nM, Vmax = 3.6 ± 0.6 pmol/min/µg 15 ECF FolT2 Lactobacillus delbrueckii Changing [3,5,7,9 -3H]folate concentrations Proteoliposomes Km 58.8 nM, Vmax 0.771 pmol/min/µg, kcat 0.00148 s -1 This study ECF PanT Lactobacillus delbrueckii Changing D-[2,3-3H]pantothenate concentrations Proteoliposomes Km 46.1 nM, Vmax 2.2 pmol/(min*µg), kcat 0.00429 s -1 This study

*All transport kinetic parameters were measured with radiolabeled substrate uptake assay

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Supplementary

Table 5

ATP

analogues used in this study

Analogue Structur e State Differ ence vs. A TP Refer ence AT P Pre-hydrolysis (ground) state -36 ADP

Post hydrolysis (product)

state No γ-phosphate 25,37,38 ATP-γ-S Pre-hydrolysis (ground) state

Slowly hydrolyzed analogue with a sulfur atom instead of oxygen in a terminal phosphate. Cannot _{form pentacovalent conformation, mimicking ground}

state.

39

AMP-PNP

(Adenylyl-imidodiphosphate)

Ground state

Slowly-hydrolyzed analogue that contain imido group instead of oxygen between the β- and γ-phosphorus atom.

Tetrahedral geometry . 12,26,40–42 Beryllium fluoride + ADP Pre-hydrolysis (ground) state Tetrahedral geometry 41,43 Vanadate (VO 3 3−) + ADP Post-hydrolysis (transition) state An analogue of inor ganic phosphate. The covalently

linked oxygen forms trigonal bipiramid, mimicking a

transition state.

41,44

Aluminum fluoride +

ADP

Transition state

Octahedral geometry that resemble conformation γ-phosphate under the water nucleophilic attack

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