Supplemental Data

Global Conformational Change Associated with the Two-step Reaction Catalyzed by Escherichia coli Lipoate-protein Ligase A

Kazuko Fujiwara, Nobuo Maita, Harumi Hosaka, Kazuko Okamura-Ikeda, Atsushi Nakagawa, and

Hisaaki Taniguchi

Supplemental TABLE S1 Primers used for mutagenesis. Mutant Sequence

LplA-K133A sensea 5’-GGCGACCGCGCAGTCTCAGGCTC-3’ LplA-N121A sensea 5’-GTCCGGACGTGCCGATCTGGTGG-3’ LplA-N122A sensea 5’-CGGACGTAACGCTCTGGTGGTGA-3’ H-protein-K64A sensea 5’-CGAATCGGTAGCAGCGGCGTC-3’

LplA-H149A sense 5’-GCTTCCACGCCGGCACCTTGCTACTC-3’ anti-sense 5’-TGCCGGCGTGGAAGCCGCGATCTTTG-3’ aA sense and complementary anti-sense primer set was used. Modified bases are shown in bold.

1

Supplemental TABLE S2 Potential hydrogen bond and electrostatic interactions between LplA and octyl-AMP or apoH-protein in the LplA·octyl-AMP·apoH-protein ternary complex LplA (molA) OAMa (molA) distance (Å) LplA (molC) OAMa (molC) distance (Å) F78/O — AMP/N6 2.9 F78/O — AMP/N6 2.9 N83/Oδ1 — AMP/N6 2.9 N83/Oδ1 — AMP/N6 3.1 T151/Oγ1 — AMP/ N1 2.7 T151/Oγ1 — AMP/N1 2.9 S182/N — AMP/O2* 3.3 S182/N — AMP/O2* 3.3 V180/N — AMP/O2P 2.8 G75/N — AMP/O2P 2.9 K133/Nζ — AMP/O4* 3.0 K133/Nζ — AMP/O5* 3.1 G75/N — AMP/O3P 2.9 LplA (molA) apoHb (molB) distance (Å) LplA (molC) apoHb (molD) distance (Å) R47/NH1 — D43/Oδ2 2.6 R47/NH1 — D43/Oδ2 3.6 R47/NH2 — D43/Oδ2 3.0 G74/N — E61/Oε2 3.6 G74/N — E61/Oε2 2.9 Y139/O — A66/N 3.0 Y139/O — A66/N 2.9 R140/Nε — A66/O 3.0 R140/NH1 — D68/Oδ1 3.4 R140/Nε — D68/Oδ2 3.0 R140/NH1 — D68/Oδ2 3.6 R140/NH2 — D68/Oδ2 3.3 E141/N — A66/O 2.9 E141/N — A66/O 3.1 E141/O — D68/N 2.9 E141/O — D68/N 3.3 K143/N — Y70/Oη 2.9 K143/Nε — D68/O 3.1 K175/Nζ — D86/Oδ1 2.6 K143/N — Y70/Oη 3.5 K175/Nζ — D86/Oδ2 3.5 K175/Nζ — S85/O 3.2 K272/Nζ — E46/Oε1 3.1 K175/Nζ — D86/Oδ1 2.2 K272/Nζ — E46/Oε2 3.1 aOAM: octyl-AMP bapoH: apoH-protein

2

Ec LplA-lip-AMP S T L R L L I S D S Y D PWF N L A V E E C I F R QM P A T Q R - V L F LWR N S P T V V I G R A Q N PWK E C N T 57Ec LplA S T L R L L I S D S Y D PWF N L A V E E C I F R QM P A T Q R - V L F LWR N S P T V V I G R A Q N PWK E C N T 57Sp LplA MK Y I I N H S N D T A F N I A L E E Y A F K H L L D E D Q - I F L LW I N K P S I I V G R H Q N T I E E I N R 55Ta LplA ME G R L L L L E T P G N T RM S L A Y D E A I Y R S F Q Y G D K P I L R F Y R H D R S V I I G Y F Q V A E E E V D L 59bLT T V K S G L I L Q S I S N D V Y H N L A V E DW I H D HMN L E G K P V L F LWR N S P T V V I G R H Q N PWQ E C N L 60

Ec LplA-lip-AMP R RME E D N V R L A R R S S G G G A V F H D L G N T C F T FMA G K P E - - - - Y D K T I S T S I V L N A L N A L G - 112Ec LplA R RME E D N V R L A R R S S G G G A V F H D L G N T C F T FMA G K P E - - - - Y D K T I S T S I V L N A L N A L G - 112Sp LplA D Y V R E N G I E V V R R I S G G G A V Y H D L N N L N Y T I I S K E D E N K A - F D F K S F S T P V I N T L A Q L G - 113Ta LplA D YMK K N G I ML A R R Y T G G G A V Y H D L G D L N F S V V R S S D DMD I T S M F R TMN E A V V N S L R I L G - 118bLT N LMR E E G V K L A R R R S G G G T V Y H DMG N I N L T F F T T K K K - - - - Y D RME N L K L V V R A L K A V H P 116

Ec LplA-lip-AMP - V S A E A S G R N D L V V K T - - - - - - V E G D R K V S G S A Y R E T K D R G F H H G T L L L N A - D L S R L A N Y 164Ec LplA - V S A E A S G R N D L V V K T - - - - - - V E G D R K V S G S A Y R E T K D R G F H H G T L L L N A - D L S R L A N Y 164Sp LplA - V K A E F T G R N D L E I D - - - - - - - - - - G K K F C G N A Q A Y I N G R I MH H G C L L F D V - D L S V L A N A 161Ta LplA - L D A R P G E L N D V S I P V N K K T D I MA G E K K I MG A A G AMR K G A K LWH A AML V H T - D L DML S A V 176bLT H L D V Q A T K R F D L L L D - - - - - - - - - G Q F K I S G T A S K I G R N A A Y H H C T L L C G T - D G T F L S S L 166

Ec LplA-lip-AMP L N P D K K K L A A K G I T S V R S R V T N L T E L L P G I T H E Q V C E A I T E A F F A H Y G E R V E A E I I S P N K 224Ec LplA L N P D K K K L A A K G I T S V R S R V T N L T E L L P G I T H E Q V C E A I T E A F F A H Y G E R V E A E I I S P N K 224Sp LplA L K V S K D K F E S K G V K S V R A R V T N I I N E L P K K I T V E K F R D L L L E YMK K E Y P EMT E Y V F S E E E 221Ta LplA L K V P D E K F R D K I A K S T R E R V A N V T D - F V D V S I D E V R N A L I R G F S E T L H I D F R E D T I T E K E 235bLT L K S P Y Q G I R S N A T A S T P A L V K N LME K D P T L T C E V V I N A V A T E Y A T S H Q I D N H I H L I N P T D 226

Ec LplA-lip-AMP T P D L P N F A E T F A R Q S S WEWN F G Q A P A F S H L L D E R F TWG G - - - - V E L H F D V E K G - - H I T R A 278Ec LplA T P D L P N F A E T F A R Q S S WEWN F G Q A P A F S H L L D E R F TWG G - - - - V E L H F D V E K G - - H I T R A 278Sp LplA L A E I N R I K D T K F G - - TWDWN Y G K S P E F N V R R G I K F T S G K - - - - V E V F A N V T E S - - K I Q D I 273Ta LplA E S L A R E L F D K K Y S T E EWNMG L L R K E V V 262bLT E T V F P G I N S K A I E L Q TWEW I Y G K T P K F S V D T S F T V L H E Q S H V E I K V F I D V K N G R I E V C N I 286

Ec LplA-lip-AMP Q V F T D S L N P A P L E A L A G R L Q G C L Y R A DML Q Q E C E A L L V D F P E Q E K E L R E L S AWMA G A V R 337Ec LplA Q V F T D S L N P A P L E A L A G R L Q G C L Y R A DML Q Q E C E A L L V D F P E Q E K E L R E L S AWMA G A V R 337Sp LplA K I Y G D F F G I E D V A A V E D V L R G V K Y E R E D V L K A L K T I D I T R Y F A G I S R E E I A E A V V G 329bLT E A P D HWL P L E I C D Q L N S S L I G S K F S P I E T T V L T S I L H R T Y P G D D E L H S KWN I L C E K I K G I 346

bLT M 347

Supplemental Figure S1

α10α9 α11

α7 β13α8 β15β14

α1β1 α2β3β2

α3 β4 β5 β6 α4

α5β8β7 β9 β10

β12β11 α63101

3

FIGURE S1. Amino acid sequence alignment of E. coli LplA with secondary structural elements. Amino acid sequence of E. coli LplA is aligned with those from Streptococcus pneumoniae (Sp) (1VQZ), T. acidophilum (Ta) (2ART, 2C8M), and bovine lipoyltransferase (bLT) (2E5A). Sequence alignment was carried out using ClustalW (http://clustalw.genome.jp). Identical residues among four proteins are shown in red. The regions of α-helix, β-strand, and 310-helix are shown against yellow, blue, and orange backgrounds, respectively. Residues with red and green arrowheads interact with lipoyl-AMP with hydrophobic and hydrogen-bonding interactions, respectively. The lipoate-binding loop and adenylate-binding loop are shown by bars in magenta and yellow, respectively. The connecting loops between N- and C-terminal domains are shown by green bars. Ec LplA-lip-AMP and Ec LplA represent E. coli LplA in complex with lipoyl-AMP and in its unliganded form, respectively. The secondary structural elements were assigned by the program DSSP (1).

REFERENCE1. Kabsch, W., and Sander, C. (1983) Biopolymers 22, 2577-2637

Supplemental Figure S2

2.89

2.36

2.81

2.65

2.99

2.83

2.69

2.75

3.25

2.87

3.16

2.752.63

3.30

3.04

2.79

3.012.99

3.06

2.75

2.76

3.20

2.91

2.98

3.09

3.12

2.87

3.20

C16 C17

C15 S17

S15

C14

C13

C12

C11 C10

O10

O3P P

O2P

O1P

O5*

C5*

C4*

O4*

C3*

C1*

O3*

C2*

O2* N9

C8

C4

N7

C5 C6

N3 C2

N1

N6

N

CA CB

C CG CD

CE NZ

O

N

CA

CB

C

CG OD1

ND2

O

N

CA

CB

C

OG

O

N CA

CB

C

CG1

CG2

O

N

CA

C O

N

CA CB

C

CG

CD1 CD2

CE1

CZ

CE2

O

N

CA

CB

C

CG

CD

NE CZ

NH1

NH2

O

N

CA

CB

C

CG

OD1

ND2

O

N

CA

CB

C

OG1

CG2

O

N

CA CB

C OG1

CG2

O

N

CA

CB

C O

Val 77(A)

Trp 37(A)

Val 44(A)

His 149(A)

Arg 70(A)

His 79(A)

Gly 150(A)

Leu 153(A)

Ser 135(A)

Gly 136(A)

Ser 137(A)

Leu 161(A)

Leu 165(A)

Val 184(A)

Laq 401(A)Lys 133(A)

HOH 1092(A)

MG 1(B)

Asn 121(A)

Ser 182(A)

HOH 663(A)

Val 180(A)

Gly 75(A)

HOH 601(A)

HOH 751(A)

HOH 777(A)

HOH 1036(A)

Phe 78(A)

Arg 181(A)

Asn 83(A)

Thr 151(A)

Thr 178(A)

Ala 76(A)

4

2.50HOH 935(A)

FIGURE S2. Schematic diagram showing interactions between lipoyl-AMP and LplA. Lipoyl-AMP and LplA residues interacting with lipoyl-AMP are shown in a flattened form. Lipoyl-AMP atoms with short red lines represent hydrophobic interactions pointing toward the LplA residues also outlined with red lines. Green dashed lines show potential hydrogen bonds between atoms with distances in Å.

SNVPAEL 7ALRMWASSTANALKLSSSSRLHLSPTFSISRCFSNVLDGL 40

KYSKEHEWLRKEADGTYTVGITEHAQELLGDMVFVDLPEV 47KYAPSHEWVKHEGS-VATIGITDHAQDHLGEVVFVELPEP 80

GATVSAGDDCAVAESVKAASDIYAPVSGEIVAVNDALSDS 87GVSVTKGKGFGAVESVKATSDVNSPISGEVIEVNTGLTGK 120

PELVNSEPYAGGWIFKIKASDESELESLLDATAYEALLED 127 PGLINSSPYEDGWMIKIKPTSPDELESLLGAKEYTKFCEE 160

E 128 EDAAH 165

β1

β9

β8β7β6β5

β4β3β2

α5α4α3

α2

α1

Ec apoHPea H

Ec apoHPea H

Ec apoHPea H

Ec apoHPea H

Ec apoHPea H

Supplemental Figure S3

A

5

FIGURE S3. Structure of E. coli apoH-protein. A, amino acid sequence alignment of E. coli apoH-protein (Ec apoH) with pea H-protein (pea H). Residues in red are identical in two proteins. Orange rectangles and blue arrows represent α-helices and β-strands, respectively. Red arrowhead shows the lysine residue which accepts lipoic acid. B, apoH-protein of molB (orange), molD (cyan), and in its free form (gray) are superimposed and shown in stereo form. Lys64 is shown as a stick model. Secondary structural elements and N- and C-termini are labeled.

CC

N α1

β8

β7

β6

β5β4

β3

β2

β1

α5

α4α3

α2

N α1

β8

β7

β6

β5β4

β3

β2

β1

α5

α4α3

α2

K64K64

β9 β9

B

Supplementa1 Figure S4

A

B

FIGURE S4. Structure of LplA-octyl-AMP-apoH-protein complex. A, stereo view of the ternary complex (molA-molB). LplA is colored in cyan, and apoH-protein is in purple. Octyl-AMP is in orange (bond) and atom-type colors. Secondary structural elements of apoH-protein and N- and C-termini are labeled. B, schematic diagram showing interactions between LplA and octyl-AMP (molA). Potential hydrogen bonds and hydrophobic interactions are shown as in Fig. S2.

3.33

2.80

2.72

2.97

3.14

2.94

2.87

2.91

C1 O3P

C8

C7

C6

C5 C4

C3 C2

P

O2P

O1P O5*

C5*

C4*

O4*

C3*

C1*

O3*

C2* O2*

N9 C8

C4 N7

C5

C6

N3

C2

N1 N6

N

CA

CB

C

CG

CD

CE

NZ

O

N

CA

CB C

OG1

CG2

O

N

CA CB

C CG

CD1

CD2

CE1 CZ

CE2

O

N

CA

CB

C

CG

OD1

ND2

O

N

CA

CB

C

CG1

CG2

O

N

CA

CB C

OG O

N

CA

C

O

Val 77(A)

Trp 37(A)

His 149(A)

Arg 70(A)

His 79(A)

Ser 135(A)

Gly 136(A)

Leu 153(A)

Leu 165(A)

Val 184(A)

Oct 401(A)

Amp 402(A)

Lys 133(A)

Thr 151(A)

Phe 78(A)

Asn 83(A)

Val 180(A)

Ser 182(A)

Gly 75(A)

6

LplA

N

C

N

C N

C

N

C

apoH-proteinapoH-protein

LplA

β7 β7

β8

β7

α4

α5 α5

β4α2α2

β4

β7

β8α1

α3α3

α1

β8β5 β5

β3β3

β1β2β2

β9

β8

β9

β1

β6β6

α4

Supplemental Figure S5

α1

β1

α11

α10

α9

α8

α7

α6

α5

α4

α3

α2

β13

β12

β11

β10

β9

β8

β7

β6

β5β4

β3

β2

N

C

K168

K187α1

β1

α11

α10

α9

α8

α7

α6

α5

α4

α3

α2

β13

β12

β11

β10

β9

β8

β7

β6

β5β4

β3

β2

N

C

K168

K187

7

FIGURE S5. Structure of bovine lipoyltransferase in unliganded form. The structure of bovine lipoyltransferase in its unliganded form (green) is superimposed onto that in complex with lipoyl-AMP (gray, PDB ID: 2E5A) with the root mean square deviation of 0.93 Å (for 267 Cα atoms of 311). Lipoyl-AMP in the complex is in gray (bond) and atom-type colors. The loop in the complex form (residues 169−186), which is disordered in the unliganded form, is in magenta. N- and C-termini, positions of K168 and K187, and secondary structural elements are labeled.

Supplemental Figure S6

FIGURE S6. HPCL analysis of lipoyl-AMP produced by lipoate-adenylation reaction. A, standard lipoyl-AMP (3.99 nmol). B, C, D, E, F, and G, reaction products by wild-type LplA, K133A, N121A, D122A, S72A, and H149A mutant-LplA were resolved on a C18 column, respectively. Experiments were carried out two or three times, and representative data are shown. Line, absorbance at 258 nm; dashed line, concentration of acetonitrile. Results were summarized in Table 2.

Abs

orba

nce

at 2

58 n

m

Ace

toni

trile

(%

)

1.000

40

80

Lipoyl-AMP

1.000

1.000

0 15 30 45Retention time (min)

A: STD

0

0

0

0

1.000

0

8

1.000

0

G: H149A

F: S72A

E: D122A

1.000 D: N121A

C: K133A

1.000LplA

0

B: Wild-type