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Architecture of the Type IV coupling protein complex of

Legionella pneumophila

Mi-Jeong Kwak1,#, J Dongun Kim1,#, Hyunmin Kim1, Cheolhee Kim2, James W. Bowman3,

Seonghoon Kim1, Keehyoung Joo4, Jooyoung Lee4, Kyeong Sik Jin5, Yeon-Gil Kim5, Nam Ki

Lee2, Jae U. Jung3, Byung-Ha Oh1,*

1Department of Biological Sciences, KAIST Institute for the Biocentury, Korea Advanced

Institute of Science and Technology, Daejeon 305-701, Korea 2Department of Physics, Pohang University of Science and Technology, Pohang, Kyungbuk

790-784, Korea 3Department of Molecular Microbiology and Immunology, Keck School of Medicine,

University of Southern California, Los Angeles, California, USA 4Center for in Silico Protein Science, Korea Institute for Advanced Study, Seoul 130-722,

Korea 5Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang,

Kyungbuk, 790-784, Korea

#Co-first authors; *Corresponding author (e-mail: bhoh@kaist.ac.kr)

Supplementary information This file contains Supplementary Figures 1-7 and Supplementary Table 1.

Architecture of the type IV coupling proteincomplex of Legionella pneumophila

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONVOLUME: 2 | ARTICLE NUMBER: 17114

NATURE MICROBIOLOGY | DOI: 10.1038/nmicrobiol.2017.114 | www.nature.com/naturemicrobiology 1

b Contact 1V729 Q137

L718E716

Q711T708

F737

D736I715L147

L144

V143

L140

F135 L131

M712

L733

A719

V83

P81

L87

L84

Y56

L54

IcmW

IcmS

DotL

L86

Contact 2

L691L691

F698

L702

I677L675

Y743P744

P745

I690

I683

L681

F14

R23

Y22

F27V24

L131

L128 M28

V124

V31

W34

F117

R121

Contact 3V753

I751

L756 T757

I759

I760

S764

L763

I767

K766R771

L36

L42

A45

M46

I73L49

F70

E66

L64

I56

I59

D52L53

L601L605

F608

L612

I611

L617

I619

V623

Q168

L169

M170

V175

I180

L184F156

F166

V152

I188

I151

Y108

P110

Y597

DotN

Contact 4N625

E627

I628

H655

F654

I652V648

L635

L651

I631

A650

L109

F145

S144

L112

I141

S137

L120

L124

A138

V123 A127

Y134

Contact 5

α1

N

α2

α3

β1

β2

α8

α6

α7

α4

α5

α4

α8

α2

α1

β1

β2

β3

β3

α9

α10

C

N α3

α6

α7

α9

α10

C

90°

N

α1

α2

β1 β2α3

β3

α4α5

α6

α7

β4

α8

α4α1

α2

β1α3β2

β3β4

α8

N

α10

α9

α6

α7

α9

α10

C

Ca

C87

C84

C52

C55

Zn2+

α4

β2

α10

α6

Zn2+

C87

C84

C52

C55

30

40

50

60

70

0

0.2

0.4

0.6

0.8

1

1.2

13 15 17 19 21

Ab

sorb

anc

e (a

.u.)

Mw

(kD

a)

Retention time (min)

Molar mass 47710 (± 0.718 %)

Polydispersity 1.007 (± 1.031 %)

Supplementary Figure 1. Crystal structure of free DotN and intersubunit interactions of

DotL(590-783) with IcmSW and DotN

(a) Structure of free DotN. (Left) Two perpendicular views are shown with the two monomers in

different colors and the bound Zn2+ ions shown as gray spheres. Two antiparallel α-helices (α6, α7)

of one monomer are stacked onto the equivalent α-helices of the other monomer, as if they form a

four-helical bundle. The 62-residue segment structurally similar to the HNH superfamily nucleases

is indicated by red color. (Right, Top) Two views of the zinc cage. Shown are a ribbon drawing with

the four Zn2+-coordinating cysteines in sticks and a cut-away view demonstrating the complete

burial of Zn2+. (Right, Bottom) Molecular mass analysis of free DotN by size-exclusion

chromatography coupled with multi-angle light scattering analysis.

(b) Detailed intersubunit interactions. Residue-residue contacts are shown for the five interfaces

marked in Figure 1a,b. Hydrogen bonds are indicated by dashed lines.

90°

90°

F118

A97 L101

I102

I123

E126

L52

Q48

R45

D114

W45

W42

W43

Y39

A46

V38

I41

L40

H44R37

IcmW

IcmSLvgA

α1

S51A97

L101

F118

L119

I102

R45

D114

Q48

W45 W43

W42

V38

I34

A33V35

R37

Y39

L40

α1

S51

LvgA

IcmS

α4

V27

L22

Y113M112

E31

L69

L70

Y185

Y173

W169

A181

V177

N176K183

R179

Y113

M112

L22V27

E31L70 L69

W169

Y173

Y185

V177N176 R179

A181L180

K183

K172

I168

α4

L86

L86

Intermolecular interaction of α1 of LvgA

Intermolecular interaction of α4 of LvgA

Supplementary Figure 2. Highlighted hydrophobic interactions between LvgA and IcmSW

Intersubunit residue-residue contacts are shown. Hydrogen bonds are indicated by dashed lines.

0

0.005

0.01

0.015

0.02

0 50 100

P(r

) [a

.u.]

r [Å]

ExperimentalCrystal structure

0.001

0.010

0.100

1.000

10.000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

I(q

) [a

.u.]

q [Å-1]

ExperimentalCrystal structure

b

80

90

100

00.10.20.30.40.50.60.70.80.9

11.1

4 6 8 10 12

Ab

sorb

ance

(a.

u.)

Mw

(kD

a)

Retention time (min)

a

Molar mass 91190 (± 0.456 %)

Polydispersity 1.000 (± 0.644 %)

c

Supplementary Figure 3. SAXS analysis of DotL(590-783)‒DotN‒IcmSW‒LvgA

(a) Molecular mass analysis of the complex by AF4-MALS.

(b) The SAXS curves showing the experimental and the calculated X-ray scattering. The plot

shows the scattering intensity I(q) as a function of q (q = 4πsinθ/λ, where 2θ is the scattering angle

and λ is the wavelength). The scattering data were extrapolated to zero concentration and

normalized by zero angle scattering intensity I(0). The experimental scattering intensities are the

average of six successive frames of 5 to 10 s exposure, indicating no sign of radiation damage.

(c) The P(r) functions showing the experimental and the calculated distance distributions.

Distribution of inter-atomic distances, P(r), is plotted as a function of distance (r).

SidJ + +Lpg0393 + +

VpdB + +SetA + +

Subcomplex + - + - + - + - +

a b

c

d

e

PieA + + + + + +Subcomplex 1 + +Subcomplex 2 + +Subcomplex 3 + +

+ ++ +

+ ++ +

+ - + - + - + - +

SidH CTD + + + + + +Subcomplex 1 + +Subcomplex 2 + +Subcomplex 3 + +

* IcmSW * IcmS–LvgA

*

-

* * * *

* * * * *-

--

-

-

--

1: DotL(590-783)–DotN–IcmSW‒LvgA2: DotL(590-783)–DotN–IcmSW3: DotL(590-783)–IcmSW‒LvgA

◀

1

-

23

- -

◀

12

3

◀ ◀

- - -

f RalF + +Subcomplex + - +

Subcomplex: DotL(590-783)–DotN–IcmSW‒LvgA

VpdB(11-485) + +

VpdB + +

Subcomplex + - + - +

Subcomplex: DotL(590-783)–DotN–IcmSW‒LvgA

◀◀

I R I R I R PieA SidH RalF

Empty resin

25

70

55

40

35

15

-

--

SetA

1: (His)10MBP-DotL(590-783)–DotN–IcmSW‒LvgA2: (His)10MBP-DotL(590-783)–DotN–IcmSW

I R I R I R Empty 1 2

(His)10

MBP-DotL(590-783)

DotN

IcmWIcmS

LvgA25

70

55

40

35

15

- -

I R I R I R PieA SidH RalF

(His)10MBP-DotL(590-783)–DotN–IcmSW‒LvgA

25

70

55

40

35

15

(His)10

MBP-DotL(590-783)

LvgADotN

IcmWIcmS

-

- -

I R I R I R PieA SidH RalF

(His)10MBP-DotL(590-783)–DotN–IcmSW

(His)10

MBP-DotL(590-783)

DotN

IcmWIcmS

-

- -

Supplementary Figure 4. Protein binding assays

(a-b) Interaction of IcmSW and IcmS‒LvgA with effector proteins. The four indicated effector

proteins (6 µM) were incubated with IcmSW or IcmS‒LvgA at 1:1 molar ratio, and the mixtures

were analyzed by native PAGE. (a) The migration of the four effectors did not change upon addition

of IcmSW. (b) Tailing of the VpdB or SetA protein band was observed upon addition of IcmS‒LvgA.

(c) Native PAGE analysis for PieA and SidH(1830-2225). The two proteins (6 µM) were incubated

with the indicated subcomplexes at 1:1 molar ratio. The effector proteins are indicated by ‘-’.

Triangles indicate newly formed protein bands, which are smeared for both effectors. The reason

for this migration behavior is unknown. PieA exhibits smeared bands probably due to its basic

property (theoretical pI= 8.57).

(d) (His)10 pull-down assay for PieA and SidH(1830-2225). Each protein (200 µM) was incubated

with the indicated subcomplex (100 µM) at room temperature for 30 min and mixed with 70 µL of

Co2+ resin. The resin was washed two times with a buffer solution containing 20 mM Tris-HCl (pH

7.5) and 100 mM NaCl, and then two times with the same buffer containing additional 10 mM

imidazole. Input proteins (I) and Co2+ resin-bound proteins (R) were visualized on a denaturing gel.

RalF and SetA served as a negative and a positive control, respectively.

(e) Native PAGE analysis for VpdB(11-485). VpdB(11-485) (6 µM) was incubated with DotL(590-

783)–DotN–IcmSW‒LvgA at 1:1 molar ratio. Newly formed protein bands are indicated by

triangles. Lines are drawn to distinguish the new protein band from VpdB(11-485) bands.

(f) Native PAGE analysis for RalF. RalF (6 µM) was incubated with DotL(590-783)–DotN–

IcmSW‒LvgA at 1:1 molar ratio. No detectable interaction is observable.

Throughout the figures, a representative image from more than three replicate experiments are

shown.

90°N

α1

C

α2

α3

α4

α5 α6

α7α8

α9

α10

α11

α12

α13

42 Å

N

α1

α2

α3

α4

α5

α6

α7

α8

α9α10

α11

α12

α13

C

62 Å

28 Å

DotM1 380

371161

Supplementary Figure 5. Crystal structure of DotM(161-371)

Two perpendicular views are shown. A schematic drawing of the construct is shown at the top.

Crystallographic data statistics are summarized in Supplementary Table 1.

1 2 3 4 5 6 8 9 10 11 TrwC TrwBE.c. VirB

TraJ11109865431S.f. VirB

4443TrwBTrwC109865 1P.s. VirB

FIJLMVirB10O VirD4N.u. Tra

MLKJIGE.c. Tra

D411108654321 9A.t. VirB

6TraK10984321 11E.c. VirB

VPKELA B NUC W DGHFE.c. Tra

E.f. Prgf LKJIEDCBA H FEDCA B G

B7

TraG

F G M

Pcf

D

N

R

IM L PEGH F IcmFON BCD AL.p. Dot

HGCBLMNOI DP.s. Dot

HGEPNO F MII L CD ABC.b.. Dot

M L N F G H IOP.p. Dot

L M G H I D COM.a. Dot

A H N O LGY.p. Dot

X.c. Dot L O G H IN M

A.R. Dot M I G N OH L

X.c. Dot C B L F GM H ID

B.v. Dot I D M LCG

R.g. Dot NOCB D HGEP F MLI A

IcmS QR IcmM IcmC IcmV XW

IcmT S

IcmV IcmW IcmX

IcmT

D411108765432 9B.h. VirB

T4ASS genes

T4BSS genes

46 8 4A.p. VirB D4 11 10 9 8

B6B2

(ref:16)

(ref:68)

(ref:69)

(ref:16)

(ref:70)

(ref:16)

(ref:16)

(ref:71)

(ref:16)

(ref:72)

(ref:73)

(ref:74)

(ref:75)

(ref:76)

(ref:76)

(ref:76)

Supplementary Figure 6. Genetic organizations

Shown are the genetic organizations of T4ASS and T4BSS associated with the T4CPs listed in

Figure 6. The open-reading frames were identified and annotated by using the RAST server67.

Homologous genes are color-coded (pink: components of the secretion channel, orange: coupling

proteins). The shown references identify between the two subtypes of T4SS. Accession numbers

are E.c.: Escherichia coli (R388 plasmid) (BR000038.1), S.f.: Shigella flexneri 4c (1205p3 plasmid)

(CP012143.1), P.s.: Pseudomonas syringae (NCPPB880-40 plasmid) (JQ418534.1), E.f.:

Enterococcus faecalis (CF10 plasmid) (AY855841.2), N.u.: Nitrosomonas ureae

(FNUX01000024.1), E.c.: Escherichia coli (IncP-alpha RP4 plasmid) (X54459.1), B.h.: Bartonella

henselae (JQ701698.1), A.t.: Agrobacterium tumefaciens (Ti plasmid) (J03320.1), E.c.: Escherichia

coli (O157_Sal plasmid) (CP001927.1), E.c.: Escherichia coli (F plasmid) (AP001918.1), A.p.:

Anaplasma phagocytophilum (NZ_APHI01000002.1), L.p.: Legionella pneumophila

(NZ_CP013742.1), P.s.: Piscirickettsia salmonis (CP012413.1), R.g.: Rickettsiella grylli

(NZ_AAQJ02000001.1), C.b.: Coxiella burnetii (CP018150.1), P.p.: Pseudomonas putida

(CP003589.1), B.v.: Burkholderia vietnamiensis (LPCP01000001.1), X.c.: Xanthomonas campestris

pv. vesicatoria str. 85-10 (AM039951.1), M.a.: Micavibrio aeruginosavorus (CP002382.1), A. :

Acidovorax sp. Root70 (LMHQ01000001.1), Y.p.: Yersinia pseudotuberculosis (CP000719.1), X.c.:

Xanthomonas citri (CCXZ01000025.1).

Figure 4a

Left Middle Right

Supplementary Figure 4

a b

c

Figure 2a

d

Supplementary Figure 4

e f

Supplementary Figure 7. Full blots of all gels shown in this manuscript

Rectangled boxes indicate the regions shown in the indicated figures.

Supplementary Table 1. X-ray data collection and structure refinement statistics.

Data Collection DotL(656-783)‒IcmSW (SelMet)

DotN DotL(590-659)‒DotN DotL(656-783)‒IcmSW‒LvgA

DotM(161-371)(SelMet)

Space group P212121 P6522 P212121 P32 P21

Unit cell dimensions

a, b, c (Å) 67.597, 75.803, 150.637155.357, 155.357, 527.711

50.683, 72.220, 170.435152.325, 152.325, 74.475

50.529, 72.021, 65.691

α, β, γ (°) 90, 90, 90 90, 90, 120 90, 90, 90 90, 90, 120 90, 102.154, 90

Wavelength (Å) 0.9796 1.2828 1.2828 1.0000 0.9796

Resolution (Å) 50-2.0 50-3.0 50-1.8 50-2.8 50-1.8

Rsym 10(28.9)a 10.4(31.7)a 6.4(27.2)a 8.1(37)a 7.9(17.9)a

I/σ(I) 28.6(4) 18.8(2.86) 42.2(2.85) 18.8(1.56) 21.3(3.89)

Completeness (%) 92(85.5) 85.4(60.9) 97(91.2) 97.9(90.4) 91(72)

Redundancy 5.4(3.1) 12(1.8) 8.8(3.4) 4.2(2.5) 4.3(2.6)

Refinement

Resolution (Å) 50-2.0 50-3.0 50-1.8 50-2.8 50-1.8

No. of reflections 86494 109241 104008 46584 70306

Rwork / Rfree (%) 22.1/26.9 25.6/29.7 21.0/25.9 25.0/29.0 17.6/19.8

R.m.s deviations

bond (Å) / angle (º) 0.008/0.966 0.002/0.408 0.008/0.84 0.01/1.185 0.007/0.83

Average B-values (Å2) 16.37 55.68 26.31 64.94 14.69

Ramachandran plot (%)

Favored / Additional allowed

94.7/5.0 86.3/13.5 92.4/7.6 87.3/12.4 91.5/8.3

Generously allowed 0.2 0.1 0 0.3 0.3

aThe numbers in parentheses are the statistics from the highest resolution shell.

Supplementary References 67. Brettin, T. et al. RASTtk: a modular and extensible implementation of the RAST

algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep 5, 8365 (2015).

68. Zhao, Y. F., Ma, Z. H. & Sundin, G. W. Comparative genomic analysis of the pPT23A plasmid family of Pseudomonas syringae. J Bacteriol 187, 2113-2126 (2005).

69. Goessweiner-Mohr, N., Arends, K., Keller, W. & Grohmann, E. Conjugative type IV secretion systems in Gram-positive bacteria. Plasmid 70, 289-302 (2013).

70. Schulein, R. & Dehio, C. The VirB/VirD4 type IV secretion system of Bartonella is essential for establishing intraerythrocytic infection. Molecular Microbiology 46, 1053-1067 (2002).

71. Rikihisa, Y., Lin, M., Niu, H. & Cheng, Z. Type IV secretion system of Anaplasma phagocytophilum and Ehrlichia chaffeensis. Ann N Y Acad Sci 1166, 106-111 (2009).

72. Gomez, F. A. et al. Evidence of the presence of a functional Dot/Icm type IV-B secretion system in the fish bacterial pathogen Piscirickettsia salmonis. PLoS One 8, e54934 (2013).

73. Leclerque, A. & Kleespies, R. G. Type IV secretion system components as phylogenetic markers of entomopathogenic bacteria of the genus Rickettsiella. FEMS Microbiol Lett 279, 167-173 (2008).

74. Zamboni, D. S., McGrath, S., Rabinovitch, M. & Roy, C. R. Coxiella burnetii express type IV secretion system proteins that function similarly to components of the Legionella pneumophila Dot/Icm system. Mol Microbiol 49, 965-976 (2003).

75. Ye, L. M. et al. Draft Genome Sequence Analysis of a Pseudomonas putida W15Oct28 Strain with Antagonistic Activity to Gram-Positive and Pseudomonas sp Pathogens. Plos One 9 (2014).

76. Nagai, H. & Kubori, T. Type IVB secretion systems of Legionella and other Gram-negative bacteria. Front Microbiol 2 (2011).