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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).
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