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752.4128
1427.6366
1458.7615
Probing protein interactions in living cells of
Pseudomonas aeruginosa by chemical cross-linking
Arti Navare, Richard Siehnel, Kirsten Beck, Alejandro Wolf-Yadlin, Pradeep Singh,
James E. Bruce
University of Washington, United States
ASMS 2014 1
Pseudomonas aeruginosa: An opportunistic pathogen
• Gram negative bacteria
• Widely found in the environment
• Causes serious infections in patients with weakened immune system
• Prevalent in Cyctic Fibrosis patients causing chronic infection
• 51,000 hospital-acquired cases/year within US
1 µm
2http://www.cdc.gov/drugresistance/threat-report-2013/pdf/ar-threats-2013-508.pdf#page=69
• 13% infections caused by Multidrug resistant strains of P. aeruginosa
Membrane proteins play multifunctional role in
bacteria
Outer
membrane
Inner
membrane
Peptidoglycanperiplasm
Drug resistance
Translocation
Cell shapeDiffusion of
molecules
3
Formation of
membrane vesicles
4
Knowledge of membrane proteins interactions of P.
aeruginosa is limited
Only 40 manually curated P. aeruginosa protein interactions
are available on the MPIDB database
Rajagopala S.V. et al, PLoS one, 2008, 24, 2622-2627
Membrane protein purification is
challenging
- Native protein complexes and
interactions are not stable ex vivo
Ex vivo
Native state
Isolation
5
Protein Interaction Reporter (PIR) crosslinking can
help identify membrane PPIs in vivo
Weisbrod et al, J. Proteome Res, 2013, 12, 71569-71579
Use the PIR-crosslinking approach to identify membrane protein
interactions of Pseudomonas aeruginosa in their native stateGoal
6
Introduction to Protein Interaction Reporter (PIR)
crosslinking technology
Biotin affinity tag
Mass encoded
reporter
Cleavable bonds
Primary amine reactive
groups
Tang et al, Anal Chem, 2005, 77, 311-318
7
Workflow of in vivo Protein Interaction Reporter (PIR)
crosslinking technology
LC-MSn
Real-time analysis for crosslinked peptide technology (ReACT)
8
ReACT allows on-the-fly detection of crosslinked
peptide pairs
MS1
inte
nsity
m/z
High resolution MS1 scan
+4 MS2
Cleave PIR bonds to release peptides
MS3 Identify released peptidesMS3
Weisbrod et al, J. Proteome Res, 2013, 12, 71569-71579
PIR crosslinking detects proteins close to one another
in vivo
9
How close? Distribution of distances between
cross-linked sites mapped to known
Protein structures
95%
Distance (Å)
60
30
10
50
40
20
00 32 6416 48
frequency
35 Å
10
PPI in the living cells of P. aeruginosa derived by PIR
crosslinking
613 peptide pairs
224 crosslinked proteins
11
Membrane proteins
Periplasmic Cytoplasmic
Unknown
Extracellular
PPI in the living cells of P. aeruginosa derived by PIR
crosslinking
12
Highly crosslinked membrane proteins = lipoproteins
oprI (lipoprotein)E. Coli homolog: LPP
oprF (major porin)E. Coli homolog: ompA
oprL(peptidoglycan-associated lipoprotein)
E. Coli homolog: PAL
Crystal structures of oprI, oprL, oprF are unknown
13
C-termini of the major lipoproteins were involved in
inter-protein interactions
C-termini of the major lipoproteins are solvent accessible
14
oprL-oprF-oprI : Role in structural stability?
Cascales et al, J. Bacteriology, 2002, 184, 754-759
15
Membrane proteins
Identification of novel interactions of bacterial
pro-inflammatory factors
PA3691
LptFoprI
PA3691
16
Firoved, A.M., Infect Immun, 2004. 72, 5012-8, Darmon et al, Microbiology, 2009, 155,1029-38
LptF: multifunctional outer membrane lipotoxin
• Triggering of host immune response by signal transduction
• Protection against oxidative stress during infection
lptF
PA
36
91
PA3691LptFoprI
PA3691
17
Crosslinking derived structure prediction for LptF-PA3691 complex
LptF
18Å
15Å
PA3691
12Å
LptF
PA3691
Roy et al Nature Protocols, 2010, 5, 725-738 Nucl. Acids. Res. 2005, 33, W363-367
N
C
18
Crosslinking derived structure prediction for LptF-PA3691 complex
LptF
35Å
LptF-PA3691 complex
19
Interaction sitesop
rL
op
rF
op
rI
What else can the In vivo
crosslinking data reveal?
20
Major outer membrane lipoproteins exists as
multimers in vivo oprI
MNNVLKFSALALAAVLATGCSSHSKETEARLTATEDAAARAQARADE
AYRK50ADEALGAAQK60AQQTADEANERALRMLEK78ASRK
K50K60
K78
MNNVLKFSALALAAVLATGCSSHSKETEARLTATEDAAARAQARADE
AYRK50ADEALGAAQK60AQQTADEANERALRMLEK78ASRK
N
C
OprI monomer
21
K’50K50
K’60
K60
K78
K’78
77Å
14Å
30Å
Dimer model
No distance constraints
Dimer model
distance constraints
K’50
K50
K’60
K60
K78 K’78
18Å
8Å
2Å
Crosslinking-derived distance constraints aid
molecular docking
22
PIR-crosslinking in the living cells of P. aeruginosa
Outer
membrane
Inner
membrane
periplasm
cytoplasm
• Detected novel PPIs in vivo
• Identified PPI Interaction sites and oligomeric
complexes
• Guided structural prediction of multimeric
membrane protein complexes
Acknowledgements
Bruce LabJames E. Bruce, P.I.
Juan Chavez
Chad Weisbrod
Jake Zheng
Rick Harkewicz
Xia Wu
Devin Schwepe
Singh LabRichard Siehnel
UWPRUniversity of Washington’s
Proteomics Resource
(UWPR95794)
Funding grantsSupport provided by NIH grants 5R01HL110879, 7S10RR025107
and 5R01AI101307