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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