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 752.4128 1427.6366 1458.7615 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 ASMS 2014 1
Pseudomonas aeruginosa: An opportunistic pathogen 1 µm 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 13% infections caused by Multidrug resistant strains of P. aeruginosa http://www.cdc.gov/drugresistance/threat-report-2013/pdf/ar-threats-2013-508.pdf#page=69 2
Membrane proteins play multifunctional role in bacteria Outer membrane Diffusion of molecules Cell shape Formation of membrane vesicles Peptidoglycan periplasm Inner membrane Drug resistance Translocation 3
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 Isolation Ex vivo Native state 4
Protein Interaction Reporter (PIR) crosslinking can help identify membrane PPIs in vivo Goal Use the PIR-crosslinking approach to identify membrane protein interactions of Pseudomonas aeruginosa in their native state Weisbrod et al, J. Proteome Res, 2013, 12, 71569-71579 5
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 6
Workflow of in vivo Protein Interaction Reporter (PIR) crosslinking technology LC-MS n Real-time analysis for crosslinked peptide technology (ReACT) 7
intensity ReACT allows on-the-fly detection of crosslinked peptide pairs High resolution MS 1 scan Cleave PIR bonds to release peptides MS 1 +4 MS 2 MS 3 m/z Identify released peptides MS 3 Weisbrod et al, J. Proteome Res, 2013, 12, 71569-71579 8
frequency PIR crosslinking detects proteins close to one another in vivo How close? Distribution of distances between cross-linked sites mapped to known Protein structures 60 50 40 30 20 10 35 Å 95% 0 0 16 32 48 64 Distance (Å) 9
PPI in the living cells of P. aeruginosa derived by PIR crosslinking 613 peptide pairs 224 crosslinked proteins 10
PPI in the living cells of P. aeruginosa derived by PIR crosslinking Membrane proteins Periplasmic Cytoplasmic Extracellular Unknown 11
Highly crosslinked membrane proteins = lipoproteins Crystal structures of opri, oprl, oprf are unknown oprl(peptidoglycan-associated lipoprotein) E. Coli homolog: PAL opri (lipoprotein) E. Coli homolog: LPP oprf (major porin) E. Coli homolog: ompa 12
C-termini of the major lipoproteins were involved in inter-protein interactions C-termini of the major lipoproteins are solvent accessible 13
oprl-oprf-opri : Role in structural stability? Cascales et al, J. Bacteriology, 2002, 184, 754-759 14
Identification of novel interactions of bacterial pro-inflammatory factors PA3691 Membrane proteins opri LptF PA3691 15
PA3691 lptf LptF: multifunctional outer membrane lipotoxin Triggering of host immune response by signal transduction Protection against oxidative stress during infection opri PA3691 LptF PA3691 Firoved, A.M., Infect Immun, 2004. 72, 5012-8, Darmon et al, Microbiology, 2009, 155,1029-38 16
Crosslinking derived structure prediction for LptF-PA3691 complex PA3691 LptF N PA3691 18Å LptF C 15Å 12Å Roy et al Nature Protocols, 2010, 5, 725-738 Nucl. Acids. Res. 2005, 33, W363-367 17
Crosslinking derived structure prediction for LptF-PA3691 complex LptF LptF-PA3691 complex 35Å 18
oprl opri oprf What else can the In vivo crosslinking data reveal? Interaction sites 19
Major outer membrane lipoproteins exists as multimers in vivo opri K 60 K 50 K 78 MNNVLKFSALALAAVLATGCSSHSKETEARLTATEDAAARAQARADE AYRK 50 ADEALGAAQK 60 AQQTADEANERALRMLEK 78 ASRK MNNVLKFSALALAAVLATGCSSHSKETEARLTATEDAAARAQARADE AYRK 50 ADEALGAAQK 60 AQQTADEANERALRMLEK 78 ASRK 20
Crosslinking-derived distance constraints aid molecular docking OprI monomer N Dimer model No distance constraints K 78 Dimer model distance constraints C K 60 18Å K 50 14Å 30Å K 50 K 50 K 50 77Å K 60 K 60 8Å K 60 K 78 K 78 K 78 2Å 21
PIR-crosslinking in the living cells of P. aeruginosa Detected novel PPIs in vivo Identified PPI Interaction sites and oligomeric complexes Guided structural prediction of multimeric membrane protein complexes Outer membrane periplasm Inner membrane cytoplasm 22
Acknowledgements Bruce Lab James E. Bruce, P.I. Juan Chavez Chad Weisbrod Jake Zheng Rick Harkewicz Xia Wu Devin Schwepe Singh Lab Richard Siehnel UWPR University of Washington s Proteomics Resource (UWPR95794) Funding grants Support provided by NIH grants 5R01HL110879, 7S10RR025107 and 5R01AI101307