Towards Systematic Identification of cdiGMP Binding Proteins

1. Towards Systematic Identification of cdiGMP Binding Proteins Kevin Roelofs Laboratory of Dr. Vincent Lee University of Maryland, College Park

2. What is the total regulatory potential of a second messenger? Receptors: Nucletoide Second Messengers: cAMP ppGpp(p) cdiGMP cdiAMP c-AMP/GMP Membrane Proteins Cytosolic Enzymes Transcription Factors Phenotypes

3. CdiGMP in Vibriocholerae • In V. cholerae cdiGMP regulates: • Biofilm Formation • Host Colonization • Virulence • Environmental dissemination • 51 proteins with domains for synthesis and degradation of cdiGMP • 4 Currently Identified Receptors Tamayo Ann Rev Microbiol2007

4. Current approaches in second messenger receptor identification 1) Genetics 2) Biochemistry 3) Bioinformatics Pull down proteins with second messenger as bait Identify second messenger in crystal structures of purified proteins Identify receptors with conserved binding domains Identify target genes required for phenotype regulated by second messenger Systematically purify and test target proteins for binding to second messenger

5. Problems with current approaches • Genetics – Receptors not always associated with observable phenotype • Biochemistry – Receptor must be expressed • Bioinformatics – Limited to identification of previously described binding motifs None of these approaches systematically identify all genomically encoded receptors

6. Differential Radial Capillary Action of Ligand Assay (DRaCALA) Protein and bound ligand are immobilized on nitrocellulose membrane while free ligand is distributed uniformly by capillary action

7. DRaCALA detects specific binding interactions Proof of Principle: Purify 10 µM protein Mix with nM amounts of radiolabeled nucleotide

8. Quantitative Detection of Binding Interactions in E. coli Whole Cell Lysate Mix with 32P cdiGMP: Purified Alg44 Purified Alg44 added to BL21 E. coli cell lysate BL21 E. coli cell lysate with endogenously expressed Alg44 Plot Fraction Bound vs Protein Concentration

9. Design of DRaCALA ORFeome Screen • Obtain Open Reading Frame (ORF) library • Contains all annotated ORFs from individual organism • V.choleraeORF library contains 3866 unique clones • Express individual V. cholerae ORFs in E. coli T7IQ • 2 libraries: N-terminal His- or HisMBP- • Screen whole cell lysates in 96-well format for increased cdiGMP binding

10. Results of Partial (938 ORFs) Screen Buffer control Positive Control Positive Clone 3 standard deviation positive cutoff Average Fraction Bound • PilZ domain • EAL domain Other

11. Biochemical Classification of Positive Hits Vector control ORF expressed cAMP 1 mM Competitor NC NC cdiGMP cGMP GMP GDP GTP CTP ATP UTP His-PlzE His-VC1641 2x Serial Dilutions of E. coli WCL into binding buffer His-PlzE His-VC1641

12. DRaCALA ORFeome Screens • A screen of Staph aureusORFeome has identifed 4 novel cdiAMP receptors • Systematically identify genomically encoded receptors of metabolites • Identify processes regulated by second messenger systems • Systems-level understanding of regulatory potential of second messenger

13. Acknowledgements Funding University of Maryland NIH R21 NIH T32 Training Grant DOD DTRA Cystic Fibrosis Foundation Lee Lab Members

14. DRaCALA detects specific binding of soluble and insoluble proteins in WCL PAGE of Whole Cell Lysate (W) and Soluble Fraction (S)

15. Nucleotide Second Messengers Regulate Bacterial Metabolism Nucletoides: cAMP cdiGMP pGpG cdiAMP c-AMP/GMP ppGpp(p) Receptors: Protein RNA What is the total regulatory potential of a second messenger?

16. What is the biological function of any small molecule? cAMP has one known protein receptor in E. coli: CRP Glucose cAMP ? CRP Alternative Carbohydrate Utilization Mona Wu “Intracellular cAMP levels affect bacterial chemotaxis responses by inhibiting CheA” Poster # _______________ E. coli whole cell lysates from strains lacking CRP still bind cAMP

17. SM – less story (use other slide) Detect – put this in comparisons DRaCALA – no animation Example - ORF Staph (use figure that was made remove names thank grundling lab) - Vibrio

18. Differentiating ORFs that directly and indirectly increase cdiGMP binding • CdiGMP Binding Proteins • Receptors, Diguanylate Cyclases, Phosphodiesterases • 2. Conserved non-binding regulators of cdiGMP signaling • Transcription factors ORF ORF Mills et al. 2012 Cell Micro