Towards Systematic Identification of cdiGMP Binding Proteins

Kevin RoelofsLaboratory of Dr. Vincent Lee

University of Maryland, College Park

What is the total regulatory potential of a second messenger?

Nucletoide Second Messengers:cAMPppGpp(p)cdiGMPcdiAMPc-AMP/GMP

Membrane Proteins

Cytosolic Enzymes

Transcription Factors

Phenotypes

Receptors:

CdiGMP in Vibrio cholerae

• 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 Microbiol 2007

Current approaches in second messenger receptor identification

Identify target genes required for phenotype regulated by second messenger

Systematically purify and test target proteins for binding to second messenger

Pull down proteins with second messenger as bait

Identify second messenger in crystal structures of purified proteins

Identify receptors with conserved binding domains

1) Genetics 2) Biochemistry 3) Bioinformatics

Problems with current approaches

1. Genetics – Receptors not always associated with observable phenotype

2. Biochemistry – Receptor must be expressed

3. Bioinformatics – Limited to identification of previously described binding motifs

None of these approaches systematically identify all genomically encoded receptors

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

DRaCALA detects specific binding interactions

Proof of Principle: Purify 10 µM protein

Mix with nM amounts of radiolabeled nucleotide

Quantitative Detection of Binding Interactions in E. coli Whole Cell Lysate

Mix with 32P cdiGMP:1. Purified Alg442. Purified Alg44

added to BL21 E. coli cell lysate

3. BL21 E. coli cell lysate with endogenously expressed Alg44

Plot Fraction Bound vs Protein Concentration

Design of DRaCALA ORFeome Screen

1. Obtain Open Reading Frame (ORF) librarya. Contains all annotated ORFs from individual organismb. V. cholerae ORF library contains 3866 unique clones

2. Express individual V. cholerae ORFs in E. coli T7IQa. 2 libraries: N-terminal His- or HisMBP-

3. Screen whole cell lysates in 96-well format for increased cdiGMP binding

Results of Partial (938 ORFs) Screen

Positive Clone Positive Control

Buffer control

-0.10.00.10.20.30.40.50.6

VCA0681

VCA0593

VCA0753

VCA0042

VC2681

VC1641

His-ORF Library

Frac

tion

Boun

d

-0.1

0.1

0.3

0.5

0.7

0.9 VCA0681 VCA0593

VCA0753

VCA0042

VC1641

HisMBP-ORF Library

Frac

tion

Boun

d

Other

PilZ domainEAL domain

3 standard deviation positive cutoffAverage Fraction Bound

1 mM Competitor

His-PlzE

His-VC1641

NC NC cdiGMP cGMP cAMP GMP GDP GTP CTP ATP UTP

Vectorcontrol ORF expressed

Biochemical Classification of Positive Hits

His-PlzE

His-VC1641

2x Serial Dilutions of E. coli WCL into binding buffer

DRaCALA ORFeome Screens• A screen of Staph aureus ORFeome 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

FundingUniversity of MarylandNIH R21NIH T32 Training GrantDOD DTRACystic Fibrosis Foundation

Lee Lab Members

Acknowledgements

DRaCALA detects specific binding of soluble and insoluble proteins in WCLPAGE of Whole Cell Lysate (W) and Soluble Fraction (S)

Nucleotide Second Messengers Regulate Bacterial Metabolism

Nucletoides:cAMPcdiGMPpGpGcdiAMPc-AMP/GMPppGpp(p)

Receptors:ProteinRNA

What is the total regulatory potential of a second messenger?

What is the biological function of any small molecule?

Glucose cAMP

Alternative Carbohydrate Utilization

CRP?

E. coli whole cell lysates from strains lacking CRP still bind cAMP

Mona Wu“Intracellular cAMP levels affect bacterial chemotaxis responses by inhibiting CheA”Poster # _______________

cAMP has one known protein receptor in E. coli: CRP

SM – less story (use other slide)Detect – put this in comparisonsDRaCALA – no animationExample - ORFStaph (use figure that was made remove names thank grundling lab) - Vibrio

1. CdiGMP Binding ProteinsReceptors, Diguanylate Cyclases, Phosphodiesterases

Mills et al. 2012 Cell Micro

ORFORF

Differentiating ORFs that directly and indirectly increase cdiGMP binding

2. Conserved non-binding regulators of cdiGMP signaling Transcription factors