Transcript 3*, 3*-cGAMP

Hybrid promiscuous (Hypr) GGDEF enzymes
produce cyclic AMP-GMP (3′, 3′-cGAMP)
Proc Natl Acad Sci U S A. 2016 Feb 16;113(7):1790-5.
Zachary F. Hallberg, et al.
Student: Yi-Wen Lin
Advisor: Shan-Ho Chou
Cyclic dinucleotides (CDNs)
CDNs
bacteria
Secondary
messenger
mammalian
Agonists of the
innate immune
response
Secondary
signaling
molecules
2
Cyclic dinucleotides (CDNs)
1. Motility
2. Biofilming
-forming
In bacterial cell
wall
1. Homeostasis
2. sporulation
In Prokaryotic
(DncV)
1. motility
2. Intestinal
colonization
In Eukaryotic
(cGAS)
1. Innate
immune
response
3
Cyclic AMP-GMP(3’,3’-cGAMP, cAG)
2
(1) Induce ToxT activity
→transcription of TarB
(2) Stabilized by Hfq
(3) DncV activity increases
cellular concentration of cAMP-GMP
1
3
4
(4) colonization
Davies BW, et al. (2012) Cell 149(2):358–370.
4
Geobacter sulfurreducens
deltaproteobacteria
cAG-sensing riboswitch
• Extracellular electron
transfer
• Bacterial colonization
• Gain function via
GEMM-I riboswitches
Production of cAG
• The synthase enzyme
remained a mystery.
• Geobacter genomes have
no homologs to DncV or
cGAS.
Typically bind cdiG
cAG signaling may have evolved in
Geobacter from the cdiG signaling
pathway.
Production of cAG may be
associated with production
of cdiG.
5
Cyclic di-GMP (cdiG)
Hengge R (2009) Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol 7(4):263–273.
6
GGDEF Enzymes in Geobacter sulfurreducens
Hypothesize that one or more GGDEF domains
had gained cAG synthase activity.
7
cAG synthase activity of GGDEF domains
• In vivo flow cytometry screen using fluorescent riboswitchbased biosensors
• Detect production of cAG and cdiG
GM0970
Kellenberger CA, et al. (2015) Proc Natl Acad Sci USA 112(17):5383–5388.
8
cAG synthase activity of GGDEF domains
• DncV and GSU1658 share no sequence homology.
• DncV and GSU1658 had similar results.
→ GSU1658 may have cAG synthase activity.
9
cAG synthase activity of GGDEF domains
→The activity of GGDEF enzyme may be associated
with cAG synthase activity.
→cAG
→cdiG
10
GSU1658 activity in vitro
Method
1. GSU1658 incubated with
radiolabeled ATP and GTP
2. TLC(thin layer chromatography)
I-site mutant(R393A)
CDNs may bind I-site to inhibit
enzyme activity.
Contradicts the cell lysate data
• In vivo→ the main product
was cAG
• In vitro→ cAG wasn’t the
main product; it also produced
cdiA and cdiG
11
The discrepancy between in vivo and in vitro
In vivo
• cdiG-specific phosphodiesterase
• No cdiA- or cAG-specific phosphodiesterases
In vitro
• In vitro assays carried out with 1:1 ATP to GTP
• In cells, ATP usually found in excess relative to
GTP
12
The discrepancy between in vivo and in vitro
cAG
cdiG
cdiG
cAG
cdiA
cdiA
13
The discrepancy between in vivo and in vitro
The result is close to in vivo.
A GGDEF enzyme with dinucleotide synthase activity that produces
different CDNs depending on the ratio fo ATP to GTP.
14
Identification of specificity position in GSU1658 active site
Phosphorylation
Phosphorylation of the Rec domain is known to activate
homodimer formation of canonical GGDEF enzymes
such as PleD.
Phosphomimic compound
• Phosphorylation site knock-out(D52A) and
mimic(D52E) mutation (this work)
15
Identification of specificity position in GSU1658 active site
Phosphorylation
• D52A and D52E had little to no effect on overall enzyme
activity.
→These mutations may not adequately recapitulate the
phosphorylated state of the Rec domain in GSU1658.
16
Identification of specificity position in GSU1658 active site
• GSU1658 may harbor a Ser
residue (S347) in place of
the Asp residue(D344) that
interacts with the nucleobase
of the GTP substrate.
Nuleotide binding region of PleD
in complex with non-hydrolysable
GTP analog.
17
Identification of specificity position in GSU1658 active site
• S347A maintained dinucleotide cyclase activity
The Ser side chain is not strictly necessary for recognition of ATP or
GTP
• S347D only makes cdiG
The Asp side chain restores specificity for GTP
This position(S347) strongly influences recognition of ATP or
GTP.
18
Identification of specificity position in GSU1658 active site
• The inverse D344S mutant of PleD was inactive.
• The ratio of cdiG to cAG produced by the protein chimera is
higher than for GSU1658.
Features outside of the GGDEF domain also affect product
ratios.
cdiG (m/z = 691), cAG (m/z = 675), and cdiA (m/z = 659)
19
The function and evolution of cAG signaling
20
Conclusion
• GGDEF enzyme
• A family of dinucleotide cyclases
• Besides synthesizing cAG and cdiG, GGDEF domains
can make cdiA.(this work)
• GSU1658
• The founding member of a distinct subfamily of GGDEF
enzymes (Be called hybrid, promiscuous (Hypr) GGDEF
enzyme)
• Make hybrid CDNs
• Are promiscuous for ATP and GTP substrates
• Be renamed HyprA
21
To survey this newfound Hypr subfamily
Bioinformatics analysis of 32,587 predicted active GGDEF
enzymes
Identify sequences D-to-S or D-to-T variation (related to Hypr
activity)
Result
These two variants are rare and comprise only 0.17% of all
GGDEF domain
Geobacter and Pelobacter species
• At least one Hypr enzyme
• Have riboswitch effectors that regulate genes in response to
cAG
22
Bacteria with no cAG-selective riboswitches
• Appear to encode candidate Hypr enzymes in their
genomes
Bacteria with no
cAG-selective
riboswitches
Myxococcus
xanthus
MXAN_4463
MXAN_2643
Bdeullovibrio
bacteriovorus
Bd0367
(DgcA)
23
Bacteria with no cAG-selective riboswitches
24
M. Xanthus surface sensing
• cAG was an endogenous signaling molecule
• Found cdiG but not cAG in cell extracts of wildtype M. xanthus cultured in solution
• cAG signaling in processes related to surface
sensing
Hypothesized that cAG was similarly associated
with M. xanthus growth on solid surfaces
25
M. Xanthus surface sensing
• Analyze cyclic dinucleotide content of M. xanthus
grown in solution versus on 1.5% agar
 Revealed that cAG is produced at higher levels
upon surface growth
26
Conclusion
• The large abundance and redundancy of GGDEF genes
in bacterial genomes have allowed this enzyme family
to diverge and evolve toward new synthase activity.
• Besides synthesizing cAG, GGDEF domains can make
cdiA.
• Hypr activity is more widespread in bacteria than the
distribution of cAG riboswitches.
27
Cyclic AMP-GMP(3’,3’-cGAMP, cAG)
• cAG Synthases DncV or cGAS
oligoadenylate synthase-like domains
Produce structurally distinct isomers of cAG(3′, 3′-cGAMP and 2′,
3′-cGAMP).
28
Cyclic AMP-GMP(3’,3’-cGAMP, cAG)
29
Diguanylate cyclases
A C2-symmetric active site
formed by the homodimeric
association of two GGDEF
domains, with one GTP
bound per monomer.
30
Chan C, et al. (2004) Proc Natl Acad Sci USA 101(49):17084–17089.
GSU1658
PleD
Activation of enzyme dimerization or oligomerization
likely occurs through the receiver (Rec) domain.
31
If GSU1658 can be self-associates
PleD Mutant R313A
Elution profiles of nonactivated PleD at a
protein concentration of 33 mM (gray) and
66 mM (black)
32
Wassmann P, et al. (2007) Structure 15(8):915–927.
If GSU1658 can be self-associates
2 c-di-GMP
GSU1658
I-site
inhibition
I-site
↑ The reason of wild type protein not
measuring relevant dimerization
Size-exclusion chromatography
33
GSU1658 activity in vitro
In vitro activity assay
Enzyme
50mM Tris-HCl
100mM NaCl
10mM MgCl2
5mM dithiothreitol
ATP GTP
[α-32P] –ATP
[α-32P]-GTP
Heating
95°C 30 sec
Spotted onto a
PEI-cellulose F ThinLayer
chromatography(TLC)
Incubated
28°C 1 hr
Treated with
Calf Intestinal Alkaline Phosphatase
At 28°C 30 min
to digest unincorporated NTPs
Phosphor-image screen
Typhoon scanner
1M KH2PO4
pH 3.6
34
Mutational analysis of GSU1658
Phosphorylation
E, Glutamate
D, Aspartate
Phosphorylation
mimic
Phosphorylation site
Knock out
phosphorylation
A, Alanine
35
Flow cytometry
36
Flow cytometry
waste
37
Thin layer chromatography (TLC)
38
LC-MS
39
Size exclusion chromatography
40