Download Structural biology of second messenger signaling: simple principles

Structural biology of second messenger signaling:
simple principles
for the regulation of c-di-GMP metabolism
1
Overview
c-di-GMP
signaling in bacteria
1-component, 2-component systems
biological processes regulated via c-di-GMP
synthesis: 2GTP ➞ c-di-GMP + 2 PPi
PleD, catalysis, regulation
degradation: c-di-GMP ➞ pGpG
YkuI, catalysis, (regulation)
2
Literature
Schirmer and Jenal. Structural and mechanistic determinants of c-di-GMP
signalling. Nat Rev Microbiol (2009) vol. 7 (10) pp. 724-35
Minasov et al. Crystal structures of YkuI and its complex with second
messenger cyclic Di-GMP suggest catalytic mechanism of phosphodiester bond
cleavage by EAL domains. J. Biol. Chem. (2009) vol. 284 (19) pp. 13174-84
Wassmann et al. Structure of BeF3- -modified response regulator PleD:
implications for diguanylate cyclase activation, catalysis, and feedback
inhibition. Structure (2007) vol. 15 (8) pp. 915-27
Chan et al. Structural basis of activity and allosteric control of diguanylate
cyclase. Proc. Natl. Acad. Sci. U.S.A. (2004) vol. 101 (49) pp. 17084-9
3
cyclic mono-nucleotide
O3’
O2’
O5’
c-GMP
4
cyclic dinucleotide
O3’
O2’
O5’
c-di-GMP
small molecule crystal structure, Egli et al., 1990
5
self-intercalated c-di-GMP dimer
small molecule crystal structure, Egli et al., 1990
6
signal transduction via c-di-GMP
diguanylate cyclase (DGC)
domain: GGDEF
receptor domain: PilZ
cellulose
GTP
c-di-GMP
c-di-GMP
GTP
7
signal transduction via c-di-GMP
cellulose
phosphodiesterase
domain: EAL
GTP
pGpG
c-di-GMP
c-di-GMP
8
signal transduction via c-di-GMP
cellulose
GTP
pGpG
c-di-GMP
c-di-GMP
DP
H
RR of Rec - DGC organisation: enzymatic
output
9
signal transduction via c-di-GMP
compare with
classical 2-component system HK : RR
RR
D P
H
HK
10
Bacteria Switch between two Different Life Styles
[c-di-GMP]
[c-di-GMP]
planktonic
motile
single cells
non-adhesive
virulence factors
acute virulence
sedentary
sessile
surface attached
matrix embedded
adhesion factors
persistence
courtesy of U. Jenal
11
synthesis of c-di-GMP
O
OO-
P
P O
O
G
5'
O-
-
P O
O
O
O O
OH
HO
3'
OH
HO
O O
O P
O
P
O
OO P
- O
O
O
G
O-
intermolecular nucleophilic
attack of O3ʼ - onto α-phosphate
12
synthesis of c-di-GMP
OP O
O
G
5'
O O
O O 5'
O P
3'
HO
OH
3'
O
G
O-
c-di-GMP
2 GTP → c-di-GMP + 2 PPi
13
DUF1/GGDEF = novel signal transduction node
SMART/pfam:
DUF1/GGDEF , >7000 entries
14
DUF1/GGDEF = novel signal transduction node
SMART/pfam:
DUF1/GGDEF , >7000 entries
PleD
D
15
PleD is an unorthodox response regulator
classical RR (transcription factor)
PleD: unorthodox RR (enzyme)
PleC
DivJ
D
PleD
P
H
Sensor
D
Kinase
GGEEF
D53
D1
D2
CheY-like
CheY-like
DGC
diguanylate
cyclase
16
structure solution by MAD
0.3mm
Experimental data
Data collection
Wavelength (Å)
Resolution (Å)
(Highest shell)
Space group
a = b (Å)
c (Å)
Unique reflections
Average redundancy
I/σ
Completeness (%)
Anom complete (%)
Rmerge (%)
Phasing
Resolution shell
Figure of merit
Mean figure of merit
Refinement
No of molecules in AU
Native
0.97950
2.7 (2.85 - 2.70)
Peak
0.97950
3.2
SeMet
Inflection
0.97970
3.0
Remote
0.97700
3.0
134.9
168.1
26249
4.1 (3.9)
6.2 (2.1)
99.9 (100)
99.7
10.5 (33)
32080
4.8 (4.9)
7.2 (1.8)
99.9 (100)
99.9
9.4 (39)
32140
4.6 (3.6)
7.3 (1.1)
99.6 (97.6)
97.5
11.0 (63)
P4
P422222111
135.9
169.2
42707
5.7 (5.5)
7.2 (1.9)
99.3 (98.7)
8.3 (36)
13.12
0.82
0.35
7.69
0.72
5.98
0.67
2
5.06
0.57
4.47
0.51
3.72
0.31
3.46
0.25
Residues in
Ramachandran core (%)
3.16
0.17
93.4
17
Experimental
data by MAD
structure
solution
Data collection
Native
SeMet
Peak
Inflection
Wavelength (Å)
0.97950
0.97950
0.97970
Resolution
2.7 (2.85
2.70) found
3.2
3.0
18 of(Å)
24 potential
Se -sites
by SHELXD
(Highest shell)
Space group
P42221
2 x 4 sites showed
crossword table)
a = b (Å)
135.9 NCS (in
134.9
c (Å)
169.2
168.1
Unique
reflections
42707(SHARP)26249
32080
phase
refinement
Average redundancy
5.7 (5.5)
4.1 (3.9)
4.8 (4.9)
I/σ
7.2 (1.9)
6.2 (2.1)
7.2 (1.8)
2-fold
averaging
(DM)
Completeness (%)
99.3 (98.7)
99.9 (100)
99.9 (100)
Anom complete (%)
99.7
99.9
Rmergerefinement
(%)
8.3 (36)
10.5 (33)
9.4 (39)
(REFMAC)
Phasing
Resolution shell
Figure of merit
Mean figure of merit
13.12
0.82
0.35
7.69
0.72
Refinement
No of molecules in AU
Resolution (Å)
R / Rfree (%)
R.m.s.d.
bond lengths a (Å)
bond angles a (o)
R.m.s.d. ΔB-factor bonded atoms (Å2)
Main chain
Side chain
a
r.m.s.d. from ideal stereochemistry
5.98
0.67
5.06
0.57
2
50.0 – 2.7
22.4 / 24.2
4.47
0.51
3.72
0.31
Remote
0.97700
3.0
32140
4.6 (3.6)
7.3 (1.1)
99.6 (97.6)
97.5
11.0 (63)
3.46
0.25
3.16
0.17
Residues in
Ramachandran core (%)
Protein atoms
Water molecules
Ligand atoms
93.4
Average B-factor (Å2)
27.1
6610
13
233
0.008
1.2
0.85
1.89
18
structure solution by MAD
Carmen Chan, PhD thesis, 2004
19
structure solution by MAD
Carmen Chan, PhD thesis, 2004
20
crystal structure of PleD
product complex
DGC
(0.8 mM c-di-GMP)
454 residues
domain linkers disordered
Mg2+
Chan et al., PNAS, 2004
21
how to activate?
loose dimer in the crystal
substrate (GTP) binding site
GGDEF motif
DGC
D1/D2 stem
D53’
D53
crystallization: [PleD] = 200 μM
22
a (physiological ?) dimer
D53
Y26
2
small interface (2 x 450 Å )
phosphorylation ->
optimization of interface by
domain rearrangement?
Y26`
D53’
23
a mechanistic model of regulation
activation by
dimerization
G
R
P
+ 2 PP
P
R
G
P
P
G R
P
P
A-site with
bound GTP
P
catalysis
P
G
P
P
P
G
Rec
P
P
activation
R
R
Rec'
P
G R
P
P
C
DG
P
P
G R
P
P
P
inhibition
P
24
PP
G RP
G
P
RP
P P
- activated
BeFactivated
PleD
PleD
3
1 mM GTPαS
0.2 mM c-diGMP
1 mM BeCl2
10 mM NaF
Wassmann et al., 2007
25
PleD
native
activated (BeF3-)
26
Rec domain (D1) of PleD, native
F102
T83
D53
K105
27
Rec domain (D1) of PleD, activated
BeF3-D53
F102
T83
K105
Wassmann et al., 2007
28
red: activated
29
catalysis
- activated
BeFactivated
PleD
PleD
3
1 mM GTPαS
0.2 mM c-diGMP
1 mM BeCl2
10 mM NaF
Wassmann et al., 2007
30
substrate analog binding
GTPαS
31
diguanylate cyclase similar to
adenylate cyclase
DGC
O3’
AC
DGC - adenylate cyclase:
#Ca 93 RMSD: 1.5 Å,
15% ident.
1CJK, Tesmer et al. (1999)
32
T7 DNA polymerase:
#Ca 63 RMS: 1.8 Å, 11% ident.
e
t
la
p
m
te
p
green: PleD
r
e
m
i
r
P-loop
O3’
β-hairpin
33
A
productive DGC dimer (model)
C
!4
D2
!1
!2
"0
O3’
E370
O3’
E371
D327
D2
C
34
a mechanistic model of regulation
activation by
dimerization
G
R
P
+ 2 PP
P
R
G
P
P
G R
P
P
A-site with
bound GTP
P
catalysis
P
G
P
P
P
G
Rec
P
P
activation
R
R
Rec'
P
G R
P
P
C
DG
P
P
G R
P
P
P
inhibition
P
35
PP
G RP
G
P
RP
P P
a second layer of control:
allosteric product inhibition
0.7
∧ 7 µM c-diGMP
=
0.6
40µM PleD
100µM GTP
OD(630)
0.5
0.4
0.3
0.2
0.1
+50µM c-diGMP
0
0
5
10
time (min)
15
20
photometric assay for PPi
36
PleD
native
activated (BeF3-)
37
the allosteric product binding site
38
a mechanistic model of regulation
G
R
IS
P
IP
G
P
P
G R
P
P
A-site with
bound GTP
P
catalysis
P
G
P
activation
R
P
R
Rec'
P
G R
P
P
IS'
C
P
DG
P
P
G
Rec
P
P
G R
P
P
P
inhibition
P
P
P
G
PP
G RP
P
PP
G RP
G
RP
P P
b) inhibition by domain
immobilization
+ 2 PP
R
a) activation by
dimerization
P
: (c-di-GMP)2
RP
P P
39
C-di-GMP specific phosphodiesterase
EAL
YkuI from Bacillus subtilis
2bas (MCSG, Argonne)
PAS
40
C-di-GMP specific phosphodiesterase
2 mM c-di-GMP
2 mM CaCl2
A35
E33
Minasov et al., JBC 2009
41
C-di-GMP specific phosphodiesterase
L35
scissile bond
E33
3.6 Å
Ca++
E209
D152
enzymatically inactive!
general base
Note: in
Tbd_1265/Mg++:
2.1 Å
D646 -----Mg++ (2r6o, MCSG)
42
C-di-GMP specific phosphodiesterase
D152
regulation?
43
outlook
how is activated PleD localized to the stalk pole ?
how are DGCs regulated in other multi-domain
arrangements?
how are PDEs regulated?
down-stream targets of signaling path (c-di-GMP
receptors) ?
DP
H
What is the role of GGDEF - EAL proteins?
c-di-GMP
GTP
pGpG
44
Structural Biology
Carmen Chan, Paul Wassmann,
Dietrich Samoray, Sivaraman
Padavattan, Arnaud Baslé, Claudia
Massa, T.S.
Midwest Center for Structural Genomics
George Minasov, Ludmilla Shuvalova,
Wayne F. Anderson
Molecular Microbiology
Ralf Paul, Matthias Christen, Urs
Jenal
Biophysics
Andreas Beck, Heiko Heerklotz
Clemens Schulze-Briese
Organic Chemistry, Organic Chemistry
Nicolas Amiot, Bernd Giese
45