Download Asp P

Heme-based NO sensors
HNOX: Heme-nitric oxide/oxygen binding domain/protein Bacterial
1
J. Inorg. Biochem. 99, 892 (2005).
Soluble guanylate cyclase (sGC) is a nitric oxide (NO) sensing
hemoprotein that has been found in eukaryotes from Drosophila to
humans. Prokaryotic proteins with significant homology to the
heme domain of sGC have recently been identified through
genomic analysis. This family of heme proteins has been named the
H-NOX domain, for Heme-Nitric oxide/OXygen binding domain. The
key observation from initial studies in this family is that some members, those proteins 2
3
[B] sensor protein - HnoX (4)
HnoX
N
N
Fe N
N
SIGNAL
NO dissociation
HnoC, HnoD or HnoB
response regulators
histidine kinase
HnoK
Histidine kinase
Asp
His
ATP
P
autophosphorylation
P
phosphotransfer
LuxU
HqsK
Histidine kinase
Asp
ATP
P
His
ATP
autophosphorylation
regulation of
transcription or
catalytic activity
LuxO
Histidine kinase
His P
OUPUT
P
Asp
P
OUTPUT
regulation of
transcription
phosphotransfer
Fig. 4
4
Sessility (biofilm formation)
Fimbrial formation
Virulence
Environmental persistence
Cell-cell communication
INPUT (signal)
Binding of signal molecules
Phosphorylation
Ion binding
DNA binding
Light
O
N
OH
O
HO
P
O
N
N
HN
OUTPUT
(phenotype)
NH2
O
O
N
N
O
O
H2N
NH
O
P
OH
O
HO
N
Phosphodiesterase
c-di GMP
O
O
N
O
P
O
O
Diguanylate cyclase
NH
OH
N
N
NH2
HO
HO
OH
N
H2N
HN
N
O
OH
O
O
P
O
N
O
HO
HO
N
pGpG or l-di GMP
OUTPUT
(phenotype)
O
Motility
Virulence
Phage resistance
Hyphae formation
Antibiotic production
OH
OH O
P
P
O
O
P
O
N
HO
HO
OH
OH
O HO
O
N
HN
N
N
O
OH O
O
H2N
NH
O
N
O
Fig. 7
P
HO
GTP
O
O
P
O
P
OH
NH2
INPUT
(signal)
O HO
Binding of signal molecules
Phosphorylation
Ion binding
DNA binding
5
Light
O
LuxN
CqsS
N
LuxPQ
LuxO
Asp
P
P His
P His
Asp
Asp
LuxU
P His
His
P
Asp
Hpt
Fur
His
P
HnoK
HqsK
Asp
P His
His
Asp
P
HnoC
HnoX
HqsK
Asp
ATP
ATP
Fe
His
P
ADP
ADP
histidine kinase
DGC
ATP
ADP
His
P
response regulator
Asp
P
?
His
P
HnoB
Asp
P
PE
transcriptional
feedback
Asp
P
HnoK
PE
HnoD
2 GTP
c-diGMP
l-diGMP
Fig. 10
Asp
P
DGC
6
The Marletta Lab. Univ. California, Berkeley, Script Research Institute
Genomic analysis has recently placed sGC within a larger family of proteins with Heme Nitric
oxide/Oxygen binding (H-NOX) domains including prokaryotic proteins with significant homology (1540% identity) to the heme domain of sGC. Predicted H-NOX domains were found in facultative aerobes,
obligate anaerobes, and thermophiles. Genomic analysis reveals that the H-NOX domains may be
linked to histidine kinases or diguanylate cyclases (obligate anaerobes) or methyl-accepting chemotaxis
proteins (obligate anaerobes). Uncovering the biological function of these H-NOX domains is currently
an area of intense investigation in our lab.
(A) Structural features of Tt H-NOX. (B) Heme binding pocket.H-NOX proteins also exhibit remarkable
diatomic ligand selectivity despite a similar protein fold. For example, the H-NOX domain from Vibrio
cholera (a facultative aerobe) binds NO in a high spin 5-coordinate complex and excludes oxygen, while
the H-NOX domain from Thermoanaerobacter tengcongensis (Tt, obligate anaerobe) has been found to
bind oxygen in a low-spin 6-coordinate complex, making it the first member of the family to bind O2.
Current research is focused on understanding the nature of this ligand selectivity from a molecular
level and how this selectivity translates into protein function as sensors in biology.
7
Fig. 2. Sequence alignment selected
members of the H-NOX family. Sequence
numbering is that of Tt H-NOX. Invariant
residues are highlighted in green and
very highly conserved residues are
highlighted in blue. Y140 of Tt H-NOX is
highlighted in red. Predicted distal
pocket tyrosine residues that may
stabilize an FeII–O2 complex in other HNOX proteins are in red. Accession
numbers are: Homo sapiens β1
[gi:2746083], Rattus norvegicus β1
[gi:27127318], Drosophila melangaster
β1 [gi:861203], Drosophila melangaster
CG14885-PA [gi:23171476],
Caenorhabditis elegans GCY-35
[gi:52782806], Nostoc punctiforme
[gi:23129606], Caulobacter crescentus
[gi:16127222], Shewanella oneidensis
[gi:24373702], Legionella pneumophila
(ORF2) [CUCGC_272624], Clostridium
acetobutylicum [gi:15896488], and
Thermoanaerobacter tengcongensis
[gi:20807169]. Alignments were
generated using the program
MegAlign.
8
Fig. 3. Speculation on prokaryotic signaling pathways involving H-NOX domains. (a) Proposed role of an
H-NOX sensor in a facultative aerobic bacterium. The H-NOX domains in facultative aerobes may have
evolved as sensors for NO derived from under conditions of low O2 concentration. The NO signal may be
transmitted via the action of a histidine kinase. Most of the predicted H-NOX ORFs from aerobic bacteria
are contained within an operons that also contains a predicted histidine kinase, and additionally, these
bacteria also contain predicted nitrate reductase proteins, consistent with this hypothesis. (b) Proposed
role of an H-NOX sensor in an obligate anaerobic bacterium. The H-NOX domain in obligate anaerobes is
fused through a transmembrane domain (shown in gray) to a MCP. Here the H-NOX domain may be used
as an O2 sensor to signal a change in O2 concentration, regulating methylation by S-adenosyl-methionine
(SAM) leading to taxis towards more favorable O2 concentrations. Aside from NO binding to the H-NOX
domain of sGC, ligand binding to an H-NOX protein has not been conclusively linked to biological
signaling processes to date.
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Fig. 4. The heme environment of the Tt H-NOX domain[38]. (a) The conserved Y-S-R
motif makes hydrogen bonding interactions with the propionic acid side chains of
the heme group, which is colored yellow (porphyrin) and red (iron). (b) The
conserved H102 is the proximal ligand to the heme. In Tt H-NOX, a distal pocket
hydrogen-bonding network, involving principally Y140, stabilizes an FeII–O2 complex.
This hydrogen-bonding network is predicted to be absent in the H-NOX proteins
from sGCs and aerobic prokaryotes, suggesting this as a key molecular factor in the
remarkable ligand selectivity against O2 displayed by these heme proteins.
10
Fig. 5. Schematic summary of the H-NOX family of heme-based
sensors. The progenitor H-NOX domain has evolved to
discriminate between ligands such as NO and O2 for specific
sensing purposes. This is the first family of related heme proteins to
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discriminate between different physiologically relevant diatomic gaseous ligands.
Mol. Cell 46 (4) 449 (2012)
Highlights
► A complex multicomponent bacterial H-NOX signaling network was mapped ► EAL
response regulator phosphorylation stimulated c-di-GMP hydrolysis activity ► A
degenerate HD-GYP response regulator inhibited the EAL response regulator ► NO induced
biofilm formation through c-di-GMP modulation by the signaling network
12
Figure 6. Model of
Multicomponent Signaling
Network for NO-Induced Biofilm
Formation as a Protection
Mechanism against NO(A) The
complex multicomponent signaling
pathway is initiated by NO binding
to the sensory H-NOX protein
(HnoX), which then inhibits HnoK
autophosphorylation.
Phosphotransfer establishes a
branching of the network to three
response regulators. HnoB and
HnoD form a feed-forward loop.
Phosphorylation controls PDE
activity of HnoB, which can be finetuned by allosteric control from
HnoD. NO-controlled repression of
the PDE activity leads to an
increase in c-di-GMP levels, which
serves as a signaling cue for
cellular attachment into
biofilms.(B) The NO signal switches
the bacterial motility pattern from
planktonic growth to increased
attachment onto surfaces. The
thick layers of cells provide a
protective barrier against diffusion
of reactive and damaging NO and
may protect cells in the lower 13
layers.