In response to environmental conditions, NO (nitric oxide) induces global changes in the cellular metabolism of Pseudomonas aeruginosa, which are strictly related to pathogenesis. In particular, at low oxygen tensions and in the presence of NO the denitrification alternative respiration is activated by a key regulator: DNR (dissimilative nitrate respiration regulator). DNR belongs to the CRP (cAMP receptor protein)–FNR (fumarate and nitrate reductase regulatory protein) superfamily of bacterial transcription factors. These regulators are involved in many different pathways and distinct activation mechanism seems to be operative in several cases. Recent results indicate that DNR is a haem protein capable of discriminating between NO and CO (carbon monoxide). On the basis of the available structural data, a suggested activation mechanism is discussed.

Introduction

NO (nitric oxide) is a cellular messenger that, at low concentration, functions as a signalling molecule, whereas at high concentration, may act as general poison owing to its capability to alter biological macromolecules. In bacteria, NO induces global changes in the cellular metabolism which are modulated by the environmental conditions (i.e. oxygen tension). Low oxygen tension is required for the colonization of most of the pathogenic organism such as Pseudomonas aeruginosa [1], suggesting that anaerobic conditions are crucial in the study of pathogenic NO resistance mechanisms.

Consequently, bacteria have evolved specific sensors, usually at the level of transcription, to monitor different redox signals such as the presence or absence of oxygen, NO, cellular redox state or ROS (reactive oxygen species). The sensing mechanisms are many and varied, and can involve redox-active cofactors or redox-sensitive amino acid side chains such as cysteine thiols [2].

In denitrifying bacteria, NO is an intermediate of the facultative respiration of nitrate [3] and the activity of the four enzymes involved in denitrification is necessarily regulated both kinetically and transcriptionally to avoid its toxic accumulation.

The NO-responsive components controlling the denitrification process, under both anaerobic and aerobic conditions, belong to the CRP (cAMP receptor protein)–FNR (fumarate and nitrate reductase regulatory protein) superfamily of transcription factors [i.e. DNR (dissimilative nitrate respiration regulator) and NnrR subgroups]. Although these regulators are widely found in bacteria and the subject of deep investigation, the general activation mechanism of the CRP–FNR class of transcription factors is still not completely clear.

The CRP–FNR transcription factors

The CRP [also referred as CAP (catabolite activator protein)] from Escherichia coli is the first and name-giving member of the CRP–FNR superfamily of prokaryotic transcription factors. This class of regulators are widespread activators that respond to a broad spectrum of intra- and extra-cellular signals, controlling a huge number of different metabolic pathways. To fulfil their role, they have evolved intrinsic sensory modules allowing direct binding of the effector molecule (signal), or through prosthetic groups [4].

Despite their low sequence identity (<25%) they share common structural features, being all homodimers with the following domain organization for each subunit: (i) an N-terminal SD (sensing domain); (ii) a dimerization helix (α-C); and (iii) a C-terminal DBD (DNA-binding domain) with an HTH (helix–turn–helix) motif (Figure 1).

Structure of CRP in the ON conformation in complex with DNA (PDB code 1RUN)

Figure 1
Structure of CRP in the ON conformation in complex with DNA (PDB code 1RUN)

On the left-hand side, a cartoon representation of one monomer showing its domain organization (the partner subunit is shown as ribbons): (i) the N-terminal SD (grey); (ii) the dimerization helix (α-C; black); (iii) the C-terminal DBD (light grey). On the right-hand side, the location of important structural elements of the CRP–FNR superfamily is highlighted.

Figure 1
Structure of CRP in the ON conformation in complex with DNA (PDB code 1RUN)

On the left-hand side, a cartoon representation of one monomer showing its domain organization (the partner subunit is shown as ribbons): (i) the N-terminal SD (grey); (ii) the dimerization helix (α-C; black); (iii) the C-terminal DBD (light grey). On the right-hand side, the location of important structural elements of the CRP–FNR superfamily is highlighted.

Binding of the effector molecule (signal) to the SD drives a conformational change from the inactive conformation (OFF conformation) to the active conformation (ON conformation) capable of binding the cognate DNA sequence thus promoting the transcription of controlled genes [5].

Activation mechanism of CRP regulators

Given the very different pathways in which they are involved and the lack of structural data (especially of ligand-free proteins), a unique activation mechanism, at a molecular level, cannot be assigned to all the members of the CRP–FNR class of regulators. Nevertheless a group of common features and key structural elements have been assessed from the few structures available (see Table 1). In the ligand-free form of the protein (OFF conformation), the DBD is able to rotate more than 160° around its junction with the dimerization helix (referred to as the hinge; Figure 1): helices α-C and α-D in this case are acting as upper-arm and forearm connected by the elbow. Binding of the signal molecule to the SD (cAMP in the case of CRP) drives a first conformational rearrangement in the hinge region, which brings the HTH motif of the DBD in the correct position for DNA recognition and binding. The ON conformation transition is then accomplished by a complex network of new interaction between the DBD and a β-hairpin of the SD (referred to as the flap; Figure 1) [69]. This agrees well with the observation that the active sites of these regulators, despite the presence or absence of a prosthetic group, are always located in the central region of each monomer, contacting not only the SD, but also the hinge and the flap regions (Figure 1).

Table 1
Most representative structures of the CRP–FNR superfamily of transcription factors

CHPA, 3-chloro-4-hydroxyphenylacetate.

ProteinOrganismEffector/cofactorMutationConformationLigandsPDB code and reference
CRP E. coli cAMP  ON 2 cAMP 1G6N [22
 E. coli cAMP  ON/DNA complex 2 cAMP 1RUN [18
 E. coli cAMP  OFF − 2WC2 [9
CooA Rhodospirillum rubrum CO/haem  OFF − 1FT9 [23
 Carboxydothermus hydrogenoformans CO/haem N127L/S128L ON CO 2HKX [24
PrfA Listeria monocytogenes Unknown  OFF − 2BEO [25
 L. monocytogenes Unknown G145S ON − 2BGC [25
CprK Desulfitobacterium dehalogenans CHPA  OFF − 2H6C [26
 Desulfitobacterium hafniense CHPA  ON 2 CHPA 2H6B [26
DNR Ps. aeruginosa NO/haem  OFF − 3DKW [17
 Ps. aeruginosa NO/haem N152stop − − 2Z69 [15
ProteinOrganismEffector/cofactorMutationConformationLigandsPDB code and reference
CRP E. coli cAMP  ON 2 cAMP 1G6N [22
 E. coli cAMP  ON/DNA complex 2 cAMP 1RUN [18
 E. coli cAMP  OFF − 2WC2 [9
CooA Rhodospirillum rubrum CO/haem  OFF − 1FT9 [23
 Carboxydothermus hydrogenoformans CO/haem N127L/S128L ON CO 2HKX [24
PrfA Listeria monocytogenes Unknown  OFF − 2BEO [25
 L. monocytogenes Unknown G145S ON − 2BGC [25
CprK Desulfitobacterium dehalogenans CHPA  OFF − 2H6C [26
 Desulfitobacterium hafniense CHPA  ON 2 CHPA 2H6B [26
DNR Ps. aeruginosa NO/haem  OFF − 3DKW [17
 Ps. aeruginosa NO/haem N152stop − − 2Z69 [15

This general mechanism could be even more complicated if we consider the recent finding associated with the only apo-CRP structure available (solved by NMR) [9]. In this OFF conformation structure, with no cAMP bound, the dimerization helix α-C is partially unfolded in the hinge region. The DBD is thus connected to the rest of the protein by an unstructured linker, with α-C and α-D in this case acting more as a forearm–wrist–hand connection rather than the more rigid upper arm–elbow–forearm one. If this is a general characteristic of the OFF form of the CRP–FNR regulators then we should consider the OFF form of these proteins more like an ensemble of inactive conformation rather than a single one [9].

The plasticity of the CRP–FNR class of transcription factors not only explains their evolutionary success, since their flexible scaffold has been easily adapted to very different signals, but also accounts for the lack of structural data, especially in the apo form, and the consequent difficulties in the fine understanding of the allosteric transition for each of these regulators; indeed it is very difficult to obtain single crystals from these proteins.

The DNR transcription factor: NO sensing in Ps. aeruginosa

Ps. aeruginosa is one of the most important human pathogens and is mainly associated with nosocomial and chronic infections such as in pulmonary infections of cystic fibrosis patients; in the latter case, Ps. aeruginosa uses denitrification as the anaerobic energy producing pathway [1].

The induction of denitrification requires two CRP/FNR transcription factors, ANR (anaerobic regulation of arginine deaminase and nitrate reduction) and DNR, belonging to the DNR subtype that shares the same signature sequence (EXXSR) for the interaction with the cognate DNA sequence (i.e. the FNR box, TTGATN4ATCAA). ANR is an FNR-like global regulator for anaerobic gene expression [10] which promotes, under low oxygen tensions, the expression of the DNR protein. In the presence of NO, DNR activates the nirS, norCB and nosR promoters, inducing the expression of the denitrification gene cluster [1113].

The mechanism whereby DNR (or its homologue) responds to NO is still elusive. Recent in vivo data demonstrated that recombinant DNR responds to NO by activating the Ps. aeruginosa norCB promoter, even if in a non-denitrifier background such as E. coli; the transcriptional activity is lost by using an E. coli mutant strain unable to synthesize haem [14].

Interestingly, a previous in vitro study demonstrated that purified protein can form a stable complex with haem both in the ferric and the ferrous state, whose stability falls in the range observed for naturally occurring specific haem proteins (Kd≈10−4–10−5 s−1) [15]. Spectroscopic and kinetic analysis of the haem–DNR complex indicate that the ferrous derivative is six-co-ordinate with protein residues [15]. Moreover, the haem–DNR complex can react differently with typical haem iron ligands such as NO and CO (carbon monoxide); this evidence suggests that the protein can discriminate between the two ligands by forming a five-co-ordinate or a six-co-ordinate complex, with NO and CO respectively [15]. The different reactivity with these two diatomic gases that involves DNR, as a NO sensor, responds differently to NO and CO in vivo; as expected, it was shown recently that DNR does not activate the norCB promoter in the presence of CO [14].

Binding experiments show that haem-containing DNR can react with CO and NO with kinetics independent of ligand concentration, relatively slowly (kobs≈17 s−1 and kobs≈7 s−1 for NO and CO respectively) [15] and is therefore rate-limited by the dissociation of a protein ligand, as expected for a six-co-ordinate haem protein [16]; however, protein residues involved in haem iron co-ordination have not yet been identified.

Structural information on DNR come from two crystallographic structures, solved recently, of a deletion mutant lacking the DBD (ΔC-DNR) and of the full-length protein, both in the apo form [15,17].

Analysis of the mutant structure reveals a putative haem-binding site positioned between the SD and the dimerization helix (α-C). This site is absent from the full-length structure of DNR that displays a peculiar OFF conformation in which both the DBD and the SD deviates from the position observed in other homologues structures, and α-C is fused with α-D in a long single helix (Figure 2A).

Putative DNR activation mechanism.

Figure 2
Putative DNR activation mechanism.

(A) The structure of DNR full-length (OFF conformation; PDB code 3DKW) and the chimaera model (ON conformation). In the chimaera model, the SD and α-C correspond to the crystal structure of the mutant ΔC-DNR (PDB code 2Z69), whereas the DBD is modelled on the structure of CRP. The inset shows an enlargement of the cavity, which is likely to be the haem-binding site. (B) Hypothetical mechanism proposed for DNR activation.

Figure 2
Putative DNR activation mechanism.

(A) The structure of DNR full-length (OFF conformation; PDB code 3DKW) and the chimaera model (ON conformation). In the chimaera model, the SD and α-C correspond to the crystal structure of the mutant ΔC-DNR (PDB code 2Z69), whereas the DBD is modelled on the structure of CRP. The inset shows an enlargement of the cavity, which is likely to be the haem-binding site. (B) Hypothetical mechanism proposed for DNR activation.

To better understand the conformational changes involved in DNR activation the structure of ΔC-DNR was completed by modelling the missing DBD in the ON form using the structure of CRP as template (PDB code 1RUN) [15,17,18]. Comparison of this chimaeric protein in the ON conformation with the OFF structure reveals that both the DBD and the SD have to undergo a dramatic rearrangement in order to switch from the OFF form to the active form of the protein. In particular, the DBD has to rotate around the hinge of 155°, whereas the SD has to rotate around the pivot connection with α-C of 60°, forming the putative haem-binding site (Figure 2A).

The position of the SD in the OFF structure is peculiar to DNR because it has never been observed for other homologues. DNR thus appears to be even more flexible than its homologues, and plasticity is probably playing a key role in the activation mechanism of this protein.

In a possible mechanism that has been proposed (Figure 2B), the haem binds to the OFF form of the protein triggering a first conformational change to a six-co-ordinate ferrous haem–DNR complex. Activation is achieved upon NO binding, which releases the two haem ligands evolving towards the active ON conformation (five-co-ordinate NO–haem–DNR complex) [17].

The current hypothesis is that haem is the NO-binding site of DNR, whereas it is not clear if haem is stably bound to the protein or it is involved in a fine regulatory mechanisms as suggested for other regulators [1921]. Haem binding may regulate DNR activation by locking the protein in a given conformation or by hindering possible NO-target residues, but how haem association triggers DNR activation is yet to be determined.

Enzymology and Ecology of the Nitrogen Cycle: A Biochemical Society Focused Meeting held at University of Birmingham, U.K., 15–17 September 2010. Organized and Edited by Jeff Cole (University of Birmingham, U.K.), Rosa María Martínez-Espinosa (University of Alicante, Spain), David Richardson (University of East Anglia, Norwich, U.K.) and Nick Watmough (University of East Anglia, Norwich, U.K.).

Abbreviations

     
  • ANR

    anaerobic regulation of arginine deaminase and nitrate reduction

  •  
  • CRP

    cAMP receptor protein

  •  
  • DBD

    DNA-binding domain

  •  
  • DNR

    dissimilative nitrate respiration regulator

  •  
  • FNR

    fumarate and nitrate reductase regulatory protein

  •  
  • HTH

    helix–turn–helix

  •  
  • SD

    sensing domain

Funding

This work was supported by the Ministero della Università of Italy [grant numbers 20074TJ3ZB and RBRN07BMCT] and the University of Rome La Sapienza.

References

References
1
Hassett
 
D.J.
Cuppoletti
 
J.
Trapnell
 
B.
Lymar
 
S.V.
Rowe
 
J.J.
Yoon
 
S.S.
Hilliard
 
G.M.
Parvatiyar
 
K.
Kamani
 
M.C.
Wozniak
 
D.J.
, et al 
Anaerobic metabolism and quorum sensing by Pseudomonas aeruginosa biofilms in chronically infected cystic fibrosis airways: rethinking antibiotic treatment strategies and drug targets
Adv. Drug Delivery Rev.
2002
, vol. 
54
 (pg. 
1425
-
1443
)
2
Green
 
J.
Paget
 
M.S.
 
Bacterial redox sensors
Nat. Rev. Microbiol.
2004
, vol. 
2
 (pg. 
954
-
966
)
3
Zumft
 
W.G.
 
Cell biology and molecular basis of denitrification
Microbiol. Mol. Biol. Rev.
1997
, vol. 
61
 (pg. 
533
-
616
)
4
Korner
 
H.
Sofia
 
H.J.
Zumft
 
W.G.
 
Phylogeny of the bacterial superfamily of Crp–Fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs
FEMS Microbiol. Rev.
2003
, vol. 
27
 (pg. 
559
-
592
)
5
McKay
 
D.B.
Steitz
 
T.A.
 
Structure of catabolite gene activator protein at 2.9 Å resolution suggests binding to left-handed B-DNA
Nature
1981
, vol. 
290
 (pg. 
744
-
749
)
6
Harman
 
J.G.
 
Allosteric regulation of the cAMP receptor protein
Biochim. Biophys. Acta
2001
, vol. 
1547
 (pg. 
1
-
17
)
7
Kim
 
J.
Adhya
 
S.
Garges
 
S.
 
Allosteric changes in the cAMP receptor protein of Escherichia coli: hinge reorientation
Proc. Natl. Acad. Sci. U.S.A.
1992
, vol. 
89
 (pg. 
9700
-
9704
)
8
Yu
 
S.
Lee
 
J.C.
 
Role of residue 138 in the interdomain hinge region in transmitting allosteric signals for DNA binding in Escherichia coli cAMP receptor protein
Biochemistry
2004
, vol. 
43
 (pg. 
4662
-
4669
)
9
Popovych
 
N.
Tzeng
 
S.R.
Tonelli
 
M.
Ebright
 
R.H.
Kalodimos
 
C.G.
 
Structural basis for cAMP-mediated allosteric control of the catabolite activator protein
Proc. Natl. Acad. Sci. U.S.A.
2009
, vol. 
106
 (pg. 
6927
-
6932
)
10
Galimand
 
M.
Gamper
 
M.
Zimmermann
 
A.
Haas
 
D.
 
Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa
J. Bacteriol.
1991
, vol. 
173
 (pg. 
1598
-
1606
)
11
Arai
 
H.
Igarashi
 
Y.
Kodama
 
T.
 
Expression of the nir and nor genes for denitrification of Pseudomonas aeruginosa requires a novel CRP/FNR-related transcriptional regulator, DNR, in addition to ANR
FEBS Lett.
1995
, vol. 
371
 (pg. 
73
-
76
)
12
Arai
 
H.
Kodama
 
T.
Igarashi
 
Y.
 
Cascade regulation of the two CRP/FNR-related transcriptional regulators (ANR and DNR) and the denitrification enzymes in Pseudomonas aeruginosa
Mol. Microbiol.
1997
, vol. 
25
 (pg. 
1141
-
1148
)
13
Arai
 
H.
Mizutani
 
M.
Igarashi
 
Y.
 
Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa
Microbiology
2003
, vol. 
149
 (pg. 
29
-
36
)
14
Castiglione
 
N.
Rinaldo
 
S.
Giardina
 
G.
Cutruzzolà
 
F.
 
The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli
Microbiology
2009
, vol. 
155
 (pg. 
2838
-
2844
)
15
Giardina
 
G.
Rinaldo
 
S.
Johnson
 
K.A.
Di Matteo
 
A.
Brunori
 
M.
Cutruzzolà
 
F.
 
NO sensing in Pseudomonas aeruginosa: structure of the transcriptional regulator DNR
J. Mol. Biol.
2008
, vol. 
378
 (pg. 
1002
-
1015
)
16
Smagghe
 
B.J.
Sarath
 
G.
Ross
 
E.
Hilbert
 
J.L.
Hargrove
 
M.S.
 
Slow ligand binding kinetics dominate ferrous hexacoordinate hemoglobin reactivities and reveal differences between plants and other species
Biochemistry
2006
, vol. 
45
 (pg. 
561
-
570
)
17
Giardina
 
G.
Rinaldo
 
S.
Castiglione
 
N.
Caruso
 
M.
Cutruzzolà
 
F.
 
A dramatic conformational rearrangement is necessary for the activation of DNR from Pseudomonas aeruginosa: crystal structure of wild-type DNR
Proteins
2009
, vol. 
77
 (pg. 
174
-
180
)
18
Parkinson
 
G.
Gunasekera
 
A.
Vojtechovsky
 
J.
Zhang
 
X.
Kunkel
 
T.A.
Berman
 
H.
Ebright
 
R.H.
 
Aromatic hydrogen bond in sequence-specific protein DNA recognition
Nat. Struct. Biol.
1996
, vol. 
3
 (pg. 
837
-
841
)
19
Hernandez
 
J.A.
Peleato
 
M.L.
Fillat
 
M.F.
Bes
 
M.T.
 
Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120
FEBS Lett.
2004
, vol. 
577
 (pg. 
35
-
41
)
20
Hickman
 
M.J.
Winston
 
F.
 
Heme levels switch the function of Hap1 of Saccharomyces cerevisiae between transcriptional activator and transcriptional repressor
Mol. Cell. Biol.
2007
, vol. 
27
 (pg. 
7414
-
7424
)
21
Hou
 
S.
Reynolds
 
M.F.
Horrigan
 
F.T.
Heinemann
 
S.H.
Hoshi
 
T.
 
Reversible binding of heme to proteins in cellular signal transduction
Acc. Chem. Res.
2006
, vol. 
39
 (pg. 
918
-
924
)
22
Passner
 
J.M.
Schultz
 
S.C.
Steitz
 
T.A.
 
Modeling the cAMP-induced allosteric transition using the crystal structure of CAP-cAMP at 2.1 Å resolution
J. Mol. Biol.
2000
, vol. 
304
 (pg. 
847
-
859
)
23
Lanzilotta
 
W.N.
Schuller
 
D.J.
Thorsteinsson
 
M.V.
Kerby
 
R.L.
Roberts
 
G.P.
Poulos
 
T.L.
 
Structure of the CO sensing transcription activator CooA
Nat. Struct. Biol.
2000
, vol. 
7
 (pg. 
876
-
880
)
24
Borjigin
 
M.
Li
 
H.
Lanz
 
N.D.
Kerby
 
R.L.
Roberts
 
G.P.
Poulos
 
T.L.
 
Structure-based hypothesis on the activation of the CO-sensing transcription factor CooA
Acta Crystallogr. Sect. D Biol. Crystallogr.
2007
, vol. 
63
 (pg. 
282
-
287
)
25
Eiting
 
M.
Hageluken
 
G.
Schubert
 
W.D.
Heinz
 
D.W.
 
The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA-binding affinity by stabilizing the HTH motif
Mol. Microbiol.
2005
, vol. 
56
 (pg. 
433
-
446
)
26
Joyce
 
M.G.
Levy
 
C.
Gabor
 
K.
Pop
 
S.M.
Biehl
 
B.D.
Doukov
 
T.I.
Ryter
 
J.M.
Mazon
 
H.
Smidt
 
H.
van den Heuvel
 
R.H.
, et al 
CprK crystal structures reveal mechanism for transcriptional control of halorespiration
J. Biol. Chem.
2006
, vol. 
281
 (pg. 
28318
-
28325
)