E. coli FNR
Transcriptional Regulatory Protein
Rachel Wolters 23' and Colton Morris 23'
Contents:
I. Introduction
The fumarate and nitrate reduction regulatory
protein (FNR) is found in the source organism Aliivibrio fischeri
[4], and is a global transcription factor in Escherichia
coli (E. coli). The protein was named FNR after it was
observed that FNR mutants could not reduce fumarate nor nitrate. This
also provided initial evidence that FNR plays a role in anaerobic
respiration [3]. FNR is related to C-Reactive Protein (CRP),
and many structural features are conserved across the two proteins. This
causes FNR to interact with CRP family promoters, as the two are
structurally similar and have somewhat similar functions. FNR
controls the expression of various target genes in response to oxygen
starvation. FNR directly regulates roughly 100 genes, and indirectly
regulates up to 1000 genes [2]. Because FNR facilitates the
expression of proteins necessary for aerobic
and anaerobic respiration processes
in E. coli, it is essential for E. coli cell’s ability
to withstand anoxia (oxygen starvation).
When under anaerobic conditions, FNR
up-regulates proteins involved in anaerobic
respiration while down-regulating proteins involved in aerobic respiration [1].
FNR utilizes N-terminal iron–sulfur clusters to recognize the
presence of oxygen in the system [2]. FNR-DNA binding is mediated
by the disassembly and reassembly of FNR’s iron-sulfur clusters. When
oxidized, the iron-sulfur clusters
degrade from [4Fe-4S] clusters to [2Fe-2S] clusters. Under aerobic
conditions where iron-sulfur clusters are oxidized, FNR converts from
a dimer into a monomeric form that cannot bind to DNA. This
monomerization also inhibits FNR from interacting with RNA polymerase
(RNAP), rendering it transcriptionally inactive (unable to regulate
expression). Exposure to oxygen completely degrades the iron-sulfate
clusters, forming apo-FNR [4]. Under anaerobic conditions,
monomeric FNR converts to dimeric FNR with two iron-sulfur [4Fe-4S]
clusters. In this form, FNR has an increased affinity for FNR binding
sites on DNA (on FNR regulons) [1]. This makes the binding of
FNR to FNR binding sites the driving force behind FNR’s ability to
regulate proteins necessary for aerobic and anaerobic respiration. Due
to the oxygen-driven conformational change in FNR, there are two FNR
structures of interest:
and
.
*The preloaded interactive 3D visualization of FNR, as well
the visualization under 'General Structure' depict one subunit of FNR
under anaerobic conditions
II. General Structure
The protein FNR is 206 amino acid residues in
length, and has two symmetric subunits. Each subunit contains seven
, two
, an N-terminal Fe-S binding domain, and a C-terminal DNA binding
domain. The largest of the alpha-helices
in each subunit is
dimerization helix. Each subunit also contains a
for DNA binding in the C-terminus. Within the N-terminus there is a
characteristic
[3]. FNR in both its monomeric
and dimeric form is bound to two
types of ligands. The monomeric FNR and dimeric FNR ligands vary:
and
[4]. The more functional of the ligands present is the
iron-sulfur cluster present in the Fe-S binding domain. The other type
of ligand present, (4S)-2-Methyl-2,4-Pentanediol (MPD), has no
observed function in FNR. There are
on each subunit that create intersubunit salt bridges which play a
role in dimerization. Volbeda et al. suggests that these salt bridges
mediate very specific reactions that contribute to the balance between
monomeric and dimeric FNR [3].
III. DNA and RNAP Binding
FNR can only bind to DNA in its dimeric form, with
. As stated in General Structure, FNR only acquires a dimeric form in
anaerobic conditions. Thus, FNR only binds to DNA during anoxia. Amino
acid residues
, which comprise the
, along with the H-T-H
motif are two components of FNR responsible for DNA binding [1].
A H-T-H
motif consists of three components:
,
, and
- hence the name 'helix-turn-helix' motif. The molecule relies on the
oxygen-sensitive [4Fe-4S] cluster, a H-T-H
motif, and the dimerization
alpha-helixC to dimerize and bind to DNA.
Specific contact between FNR and DNA is still hypothesized, as a
cocrystal structure of the protein-DNA complex has not been published
to date. However, a cocrystal structure of FixK2, a homologue of FNR,
has been published. While the position of the H-T-H motif may be
different, their DNA binding sequences are identical and thus provide
strong evidence of where and how FNR and DNA contact. According to
investigation of FixK2, FNR is very likely to make hydrogen bonds with
DNA in the major groove
residues glutamic
acid 218 and arginine
222. It is also suggested that
makes water mediated contact with the DNA phosphate backbone. These
suggestions are made about FNR, as the amino acid residue equivalents
in FixK2 are observed to undergo these interactions [3][4].
The FixK2:DNA cocrystal structure also suggests other side chain
interactions between FNR and DNA may occur. However when FNR
, which via the FixK2 model is suggested to undergo hydrophobic
interaction with the phosphate backbone, is substituted with an
agrinine, FNR can interact with both FNR and CRP promoters, suggesting
valine 217
could be essential to FNR promoter recognition [3].
Unoxidized [4Fe-4S] clusters are also required for the binding of RNA
polymerase. Transcriptional activation is dependent on contact being
made between the CRP and FNR family of transcription factors and RNA
polymerase (RNAP). There are three individual activation regions that
contact the RNAP:
,
, and
. FNR contacts the RNAP through AR1
in the upstream subunit and AR2
and AR3
in the downstream subunit. AR1
plays a minor role in RNAP binding. AR1-AR3 play
a part in transcriptional initiation. The iron sulfate cluster is
present in the AR1
binding site, which suggests it likely promotes AR1
and RNAP interactions [1]. In the absence of oxygen FNR exists
in its monomeric form and because it is a monomer it is unable to
interact with RNAP and regulate gene expression [4].
IV. FNR in Anaerobic Conditions
Under anaerobic growth conditions, iron-sulfur
cluster ligands take the [4Fe-4S] structure, which conforms FNR into
dimeric shape. These clusters
with cysteine
residues 29, 32, 38, and 131, as well as proline
residue 40. FNR in the absence of oxygen has two MPD ligands
per subunit present: MPD 302 and 303. MPD 302
with amino acid residues glutamic
acid 165, asparagine
185, and tyrosine
212. MPD 303
with amino acid residues serine
187 and histidine
245.
V. FNR in Aerobic Conditions
By observing the
of monomeric FNR, you see FNR contains a third MPD ligand (MPD 304)
under aerobic conditions. The iron-sulfur cluster degrades
into [2Fe-2S]. Further, the iron cluster ligand in dimeric FNR (SF4)
is different from monomeric FNR (FeS), and only
with amino acid residues glutamic
acid 56, arginine
81, and cysteine
131. This conformational change prevents dimerization, DNA
and RNA binding and transcription. Monomerization of the FNR dimer
undergoes end-chain depolymerization, which is the breaking of bonds
in the polymer backbone. FNR is not able to bind DNA because this
occurs in the DNA binding domain [4].
Ligands MPD 302 and 303 also interact differently with monomeric FNR residues. The
difference in interacting for MPD 302, is instead of interacting with tyrosine
212, it interacts with
. In aerobic conditions, MPD 303
with glutamine
150 in two places, and tyrosine
135, as opposed to serine 187 and histidine 245 in anaerobic
conditions. For the third, and unique to aerobic FNR, ligand; MPD 304
with water
and rests within the molecule.
VI. References
[1] fnr - Fumarate and nitrate reduction regulation protein. (1986). UniProt. Retrieved December 2, 2020. DOI: P0A9E5.
[2] Grainger, D. C., Aiba, H., Hurd, D., Browning, D. F., & Busby, S. J. (2007). Transcription factor distribution in Escherichia coli: studies with FNR protein. Nucleic acids research, 35(1), 269–278. https://doi.org/10.1093/nar/gkl1023
[3] Mettert EL, Kiley PJ. Reassessing the Structure and Function Relationship of the O2 Sensing Transcription Factor FNR. Antioxidants & Redox Signaling. 2018 Dec;29(18):1830-1840. DOI: 10.1089/ars.2017.7365.
[4] Volbeda, A., Darnault, C., Renoux, O., Nicolet, Y., & Fontecilla-Camps, J. C. (2015). The crystal structure of the global anaerobic transcriptional regulator FNR explains its extremely fine-tuned monomer-dimer equilibrium. Science advances, 1(11), e1501086. https://doi.org/10.1126/sciadv.1501086
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