Download B. Subtilis

O anche far sì che solo il metallo giusto induca una
risposta a livello trascrizionale
efflusso
uptake
Pennella & Giedroc, Biometals, 2005
a, BmrR monomer. The DNA-binding domain, -helical linker and drug-binding domain
are shown in yellow, red and green, respectively. b, BmrR dimer bound to DNA. One
monomer is coloured as in a, whereas the other monomer is shown in cyan. DNA and
TPP/TPSb are represented as balls and sticks (carbon, black; nitrogen, blue; oxygen,
red; and phosphorus/antimony, green). c, Dimerization interface between the drugbinding domain of a BmrR monomer (green) and the DNA-binding domain of its
dimeric mate (cyan). The TPP/TPSb molecule and selected drug-binding residues are
are represented as ball and sticks.
BmrR è un omologo di MerR che regola la farmacoresistanza in alcuni patogeni
Heldwein & Brennan, Nature, 2001
Modello per l’attivazione della trascrizione da parte di MerR
Copper trafficking in the periplasm. The
periplasm, a compartment of the cell
envelope of Gram-negative bacteria, is
proving to be an important site of Cu
trafficking and utilization. Cellular Cu
efflux is controlled in E. coli by the cue,
cus, and pco operons, each of which is
induced at different levels of Cu stress by
separate metalloregulatory proteins.
Recent structural insights for CueO and
PcoC are highlighted.
Overall structure of the Cu-CueR dimer
Changela et al. Science 2003
ZntR
E’ possibile che la specie
fisiologicamente rilevante
sia un sito mononucleare
CueR
Metal selectivity and sensitivity of CueR in
transcriptional regulation of copA in vitro. The
incubation buffer (pH 8.0) was treated with
Chelex to remove exogenous metals.
Concentrations used were as follows: CueR, 50
nM; RNAP, 5 nM; and DTT, 1 mM. The
percentage of induction relative to the maximal
+
induction achieved by Cu (% induction) is
plotted as a function of metal concentration
([added metal]). (A) The buffer was
–
supplemented with 1.0 mM CN to sequester
transcription induced by residual copper. The
+
+
+
solid lines for Cu , Ag , and Au represent fits to
a sigmoidal function, respectively. Half-maximal
transcription was observed at 0.7 ± 0.2 µM total
+
+
Cu , at 0.013 ± 0.009 µM total Ag and at 0.6 ±
+
+
0.3 µM total Au . (B) The free Cu concentration
+
–
([Cu ]free) was buffered by 20 mM CN (circles),
–
–
5.0 mM CN (squares), and 1.0 mM CN
(diamonds), respectively (16). The vertical line
2+
represents the free Zn concentration that
induces half-maximal transcription of the
ZntR/promoter system
La famiglia ArsR/SmtB
ArsR: regolazione operone Ars
SmtB: regolazione efflusso zinco
SmtB/ArsR family
Representation of the winged-helix
structure of SmtB dimers with helices
a3 and a4, forming the DNAbinding regions.The amino terminal,
ca. 20 amino acids of each monomer
were invisible in electron-density
maps. Four metal-binding sites are
located at dimer interfaces. The a5
allosteric sites (1 and 2) each include
Asp104, His106, plus His117 and Glu120.
Sites 3 and 4 involving Cys61 at helix
a3 plus ligands from the opposing
amino-terminal (N) region are not
obligatory for SmtB metal sensing.
Eicken et al. JMB 2003
An intersubunit hydrogen-bonding network in Zn2 α5-SmtB involved in allosteric coupling of Zn and DNA binding
sites. (a) Schematic of the intersubunit hydrogen-bonding network that links the allosteric α5 zinc-binding sites to
the DNA-binding αR helices (shaded yellow) in Zn2 α5-SmtB. Only one of the coordination chelates is shown
(the zinc ion is indicated as a black sphere). The critical intersubunit His117 Nε2–Hε2 O=C Arg87′ hydrogen
bond is encircled. (b) Another view of structural changes that occur upon Zn(II) binding to the α5 metal sites of
SmtB. Left panel, apo-SmtB; right panel, Zn2 α5-SmtB.
Eicken et al. JMB 2003
Solution structure of
cadmium-CmtR and
interaction with DNA.A,
ribbon representation of
dimeric cadmium-CmtR.
flexible
Rosso: senza DNA
Blu: + DNA
core
Banci L et al. J. Biol. Chem. 2007;282:30181-30188
Cadmium-binding dampens mobility.
A, ribbon representation of the H2O-D2O
exchange rates of amide protons in apoversus cadmium-CmtR. Colored residues
exchange faster in the apo-protein.
B, the structures and dynamic properties of
CmtR. Apo-CmtR is dynamic allowing
selection of a conformer with tight affinity
for DNA. Cadmium binds via Cys-102 plus
two Cys associated with helix αR of the
other subunit, introducing rigidity, locking
the protein into a conformer with weaker
affinity for DNA.
Representation of the alternative sensory sites, for oxyanions of
arsenite and antimonite at helix a3 in ArsR (orange), zinc at a5 in
dimeric SmtB and a5 plus a3 in ZiaR (red), cobalt and nickel
at a5C in NmtR (green), and cadmium and lead (blue) at a3N in
CadC and a4C in CmtR. b strands are indicated by arrows and
a helices are indicated by boxes. Hydrogen bonds (colored bars)
connect a zinc-SmtB a5 ligand to helix a4, thereby repositioning
the DNA-associating helices a3 and a4 (open boxes)
A phylogenetic tree of 25 sequences. The calculated distance between
each pair of sequences was used to construct the phylogenetic tree
which guides the final multiple sequence alignment. α3N and α5
sensors appear to cluster on separate nodes of the dendrogram and are
linked by a common evolutionary ancestor
Metal resistance operons regulated by SmtB/ArsR family transcriptional repressors.
General operon structures which confer metal resistance in bacteria that are regulated
in vivo by the indicated SmtB/ArsR repressors. Note that not all ars operons contain
the arsD and arsA genes.
Non solo efflusso
(A) ZiaR-represses ziaA, and this is
alleviated by zinc, while cobalt CoaR
activates coaT by a DNA-unwinding
mechanism first described for mercury
MerR. (B) Regulators were swapped. (C)
DNA encoding the amino-terminal region
of CoaT was also swapped with analogous
sequences from ZiaA. (D) Zinc and not
cobalt is transported by zinc-regulated
CoaT if its cytosolic domain is removed
and the equivalent one from ZiaA is added.
DtxR family – regolatori dell’uptake di ferro
The binding of
activated IdeR to the
mbtA-mbtB and
mbtI operators,
which occurs when
iron levels in the
cell are high,
represses the
transcription of
mbtA-J genes,
thereby limiting the
synthesis of
mycobactin and,
consequently, iron
uptake into the cell.
MntR: Mn(II)-dipendente
Il mutante è
attivato anche
da Fe(II)
Protein products of
metalloregulated genes involved in
metal homeostasis in S. cerevisiae.
Products of genes that are activated
under metal-limiting conditions (A)
and metal-replete conditions (B) by
Aft1 (green), Mac1 (blue), Zap1
(red), and Ace1 (purple) are shown.
Iron that is bound to siderophores
has been circled, and stars indicate
proteins that undergo irondependent cellular trafficking. The
metal ion specificities of proteins
required for metal uptake are
indicated. See the text for further
details of the functional roles of
each protein.
La famiglia Fur
(ferric uptake regulator)
Protein subfamily
Organism
Structural/regulatory metal ion
Function(s)
E. Coli
Zn2+/Fe2+ (Mn2+)
Fe uptake
B. Subtilis
Zn2+/Fe2+
Fe uptake
P. aeruginosa
Zn2+?/Fe2+
Fe uptake
N. meningitidis
?/Fe2+
Fe uptake
H. pylori
?/Fe2+
Fe uptake
B. japonicum
-/Fe2+
Irr protein
E. coli
Zn2+/Zn2+
Zn2+ uptake
B. subtilis
Zn2+/Zn2+
Mur
R. leguminosarum
-/Mn2+ (Fe2+)
Mn2+ uptake
Nur
S. coelicolor
?/Ni2+
Ni2+ uptake
B. subtilis
Zn2+/Fe2+ (Mn2+)
Oxidative stress
S. aureus
n.d./Mn2+ (Fe2+?)
Oxidative stress
Fur
Zur
PerRb
Zn2+ uptake
Zn2+ mobilization
DNA binding
Dimerization
Pseudomonas aeruginosa Fur
Pohl et al. Mol. Microbiol. 2003
Affinità per il Fe più bassa rispetto ai regolatori dell’efflusso – dipende da qual è la
concentrazione intracellulare tollerata
Modello per il DNA binding
Quale sito di legame per il ferro?
La proteina è tollerante a una varietà di mutazioni
Lee, Hellman, Biometals 2007
Sensing of peroxides by PerR
Mutational analyses indicate that the
Zn2+- and Fe2+-binding amino acids are
essential for repressor function.
Repression was measured using
an mrgA-cat-lacZ fusion reporter
construct and are expressed as Miller
units of b-galactosidase activity
Lee, Helmann, Nature, 2006
mM levels of H2O2
Mechanism of
sensing organic
hydroperoxides
by OhrR.
Zur and ZntR transcription assay results as a function of [Zn]free.
[RNAP] = [Zur] = [ZntR] = 50 nM, [DNA template] = 4 nM,
[TPEN] = 25 µM. The dotted line and the solid line represent the fit of the
Zur/PznuC (open triangles) and ZntR/PzntA (closed circles) data points,
respectively, to a sigmoidal function. The area highlighted in gray is the
range of [Zn]free between the half-maximal induction point on the two curves
Destinations for copper: (A) in the
cytoplasm of a typical eukaryote, with
metal transporters and chaperones
excluded for simplicity; (B) in the
periplasm (IM = inner membrane, and
OM = outer membrane) of E. coli,
plus copper sensors and transporters;
(C) Outside the cytoplasm of Gram
positive B. subtilis, plus copper
sensor, transporter, and chaperone;
(D) Copper transporters, chaperone,
and sensor of E. hirae; (E) Copperrequiring particulate methane
monoxygenase (pMMO) within
internalized membranes of
methanobacteria, plus its associated
copper siderophore, methanobactin;
(F) Cyanobacteria contain discrete
internal compartments, thylakoids,
where copper is required by
plastocyanin (PC) and also
cytochrome oxidase. PC donates
electrons to photosystem I (PSI) or
cytochrome oxidase. In the absence of
copper, electrons alternatively pass
through heme iron in cytochrome c6.
Copper must enter the cyanobacterial
cytoplasm.