Resistenza al Cadmio

Sistemi per l’efflusso di metalli
transenvelope transport-exporters.
Resistenza al Cadmio
•
Si trova in molti tipi di batteri, sia ambientali sia patogeni
• Non ci sono ossidazioni/riduzioni o altri tipi di trasformazioni
come nei sistemi di detossificazione per Hg o As
• 3 diversi sistemi di pompe per l’efflusso: CzcD, CzcCBA e
CadA.
• CzcD fa parte della famiglia delle Cation Diffusion Facilitator
(CDF) presente in molti organismi; omodimero con 6 eliche TM
• CzcCBA sono presenti solo nei batteri
• CadA: P-type ATPase largamente diffuse
Resistenza al Cadmio
CusA efflux pump, transports Cu(I), Ag(I)
F. Long et al. Nature 2010
The changes
in
conformation
of the
horizontal
helix andTM8
are shown in a
superimpositio
n of the
structures of
apo (red) and
Cu(I)-bound
(green) CusA.
The bound
Cu(I) is shown
Channel in the CusA
pump. The channel
formed by the front
protomer of apo-CusA
(red) leading through
the transmembrane and
periplasmic domains is
in gray color.
The 11 methionines
forming the relay
network are in spheres
(green, carbon; red,
oxygen; blue, nitrogen;
orange, sulfur).
Two other CusA
protomers behind the
front protomer are
Structure of the CusBA
efflux complex.
C.-C. Su et al. Nature
2011
Proposed CDF-transporter mechanism. Dimeric CDF-proteins located at the cytoplasmic
membrane bind substrate metal cations (S) from the cytoplasm, probably via histidines in
a cytoplasm-open confirmation. Protons (H+) from the periplasm bind to negatively
charged residues in trans to the substrate and a conformational change towards periplasmopen state is induced. Substrate and proton are subsequently released and the
initial cytoplasm-open conformation is restored
Structure of lactose permease (LacY) a member of the major
facilitator superfamily
P-type ATPase
• dominio N-term per legare metalli
• dominio per legare ATP
• 8 eliche TM che formano il canale per il trasferimento
Evoluzione delle P1B-type ATPases
Meccanismo catalitico generale
Resistenza al Cadmio
Resistenza allo Zinco
• Zinco a basse concentrazioni è un nutriente. Importante regolare la
sua omeostasi. Ruoli catalitici e strutturali
• Enzimi di membrana differenti per uptake e efflusso
• Alcuni batteri codificano metallotioneine; proteine periplamsatiche
ricche di gruppi tiolici che sequestrano il metallo. Diverse da quelle
eucatiotiche.
Resistenza allo Zinco
• ZnuABC: uptake. Regolata da Zur. Specifica
per Zn(II), Pb(II) e Cd(II)
• ZntA: efflusso. Regolata da ZntR. La sua
produzione ha effetto sul gene per la
produzione di Cys.
Resistenza allo Zinco
Coordinazione tetragonale (come
CadA): Cys 62, Cys 59, Asp 58 e H2O
Accessibile al solvente, mentre di
solito sono strutturali o nascosti nel
core idrofobico.
Sequence analysis of the N-terminal domain
of cadmium and zinc ATPases
Conserved Glu
CadA-like
ZntA-like
b1
a1
b1
a1
Conserved Asp
b2
b2
b3
b3
a2
b4
a2
b4
Hypothetical model that illustrates plausible pathways for the transfer of (A) Zn or (B) Cd/Pb from the cytosolic MBD region
of AztA to the transmembrane site, and on to the other side of the membrane, consistent with the data presented here. At low
cytosolic Zn loads, the metal can interact with the a-MBD, b-MBD, or transmembrane site in a manner dictated by their
relative affinities. At high intracellular Zn loads, metal interacts with the low-affinity a-MBD to maximally stimulate the
transporter. For Cd/Pb, these ions form a trapped inter-MBD complex. Metal binding sites are indicated by the filled gray
circles, within the a-MBD, b-MBD, and the transmembrane sites indicated (49). The contribution of the His-rich domains
(yellow rectangles) to Zn(II) transport is not yet known; however, they likely bind Zn(II) (61) and may well influence rates of
transport of metal through the membrane.
Zn, Co Detoxification in Cyanobacteria
Cys 22
Plastocyanin
Asp 18
Cys 19
Resistenza al Rame
Trasporto di Rame nei cianobatteri
ATP ADP
ATP ADP
CtaA
Cu(I)
PacS
PC
thylakoid
ScAtx1
cytosol
periplasm
outer membrane
Copper import and distribution in humans
A partial view
as well
Copper distribution in the body
Copper is an essential
element
ATP7B is manly expressed in the
liver, whereas ATP7A is expressed in
all other tissues with the exception of
liver
The physiological importance of Cu-ATPases in humans can be illustrated by the
deleterious consequences of the Cu-ATPase inactivation on cell metabolism.
Indeed Copper is an essential element but its excess or deficiency can be a killer
Lutsenko, S. et al. Physiol. Rev. 87: 1011-1046 2007;
Pathway of copper in the body
Dietary copper
0.6-1.6 mg
Brain
Tissues/organs
Menkes
Disease
Blood
Portal
vein
Small
intestine
Bile
Feces
Caused by mutations in
the ATP7A gene.
Ceruloplasmin
[Cu]
Menkes syndrome
Cu patients fail to absorb
copper from the
gastrointestinal tract in
quantities adequate for
Liver
nutritional needs.
The clinical features of
Menkes can be attributed
to a deficiency of several
critical cuproenzymes.
Menkes disease (No cure, fatal by age of 10)
Pathway of copper in the body
Dietary copper
0.6-1.6 mg
Brain
Tissues/organs
Blood
Portal
vein
Small
intestine
[Cu]
Ceruloplasmin
Cu
Liver
Wilson
Disease
Caused by mutations in
the ATP7B gene.
Copper builds up in the
liver and injures liver
tissue.
Bile
Feces
Wilson disease (if untreated, fatal by age of 3
Model for the homeostatic
regulation of copper in mammals
ATP7A: Menkes disease; ATP7B: Wilson disease
Copper transporting ATPases
are essential for human growth and development
We have expressed each isolated domain of N-ter region of WLN and MNK ATPases
Copper transfer to a human ATPase
post-Golgi
MNK/WLN
Hah1
Nucleus
MNK/WLN
Mitochondria
Cu(I)
Human Copper(I)-transporting ATPases
Unstructure
loop
A-domain
N-domain
Pdomai
n
TGE
DOM6
CPC
DOM1
TGN / extracellular medium
Cytosol
Cell membran
Cu(I)-chaperone
+ ATPase
Superposition of two-dimensional 15N-1H HSQC spectra (700 MHz, 298 K) of uncomplexed Cu(I)-15NAtx1
(blue) and of the Cu(I)-15NAtx1·apo-Ccc2a complex (red) at a ratio of 1:1. The inset shows backbone amide
chemical shifts as a function of apo-Ccc2a concentration for some Atx1 residues exhibiting measurable
changes.
No copper, no interaction
+
Dd = 0
+ copper(I)
Dd  0
C15
C13
Ccc2
Atx1
C18
Atx1
C16
Ccc2
Atx1
Ccc2
C15
C13
Atx1
Ccc2
C18
C16
The interaction depends on copper ( metal-mediated )
The identity of Cysteines was defined by mutagenesis
The solution structure of the
Atx1-Cu(I)-Ccc2a complex
Cu(I)
Atx1
Ccc2a
Intermolecular NOEs
Site directed mutagenesis
Banci, Bertini, Cantini, Felli, Gonnelli, Hadjiliadis, Pierattelli, Rosato, Voulgaris, Nature Chemical Biology 2006
RESISTENZA BATTERICA AL PIOMBO
I geni per la resistenza al Piombo sono stati clonati e
sequenziati da un ceppo di R. metallidurans:
prbT permeasi che media l’uptake di Pb2+
pbrR induce la trascrizione di pbrABCD (in un singolo
mRNA) in presenza di Pb2+
pbrA P-type ATPase contenente il motivo CPC
pbrB lipoproteina della membrana esterna, coinvolta nella
rimozione di Pb2+, pompato da pbrA nel periplasma
prbC signal peptidase, probabilmente rimuove la sequenza di
localizzazione periplasmatica di prbB
pbrD proteina citoplasmatica legante Pb2+, ricca in cisteine
MECCANISMI DI RESISTENZA AD ALTRI
IONI METALLICI
• Sistemi di efflusso di Ni e Co comprendono ABC ATPasi e pompe
chemiosmotiche della famiglia CBA.
Esistono batteri, Archea e funghi che necessitano di alte concentrazioni di
Ni e Co per il loro metabolismo (NiCoT: sistema di uptake per Ni e Co)
• L’uptake del cromo sottoforma del suo ione cromato altamente tossico
[CrO42-, Cr(VI)] è mediato dai meccanismi di trasporto del solfato; nella
cellula il cromo è ridotto a Cr (III) e precipitato sotto forma di Cr(OH)3 .
Viene poi pompato fuori dalla ChrA
Copper transport in P. syringae (Gram-)
outer membrane
periplasm
cytosol
CopD
CopB
Toxic copper
Essential copper
Cu(II)
Cu(I) CopC Cu(I)/Cu(II)
Fet3-like
CopS
CopR