calcium and zinc in biology zinc 2.3 g / 70 kg - uni konstanz · calcium and zinc in biology zinc...
TRANSCRIPT
Calcium and Zinc in Biology
Calcium 980 g / 70 kgZinc 2.3 g / 70 kg
Eisen 4.2 g / 70 kg
Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn
Ca2+
4s2
Zn2+
4s2 3d10
Calcium and Zinc are no Redox Metals
Ca2+
Ca2+ ist eines der häufigsten Elemente der Erdkruste
( Vergleich: Zink nur 72 ppm)
Präferenz für quadratisch bipyramidale Koordination
Auschliesslich Sauerstoff als Ligand
Abstand Ca2+ - O ~ 2.4 Angstrom
Ca2+ als Strukturion eher selten im Vergleich zu Zn2+
Hauptsächlich im Signalgeschehen involviert
Ca2+ intrazellulär 0.2 µM; extrazellulär ~ 1-2 mM
Signal Transduction via Ca2+
[Ca[Ca2+2+]][Ca[Ca2+2+]]
=> gene expression; proliferation; cell differentiation....
stimulusstimulus
48 h old zebrafish heart
CaCa2+2+ signalsignal variationvariation and and modulationmodulation
CaCa2+2+
Signal diversity , intensity, duration
Ca2+ binding DNA binding Gene expression
Ca2+ sensors
Key Ca2+ signal translators and mediators
• Calmodulin
• S100 proteins (24 different in human)
The Ca2+ sensor module: EF-hand
Kretsinger 1976
E
F
S100BCalmodulin
EF-Hand Proteine
Conformational change upon Ca2+-Binding
Ca2+ free
Ca2+ bound
IIII II
IV
III
ExposureExposure of a of a HydrophobicHydrophobic SurfaceSurfaceuponupon CaCa2+2+--BindingBinding
hydrophilichydrophobic
+ Ca2+
S100B
TargetTarget bindingbinding to S100 to S100 proteinsproteins
Heizmann, Fritz, Schäfer (2002) Front. Biosci. 7:d 1356-68
Conformational change in EF-hands of S100 proteins
Wieso Zn2+ in der Biologie
Lewis Säure
Keine feste Koordinationszahl (kann leicht von 4 zu 5,6 Liganden wechseln)
Keine Redox Chemie
Zn2+ als Kofaktor in Enzymen - katalytisches Zinc, Zinkenzyme
Zn2+ als Strukturgebendes Element – strukturelles Zinc
Methoden um Zn2+ zu charakterisieren
Methods to study Zn2+-binding sites
Zn2+ -compared to other metal ions- has rather poorspectroscopic properties (no d-d transitions, no unpaired electrons, poor NMR properties)
Replacement by other metal ions:
Co2+, Cd2+, Cu2+, Mn2+, VO2+
UV-Vis, NMR, EPR
X-ray structure
Zn2+ Enzyme
Oxidoreduktasen
Hydrolasen
Lyasen
Liganden sind Histidine, Aspartate, Glutamate, Wasser,seltener Cys
Oxidoreduktasen:
Alkoholdehydrogenase (ADH), Redoxkofaktor ist NAD+.
Cu-Zn Superoxid Dismutase: 2 O2- O2 + H2O2
Zn2+ Enzyme
Alkoholdehydrogenase (ADH):
Alkohol + NAD+ Aldehyd + NADH (Hydridtransfer)
Zn2+
positioniert Alkohol
aktiviert OH Gruppe (Lewis Säure)
stabilisiert Übergangszustand
ADH katalytischer Zyklus
O HH
HH
OH
HH
OH
HH
H
OH
E-Zn2+
NAD+
E-Zn2+
H
E-Zn2+
H
-
+
NADH + H+
E-Zn2+
Zn2+ Hydrolasen
Alkalische Phosphatase (Hydrolyse Phosphatester)R-O-P-O3 + H2O R-O-H + HPO4
Proteasen (Hydrolyse Peptidbindung)
Carboxypeptidase
Aminopeptidase
Matrixmetalloprotease (Collagenase)
Beta-Lactamase (Hydrolyse von beta-Lactam Ring in
penicillinartigen Antibiotika)
Zn2+ - Lyasen
CarboanhydraseCO2 + H2O HCO3
–
E-Zn2+-OH- + CO2 E-Zn2+ + HCO3- ( kcat = 106 sec-1)
Strukturelles Zn2+
> 1000 Proteine mit sog. Zn2+ Finger, RING, LIM Domäne
kodiert in menschlichem Genom = > 3% aller Gene
DNA - Proteine Interaktionsdomänen
Protein - Protein Interaktionsdomänen
Protein – Andere (z.B.DAG) Interaktionsdomänen
Liganden sind Cysteine, Histidine
Zn2+ Finger Proteine
Cys
Cys
His
His
Zn2+XX44 XX33
Zinc finger 2Cys 2His
• Archetype for transcription factors (Sp1-Typ)~100 KDa
TFIIIARNA –Pol. III
Xfin (D.melanogaster)→37 Zinc fingers
Zn2+ binding site in p53
Zn2+ als Botenstoff
hohe Konzentrationen an Zn2+ im Gehirn in Vesikeln.
Sekretion in Synapse, Regulation postsynaptischer Rezeptoren
( z.B. NMDA Rezeptor, GABA Rezeptor) Proteine
Regulation der Zn2+ Konzentration im Cytoplasma (0.5 –4 nM)
über Zinktransporter in der Cytoplasmamebran und
Metalllothionein.
Zn2+ signals
J. Neurosci. 17:9554 Sensi et al.(1997)
Are S100 proteins Zn2+ sensors ?
• 24 different S100 proteins with distinct expression patterns, metal-binding properties and functions
• Many different S100 proteins bind Zn2+
• Connect the Zn2+ and the Ca2+ signaling network
ZnZn2+2+ binds to cysteines in many S100 proteinsbinds to cysteines in many S100 proteins
2 4 6 8 100
2
4
6
8
10
CYS
num
ber
-log Kd for Zn2+
A2 / A4
A3
BA6
340 360 380 400 420 440 460 480 500-20
0
20
40
60
80
100
120 f06 0 mM Ca f14 8.7 mM f17 20.2 mM f19 27.8 mM f24 31.6 mM f28 75.3 mM
Fluo
resc
ence
Trp
45
Wavelength / nm
0.00 0.05 0.10 0.15 0.20
0
20
40
60
80
100
Data: Data1_GModel: HyperblChi^2 = 15.88608Max 103.62664 ±4.04309Kd 0.02013 ±0.00231
Data: Data1_GModel: HillChi^2 = 5.05907max 85 ±0Kd 0.01573 ±0.00036n 2 ±0
% C
hang
eCa2+ / M
CaCa2+2+--binding to S100A3binding to S100A3
-> Binding of 2 Ca2+ fits better to data
0
200
400
600
x 1 0
a p o Z n
A bs .
-10,000
0
10,000
Θ [d
eg M
-1 c
m-1] x
10-3
Θ [d
eg c
m2 d
mol
-1] x
10-3
x 20C D
200 220 240 260 280 300-200
0
200
400
600
800
ε [M
-1 c
m-1] x
10-3
M C D
W avelength (nm )
Fritz et al., J. Biol. Chem. 277:33092-8 & unpublished data
ZnZn2+2+ --BindingBinding to S100A3to S100A3
Metal Metal ionion affinitiesaffinities of S100A3of S100A3
KKdd (Ca(Ca2+2+) ~ 15 mM) ~ 15 mM
101066
KKdd (Zn(Zn2+2+) ~ 2 nM ) ~ 2 nM
Co2+ in the Zn2+ -Binding Sites of S100A3
S Co2+ d-d
300 350 400 450 500 550 600 650 700 750 800 8500
1x103
2x103
3x103
4x103
5x103
6x103
Extin
ctio
n co
effic
ient
(M-1cm
-1)
Wavelength (nm)
500 550 600 650 700 750 800 850
0
2x102
4x102
6x102
8x102
1x103
+ Co2+
Wavelength (nm)
Fritz et al. (1998) BBA 1448:264-76.
TwoTwo ZnZn2+2+ Ions Bind in Ions Bind in TetrahedralTetrahedral GeometryGeometry and and in a in a StrictlyStrictly CooperativeCooperative MannerManner to S100A3.to S100A3.
7 Cys residues and 1 His residue are involved.
500 550 600 650 700 750 800 8500
1x102
2x102
3x102
4x102688 nm658 nm
Ext
inct
ion
coef
ficie
nt (
M-1cm
-1)
Wavelength (nm)
Probing the two different binding sites of S100A3
I II
I II
I II
Crystal Structure of MetalCrystal Structure of Metal--free S100A3 free S100A3 at 1.7 at 1.7 ÅÅ ResolutionResolution
EFS100EFcanonical
Fritz et al. (2002) J. Biol. Chem. 277:33092-8.
ZnZn2+2+ --Binding Site of S100A3Binding Site of S100A3
ZnZn2+2+ --Binding site of S100A3Binding site of S100A3
ZnZn2+2+
Model of S100A3 dimer Localization of Cys and His residues
Zn2+-binding to S100A3 induces a structural rearrangement
MetallothioneinMetallothionein Transfers ZnTransfers Zn2+2+ to to S100A3S100A3
• Metallothionein (MT) transfers Zn2+ to zinc finger proteinsand zinc dependent enzymes, e.g. superoxide dismutase.
• MT II transfers Zn2+ to S100A3
MT A3
Fritz et al., publication in preparation
metallothionein
~ 60 aa, 20 Cys -three types: MT-1, MT-2, MT-3
-MT-1 and MT-2 are expressed in almost all tissues except CNS
-MT-3 (= GIF) exclusively expressedin CNS
Binds Zn2+, Cd2+, Cu+
MT-1 and MT-1 are known to transferZn2+ to zincfinger proteins and zinc-dependent enzymes
β
α
How to follow Zinc transfer
200 220 240 260 280 300 320
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
060801S100A3 in 1 mm cuvettebuffer 20 mM Tris-Cl, 50 mM NaCl, pH 8.0conc. S100A3 20 μM
A3 Apo 20 μM A3 + Zn A3 + MT (diff:(A3-Zn)-A3
Abso
rptio
n
Wavelength /nm
Zn2+ transfer MT -> S100A3
200 210 220 230 240 250 260-40
-30
-20
-10
0
10
20
CD
Wavelength / nm200 210 220 230 240 250 260
-70
-60
-50
-40
-30
-20
-10
0
10
20
a08 a09 Da3.zn Da3.mt
CD
Wavelength / nm
Direct Zn2+ transfer ? MT -> S100A3
200 210 220 230 240 250 260-90
-60
-30
0
diff MT 0 h 12 h 16 h diff
CD
Wavelength /nm-20 0 20 40 60 80 100 120 140 160 180 200 220
-36
-34
0.2 M Tris-Cl, pH 7.4 0.02 M Tris-Cl, 0.05 M NaCl, pH 8.0
CD
time / min
No influence of Tris concentration -> direct transfer
300 400 500 6000.0
0.2
0.4
0.6
0.8
+ Co2+
Wavelength [nm]
Co2+ -binding to S100A6
0 2 4 6 8 10
20
40
60
80
100
(S100A6)2 - no metals Q
50 = 6.7 M
(S100A6)2 - 2 Zn
2+ Q
50 = 7.8 M
% fo
lded
Urea / M
Stabilization of S100A6 by Zn2+
300 400 500 600 700 800
Co2+
Abso
rptio
n
Wavelength / nm
Co2+ -binding to S100B
190 200 210 220 230 240 250
-10,000
0
10,000
20,000
30,000
apo Zn 1 equ Zn 2 equ Ca 1 mM Ca 1mM, Zn 1 mM
Θr /
deg
cm
2 dec
imol
-1
Wavelength / nm
Zn2+-binding to S100A6 and B
Zn2+-binding site in S100B
ZnZn2+2+
ZnZn2+2+
Wilder et al. Biochemistry 2005; Ostendorp, Fritz et al.
Zn2+ Stabilizes Ca2+ Binding Loop
ZnZn2+2+ binding binding increasesincreases CaCa2+2+ affinityaffinity
Zn2+ Site at pH 6.5 at 1.55 Å
Zn2+ Site at pH 9.0 at 1.5 Å
Zn2+ Site at pH 10.0 at 1.8 Å
Zn2+ in Zn2+ Binding Sites
Cd2+ in Zn2+ Binding Sites
Co2+ in Zn2+ Binding Sites
Mutational and spectroscopic analysis
Zn2+ induces teramerization S100A2
Zn2+ decreases Ca2+ affinity of S100A2
Koch, Fritz BBA 1773:457 (2007)
Ca2+ / µM
Two different Zn2+ sites in S100A2
Koch, Fritz BBA 1773:457 (2007)