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)