Spectroscopic Studies of Copper Binding to Methionine and Histidine-Rich hCtr1 Model

PeptidesKathryn L. Haas

Department of ChemistryDuke University

April,4 2006

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• Important for redox chemistry

Cu(II) + e‾ Cu(I)

• Unregulated redox is dangerous

Fenton Chemistry

Cu+ + H2O2 Cu2+ + HO‾ + HO•

Oxidative Stress!

Copper in Human Health

Neurological functionNeurological function(dopamine (dopamine ββ hydroxylase) hydroxylase)

Connective tissue formationConnective tissue formation(lysyl oxidase)(lysyl oxidase)

Iron metabolismIron metabolism(ceruloplasmin)(ceruloplasmin)

Oxidative phosphorylationOxidative phosphorylation(cytochrome C-oxidase)(cytochrome C-oxidase)

Antioxidant activityAntioxidant activity (Cu/Zn superoxide (Cu/Zn superoxide

dismutase)dismutase)

PigmentationPigmentation(tyrosinase)(tyrosinase)

Waggoner, Neurobiol. of Disease, 1999, 6, 221

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Copper in Human Disease

Neurological functionNeurological function(dopamine (dopamine ββ hydroxylase) hydroxylase)

Connective tissue formationConnective tissue formation(lysyl oxidase)(lysyl oxidase)

Iron metabolismIron metabolism(ceruloplasmin)(ceruloplasmin)

Oxidative phosphorylationOxidative phosphorylation(cytochrome C-oxidase)(cytochrome C-oxidase)

Antioxidant activityAntioxidant activity (Cu/Zn superoxide (Cu/Zn superoxide

dismutase)dismutase)

PigmentationPigmentation(tyrosinase)(tyrosinase)

• Amyotrophic Lateral Sclerosis (ALS)1

– SOD1 mutation enhances free radical generation by Cu

• Alzheimer’s Disease2

– Cu may promote Aβ aggregation

• Prion Disease3

– Cu-binding to prion protein enhances protease stability

1. Rasia, PNAS, 2005, 102(12), 4294.2. Bush, PNAS, 2003, 100(20), 11193.3. Sigurdsson, J. Biol. Chem., 2003,

278(47), 46199

Waggoner, Neurobiol. of Disease, 1999, 6, 221.

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4

Menke’s and Wilson’s Disease

MNKP and WNDP are P-type ATPase polytopic membrane proteins and have 55% amino acid identity

Lutsenko, S. et. al., J. Membrane Biol., 2002, 191, 1.

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Biological Control of Metal-Promoted Oxidative Stress

[Cu]

%S

urv

iva

l

Oxidative StressLoss of enzyme

function

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How do cells acquire copper?

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Ctr: Copper Transporter Required for Extracellular Copper Acquisition

O’Halloran, J. Bio. Chem., 2000, 275(33), 25057.

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Aller, PNAS, 2006, 103(10), 3627.

Architecture of the Ctr Copper Transporter

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• Copper transport is passive– ATP synthesis inhibitors have no effect on Cu uptake– Na+/K+-ATPase inhibitors have no effect on Cu uptake

• Copper must always be bound to proteins to prevent toxicity– Therefore transport must be governed by exchange of copper

ions with delivery proteins, chaperones, and small chelators

Binding site affinity and structure isimportant for control

How do cells regulate Cu-uptake?

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hCtr1: Human High Affinity Copper Transporter

Delivery of Cu(I) to appropriate cuproenzyme

Cu chaperone

Mets motif = MXnMXmM n,m=1 or 2

Glycosylation on N15

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N-Terminal hCtr1 Model Peptides

Short Model Peptides.

peptide sequence

hCtr1-14 H2N M D H S H H M G M S Y M D S

hCtr7-14K Ac M G M S Y M D S K

hCtr38-45K Ac S M M M M P M T K

By standard F-moc solid phase peptide synthesis

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hCtr1-14

13

200 400 600 800 1000 1200 1400 1600 1800 2000

200 400 600 800 1000 1200 1400 1600 1800 2000

200 700 1200 1700

P+

P+

P+

P++/2

P++/2

P++/2

[PCu(I)]++/2

[PCu(II)]++/2

PCu(II)+

PCu(I)+

hCtr1-14

+ CuSO4

+ CuSO4 + H2Asc

ESI-MS (+)hCtr1-14

band typical of His-Cu(II) bindingTitration of 400μM hCtr1-14 with 0-600 μM CuSO4

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Cu(II)-Dependant Spectrophotometric Titration ofhCtr1-14

0.00

0.01

0.02

0.03

0.04

0.05

0.06

400 450 500 550 600 650 700

Wavelength (nm)

Ab

s

[Cu]

H2N M D H S H H M G M S Y M D S

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hCtr7-14K and 38-45K“Mets-only”

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ESI-MS (+)hCtr7-14K

200 400 600 800 1000 1200 1400 1600 1800 2000

hCtr7-14K + CuSO4

200 700 1200 1700

hCtr7-14K + CuSO4 + H2Asc

200 400 600 800 1000 1200 1400 1600 1800 2000

hCtr7-14K

P+

P+

P+

[PCu(I)]++/2

PCu(I)+

hCtr7-14K

+ CuSO4

+ CuSO4 + H2Asc

Mets motif MXMXXM is capable of binding Cu and is selective for Cu(I)

Ac M G M S Y M D S K

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200 400 600 800 1000 1200 1400 1600 1800 2000

200 400 600 800 1000 1200 1400 1600 1800 2000

200 700 1200 1700

P+

P+

P+[PCu(I)]++/2

hCtr38-45K

+ CuSO4

+ CuSO4 + H2Asc

Mets motif MMMMXM is capable of binding Cu and is selective for Cu(I)

ESI-MS (+)hCtr38-45K

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Ac S M M M M P M T K

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Quantitative ESI-MS: Peptide-Copper Titration

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Determination of KD by ESI-MS Peptide-Copper Titration

ESI-MS Cu Titration. Peptide peak intensity as a function of [Cu]

0 2 4 6 8 10

Cu/P

%In

tens

ity

hCtr1-14 Cu(II)hCtr7-14K Cu(I)hCtr38-45K Cu(I)

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Determination of KD by Peptide Inhibition of Copper-Catalyzed Ascorbate Oxidation

Cu chelation slows rate

Rate limiting step

HAsc‾ HAsc• Asc

•

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Determination of KD by Peptide Inhibition of Copper-Catalyzed Ascorbate Oxidation

HAsc‾ HAsc• Asc λmax = 260nm no absorbance at 260nm

0.94

0.95

0.96

0.97

0.98

0.99

1

0 0.5 1 1.5 2

Time (S)

A2

60

H2Asc

Cu

hCtr1-14

hCtr7-14K

hCtr38-45K

•

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-d[HAsc-]/dt = k[HAsc-][Cu2+]

Under excess HAsc-

kobs = k[Cu2+]

-d[HAsc-]/dt = kobs[HAsc-]

y = 0.0033x + 0.0039R2 = 0.9817

0

0.01

0.02

0.03

0.04

0.05

0.06

0 2 4 6 8 10 12 14 16

[Cu] uMk

ob

s

Pseudo 1st Order Kinetics

•

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Current Understanding

• MXmMXnM motifs are sufficient for binding Cu(I) with a KD of ~3-6μM

• His cluster HHXH contributes to Cu(II) binding with a KD ~ 1μM

• Further effort needs to be taken to understand effect of His residues on Cu(I) and Cu(II) binding

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N-Terminal hCtr1

• Current studies are limited because isolated sequences may not indicate binding of overall N-terminal hCtr1

MDHSHH MGMSYMDS NSTMQPSHHHPTTSASHSHGGGDS SMMMMPMT FYFGFKNVELLFSGLVINT

1-147-14

38-45

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Expression of 65aa N-Terminal in E.coli

Obtained from Thiele Lab

Am

pr

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Am

pr

Expression of 65aa N-Terminal in E.coliCompetent

E. coli

Expression of GST-N-hCtr1

OOH S

OH

OH

HO

GST Affinity

purification

IsolatedGST-N-hCtr1

Factor Xa

Solution of GST

+Factor Xa

+ N-hCtr1

GST Affinity

purification

XarrestAffinity

purification

Purified N-hCtr1

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So Far…

“N-hCtr1”7234Da

GST-N-hCtr1

1 2 3 4 5 6 7 8 9 10

1 blank

2 Crude induced lysate

3 Buffer

4 Purified fusion protein

5 Factor Xa cleavage RXT

6 Factor Xa

7 GST affinity purification

8 Xarrest affinity purification

9 Both affinity purifications

10 SDS-PAGE broad range standard

7Kda

37Kda

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Future Studies on N-Terminal hCtr1

Observe overall structural changes upon Cu binding using Circular Dichroism (CD) and 15N NMR

Wawick Analytical Service. Available at http://www.warwickanalytical.co.uk/circular.htm