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

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

• Kathryn L. Haas
• Department of Chemistry
• Duke University
• April,4 2006

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Copper in Human Health
Neurological function(dopamine ß hydroxylase)
Oxidative phosphorylation(cytochrome C-oxidase)

• Important for redox chemistry
• Cu(II) e? Cu(I)
• Unregulated redox is dangerous

Antioxidant activity (Cu/Zn superoxide dismutase)
Iron metabolism(ceruloplasmin)
Fenton Chemistry Cu H2O2 Cu2 HO?
HO Oxidative Stress!
Pigmentation(tyrosinase)
Connective tissue formation(lysyl oxidase)
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Waggoner, Neurobiol. of Disease, 1999, 6, 221
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Copper in Human Disease
Neurological function(dopamine ß hydroxylase)
Oxidative phosphorylation(cytochrome C-oxidase)

• Amyotrophic Lateral Sclerosis (ALS)1
• SOD1 mutation enhances free radical generation by
  Cu
• Alzheimers Disease2
• Cu may promote Aß 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

Antioxidant activity (Cu/Zn superoxide dismutase)
Iron metabolism(ceruloplasmin)
Pigmentation(tyrosinase)
Connective tissue formation(lysyl oxidase)
3
Waggoner, Neurobiol. of Disease, 1999, 6, 221.
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Menkes and Wilsons Disease
Lutsenko, S. et. al., J. Membrane Biol., 2002,
191, 1.
MNKP and WNDP are P-type ATPase polytopic
membrane proteins and have 55 amino acid identity
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Biological Control of Metal-Promoted Oxidative
Stress
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How do cells acquire copper?
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Ctr Copper Transporter Required for
Extracellular Copper Acquisition
OHalloran, J. Bio. Chem., 2000, 275(33), 25057.
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Architecture of the Ctr Copper Transporter
Aller, PNAS, 2006, 103(10), 3627.
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How do cells regulate Cu-uptake?

• 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

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hCtr1 Human High Affinity Copper Transporter
Mets motif MXnMXmM n,m1 or 2
Glycosylation on N15
Cu chaperone
Delivery of Cu(I) to appropriate cuproenzyme
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N-Terminal hCtr1 Model Peptides
Short 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
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ESI-MS ()hCtr1-14
P/2
hCtr1-14
P
H2N M D H S H H M G M S Y M D S
CuSO4
P/2
PCu(II)/2
PCu(II)
P
Cu
P/2
CuSO4 H2Asc
PCu(I)/2
P
PCu(I)
band typical of His-Cu(II) bindingTitration
of 400µM hCtr1-14 with 0-600 µM CuSO4
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14
hCtr7-14K and 38-45KMets-only
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ESI-MS ()hCtr7-14K
hCtr7-14K
P
Ac M G M S Y M D S K
P
CuSO4

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

CuSO4 H2Asc
PCu(I)
PCu(I)/2
P
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ESI-MS ()hCtr38-45K
hCtr38-45K
P
Ac S M M M M P M T K
P
CuSO4

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

P
CuSO4 H2Asc
PCu(I)/2
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Quantitative ESI-MS Peptide-Copper Titration
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Determination of KD by ESI-MS Peptide-Copper
Titration
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Determination of KD by Peptide Inhibition of
Copper-Catalyzed Ascorbate Oxidation
Rate limiting step
HAsc? HAsc
Asc
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Determination of KD by Peptide Inhibition of
Copper-Catalyzed Ascorbate Oxidation
HAsc? HAsc
Asc ?max 260nm
no absorbance at 260nm
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Pseudo 1st Order Kinetics
-dHAsc-/dt kHAsc-Cu2 Under excess
HAsc- kobs kCu2 -dHAsc-/dt kobsHAsc-
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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

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Expression of 65aa N-Terminal in E.coli
Ampr
Obtained from Thiele Lab
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Expression of 65aa N-Terminal in E.coli
Competent E. coli
Purified N-hCtr1
Expression of GST-N-hCtr1
XarrestAffinity purification
Solution of GSTFactor Xa N-hCtr1
Factor Xa
GST Affinity purification
IsolatedGST-N-hCtr1
GST Affinity purification
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So Far
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
37Kda
7Kda
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Future Studies on N-Terminal hCtr1
Wawick Analytical Service. Available at
http//www.warwickanalytical.co.uk/circular.htm

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