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Spectroscopic Studies of Copper Binding to Methionine and Histidine-Rich hCtr1 Model
PeptidesKathryn L. Haas
Department of ChemistryDuke University
April,4 2006
2
• 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
2
3
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.
3
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.
5
Biological Control of Metal-Promoted Oxidative Stress
[Cu]
%S
urv
iva
l
Oxidative StressLoss of enzyme
function
6
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?
10
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
12
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
14
hCtr7-14K and 38-45K“Mets-only”
15
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
16
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
17
Quantitative ESI-MS: Peptide-Copper Titration
18
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)
19
Determination of KD by Peptide Inhibition of Copper-Catalyzed Ascorbate Oxidation
Cu chelation slows rate
Rate limiting step
HAsc‾ HAsc• Asc
•
20
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
•
22
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
23
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
25
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
26
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
27
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