Transcript Spectroscopic Studies of Methionine and Histidine-Rich

Spectroscopic Studies of Copper
Binding to Methionine and
Histidine-Rich hCtr1 Model
Peptides
Kathryn L. Haas
Department of Chemistry
Duke University
April,4 2006
Copper in
Human Health
Neurological function
(dopamine β hydroxylase)
Oxidative phosphorylation
(cytochrome C-oxidase)
• Important for redox
chemistry
Cu(II) + e‾
Cu(I)
Iron metabolism
(ceruloplasmin)
Antioxidant activity
(Cu/Zn superoxide
dismutase)
• Unregulated redox is
dangerous
Fenton Chemistry
Cu+ + H2O2
Cu2+ + HO‾ + HO•
Oxidative Stress!
Pigmentation
(tyrosinase)
Connective tissue formation
(lysyl oxidase)
Waggoner, Neurobiol. of Disease, 1999, 6, 221
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Copper in
Human Disease
• Amyotrophic Lateral
Sclerosis (ALS)1
Neurological function
(dopamine β hydroxylase)
Oxidative phosphorylation
(cytochrome C-oxidase)
– SOD1 mutation enhances
free radical generation by Cu
• Alzheimer’s Disease2
– Cu may promote Aβ
aggregation
Iron metabolism
(ceruloplasmin)
Antioxidant activity
(Cu/Zn superoxide
dismutase)
• 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
Pigmentation
(tyrosinase)
Connective tissue formation
(lysyl oxidase)
Waggoner, Neurobiol. of Disease, 1999, 6, 221.
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Menke’s and Wilson’s 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
4
Biological Control of MetalPromoted Oxidative Stress
Loss of enzyme
function
%Survival
Oxidative Stress
[Cu]
5
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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Architecture of the Ctr Copper
Transporter
Aller, PNAS, 2006, 103(10), 3627.
8
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 is
important for control
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hCtr1: Human High Affinity Copper Transporter
Mets motif = MXnMXmM
n,m=1 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.
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
12
ESI-MS (+)
hCtr1-14
P++/2
hCtr1-14
P+
H2N M D H S H H M G M S Y M D S
200
400
600
800
P++/2
1000
1200
1400
[PCu(II)]++/2
+ CuSO4
[Cu
]
0.05
0.04
Abs
2000
P+
0.06
200
0.03
400
600
800
P++/2
0.02
1000
1200
1400
1600
1800
2000
+ CuSO4 + H2Asc
[PCu(I)]++/2
0.01
450
500
550
600
650
700
P+ PCu(I)+
Wavelength (nm)
band typical of His-Cu(II) binding
Titration of 400μM hCtr1-14 with 0-600 μM CuSO4
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1800
PCu(II)+
Cu(II)-Dependant Spectrophotometric Titration of
hCtr1-14
0.00
400
1600
13
200
700
1200
1700
hCtr7-14K and 38-45K
“Mets-only”
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hCtr7-14K
hCtr7-14K
P+
ESI-MS (+)
hCtr7-14K
Ac M G M S Y M D S K
200
400
hCtr7-14K + CuSO4
600
800
400
1200
1400
1600
1800
2000
P+
+ CuSO4
200
1000
600
800
1000
1200
1400
1600
1800
hCtr7-14K + CuSO4 + H2Asc
+ CuSO4 + H2Asc
PCu(I)+
2000
Mets motif MXMXXM is
capable of binding Cu
and is selective for Cu(I)
[PCu(I)]++/2
P+
15
200
700
1200
1700
hCtr38-45K
P+
ESI-MS (+)
hCtr38-45K
Ac S M M M M P M T K
200
400
600
800
1000
1200
1400
P+
Mets motif MMMMXM is
capable of binding Cu
and is selective for Cu(I)
200
400
600
800
[PCu(I)]++/2
1000
1800
1400
1600
1800
+ CuSO4 + H2Asc
16
16
200
700
2000
+ CuSO4
1200
P+
1600
1200
1700
2000
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]
%Intensity
hCtr1-14 Cu(II)
hCtr7-14K Cu(I)
hCtr38-45K Cu(I)
0
2
4
6
Cu/P
8
10
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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•
λmax = 260nm
Asc
no absorbance at 260nm
1
0.99
A260
0.98
0.97
H2Asc
Cu
0.96
hCtr1-14
hCtr7-14K
0.95
hCtr38-45K
0.94
0
0.5
1
Time (S)
1.5
2
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Pseudo 1st Order Kinetics
-d[HAsc-]/dt
=
0.06
k[HAsc-][Cu2+]
0.05
Under excess HAsc-
0.04
k obs
kobs = k[Cu2+]
y = 0.0033x + 0.0039
R2 = 0.9817
0.03
0.02
-d[HAsc-]/dt = kobs[HAsc-]
0.01
0
0
2
4
6
8
10
12
14
[Cu] uM
•
21
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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
7-14
1-14
38-45
MDHSHH MGMSYMDS NSTMQPSHHHPTTSASHSHGGGDS SMMMMPMT FYFGFKNVELLFSGLVINT
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Expression of 65aa N-Terminal in E.coli
Obtained from Thiele Lab
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Expression of 65aa N-Terminal in E.coli
Competent
E. coli
HO
O
OH
S
OH
OH
Xarrest
Affinity
purification
GST
Affinity
purification
Purified N-hCtr1
Solution of
GST
+
Factor Xa
+
N-hCtr1
Expression of
GST-N-hCtr1
Factor Xa
Isolated
GST-N-hCtr1
GST
Affinity
purification
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So Far…
1 blank
2 Crude induced lysate
1
3 Buffer
2 3 4 5 6 7 8 9 10
4 Purified fusion protein
5 Factor Xa cleavage RXT
37Kda
7Kda
GST-N-hCtr1
“N-hCtr1”
7234Da
6 Factor Xa
7 GST affinity purification
8 Xarrest affinity purification
9 Both affinity purifications
10 SDS-PAGE broad range
standard
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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
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