1. Spectroscopic Studies of Copper Binding to Methionine and Histidine-Rich hCtr1 Model Peptides Kathryn L. Haas Department of Chemistry Duke University April,4 2006

2. 2 Important for redox chemistry Cu(II) + e‾ Cu(I) Unregulated redox is dangerous Fenton Chemistry Cu + + H 2 O 2 Cu 2+ + HO‾ + HO Oxidative Stress! Copper in Human Health Neurological function (dopamine β hydroxylase) Connective tissue formation (lysyl oxidase) Iron metabolism (ceruloplasmin) Oxidative phosphorylation (cytochrome C-oxidase) Antioxidant activity (Cu/Zn superoxide dismutase) Pigmentation (tyrosinase) Waggoner, Neurobiol. of Disease, 1999, 6, 221 2

3. 3 Copper in Human Disease Neurological function (dopamine β hydroxylase) Connective tissue formation (lysyl oxidase) Iron metabolism (ceruloplasmin) Oxidative phosphorylation (cytochrome C-oxidase) Antioxidant activity (Cu/Zn superoxide dismutase) Pigmentation (tyrosinase) Amyotrophic Lateral Sclerosis (ALS) 1 –SOD1 mutation enhances free radical generation by Cu Alzheimer’s Disease 2 –Cu may promote Aβ aggregation Prion Disease 3 –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. 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. 5 Biological Control of Metal- Promoted Oxidative Stress Oxidative Stress Loss of enzyme function

6. 6 How do cells acquire copper?

7. 7 Ctr: Copper Transporter Required for Extracellular Copper Acquisition O’Halloran, J. Bio. Chem., 2000, 275(33), 25057.

8. 8 Aller, PNAS, 2006, 103(10), 3627. Architecture of the Ctr Copper Transporter

9. 9 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 How do cells regulate Cu-uptake?

10. 10 hCtr1: Human High Affinity Copper Transporter Delivery of Cu(I) to appropriate cuproenzyme Cu chaperone Mets motif = MX n MX m M n,m=1 or 2 Glycosylation on N 15

11. 11 N-Terminal hCtr1 Model Peptides Short Model Peptides. peptidesequence hCtr1-14H 2 N M D H S H H M G M S Y M D S hCtr7-14KAc M G M S Y M D S K hCtr38-45KAc S M M M M P M T K By standard F-moc solid phase peptide synthesis

12. 12 hCtr1-14

13. 13 P+P+ P+P+ P+P+ P ++ /2 [PCu(I)] ++ /2 [PCu(II)] ++ /2 PCu(II) + PCu(I) + hCtr1-14 + CuSO 4 + CuSO 4 + H 2 Asc ESI-MS (+) hCtr1-14 band typical of His-Cu(II) binding Titration of 400μM hCtr1-14 with 0-600 μM CuSO4 13 [Cu ] H 2 N M D H S H H M G M S Y M D S

14. 14 hCtr7-14K and 38-45K “Mets-only”

15. 15 ESI-MS (+) hCtr7-14K P+P+ P+P+ P+P+ [PCu(I)] ++ /2 PCu(I) + hCtr7-14K + CuSO 4 + CuSO 4 + H 2 Asc 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. 16 P+P+ P+P+ P+P+ [PCu(I)] ++ /2 hCtr38-45K + CuSO 4 + CuSO 4 + H 2 Asc Mets motif MMMMXM is capable of binding Cu and is selective for Cu(I) ESI-MS (+) hCtr38-45K 16 Ac S M M M M P M T K

17. 17 Quantitative ESI-MS: Peptide-Copper Titration

18. 18 Determination of K D by ESI-MS Peptide-Copper Titration

19. 19 Determination of K D by Peptide Inhibition of Copper-Catalyzed Ascorbate Oxidation Cu chelation slows rate Rate limiting step HAsc‾ HAsc Asc

20. 20 Determination of K D by Peptide Inhibition of Copper-Catalyzed Ascorbate Oxidation HAsc‾ HAsc Asc λ max = 260nm no absorbance at 260nm

21. 21 -d[HAsc - ]/dt = k[HAsc - ][Cu 2+ ] Under excess HAsc - k obs = k[Cu 2+ ] -d[HAsc-]/dt = k obs [HAsc-] Pseudo 1 st Order Kinetics

22. 22 Current Understanding MX m MX n M motifs are sufficient for binding Cu(I) with a K D of ~3-6μM His cluster HHXH contributes to Cu(II) binding with a K D ~ 1μM Further effort needs to be taken to understand effect of His residues on Cu(I) and Cu(II) binding

23. 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 1-14 7-14 38-45

24. 24 Expression of 65aa N-Terminal in E.coli Obtained from Thiele Lab Amp r

25. 25 Amp r Expression of 65aa N-Terminal in E.coli Competent E. coli Expression of GST-N-hCtr1 GST Affinity purification Isolated GST-N-hCtr1 Factor Xa Solution of GST + Factor Xa + N-hCtr1 GST Affinity purification Xarrest Affinity purification Purified N-hCtr1

26. 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. 27 Future Studies on N-Terminal hCtr1 Observe overall structural changes upon Cu binding using Circular Dichroism (CD) and 15 N NMR Wawick Analytical Service. Available at http://www.warwickanalytical.co.uk/circular.htm