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

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. Neurological function(dopamine β hydroxylase) Copper in Human Health 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) 2 Waggoner, Neurobiol. of Disease, 1999, 6, 221

3. Neurological function(dopamine β hydroxylase) Copper in Human Disease Oxidative phosphorylation(cytochrome C-oxidase) • 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 Antioxidant activity (Cu/Zn superoxide dismutase) Iron metabolism(ceruloplasmin) Pigmentation(tyrosinase) Connective tissue formation(lysyl oxidase) 3 Waggoner, Neurobiol. of Disease, 1999, 6, 221.

4. 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

5. Loss of enzyme function Oxidative Stress Biological Control of Metal-Promoted Oxidative Stress

6. How do cells acquire copper?

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

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

9. 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

10. 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

11. N-Terminal hCtr1 Model Peptides By standard F-moc solid phase peptide synthesis

12. hCtr1-14

13. ESI-MS (+)hCtr1-14 P++/2 hCtr1-14 P+ H2N M D H S H HM 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 13

14. hCtr7-14K and 38-45K“Mets-only”

15. hCtr7-14K P+ ESI-MS (+)hCtr7-14K Ac M G M S Y M D S K Mets motif MXMXXM is capable of binding Cu and is selective for Cu(I) P+ + CuSO4 + CuSO4 + H2Asc PCu(I)+ [PCu(I)]++/2 P+

16. hCtr38-45K P+ ESI-MS (+)hCtr38-45K Ac S M M M M P M T K Mets motif MMMMXM is capable of binding Cu and is selective for Cu(I) P+ + CuSO4 P+ + CuSO4 +H2Asc [PCu(I)]++/2 16

17. Quantitative ESI-MS: Peptide-Copper Titration

18. Determination of KD by ESI-MS Peptide-Copper Titration

19. Cu chelation slows rate • Determination of KD by Peptide Inhibition of Copper-Catalyzed Ascorbate Oxidation 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

21. Pseudo 1st Order Kinetics • -d[HAsc-]/dt = k[HAsc-][Cu2+] Under excess HAsc- kobs = k[Cu2+] -d[HAsc-]/dt = kobs[HAsc-]

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. 7-14 1-14 38-45 MDHSHH MGMSYMDS NSTMQPSHHHPTTSASHSHGGGDS SMMMMPMT FYFGFKNVELLFSGLVINT N-Terminal hCtr1 • Current studies are limited because isolated sequences may not indicate binding of overall N-terminal hCtr1

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

25. Ampr Expression of 65aa N-Terminal in E.coli Competent E. coli Purified N-hCtr1 XarrestAffinity purification Expression of GST-N-hCtr1 Solution of GST+Factor Xa + N-hCtr1 Factor Xa GST Affinity purification IsolatedGST-N-hCtr1 GST Affinity purification

26. 1 2 3 4 5 6 7 8 9 10 GST-N-hCtr1 “N-hCtr1”7234Da 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

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