1. Principles of Bioinorganic Chemistry - 2003

2. Metallochaperones; Metal Folding PRINCIPLES: Metallochaperones guide and protect metals to natural sites Chaperone and target receptor protein structurally homologous Metal-mediated protein structure changes affect transcription Metal-mediated protein structure changes affect translation Metal-induced protein structure changes also activate enzymes Metal-induced bending of DNA affects function Metal ionic radii and M–L water bridging are used to advantage ILLUSTRATIONS: Copper insertion into metalloenzymes Zinc finger proteins control transcription Ca 2+, a second messenger and sentinel at the synapse Cisplatin, an anticancer drug

3. Zinc Fingers - Discovery, Structures A. Klug, sequence gazing, proposed zinc fingers for TFIIIA, which controls the transcription of 5S ribosomal RNA. Zn 2+ not removed by EDTA. 9 tandem repeats. 7-11 Zn/protein. Y or F – X – C – X 2,4 – C – X 3 – F – X 5 – L – X 2 – H – X 3,4 – H – X 2,6 CCCHHHHH The coordination of two S and 2 N atoms from Cys and His residues was supported by EXAFS; Zn–S, 2.3 Å; Zn–N, 2.0 Å. T d geometry. The protein folds only when zinc is bound; > 1% of all genes have zinc finger domains.

4. X-ray Structure of a Zinc Finger Domain

5. Structure of a Three Zinc-Finger Domain of Zif 268 Complexed to an Oligonucleotide Containing its Recognition Sequence

6. The Specificity of Zinc for Zinc-finger Domains K d value:2 pM5nM2mM3mM Metal ion:Zn 2+ Co 2+ Ni 2+ Fe 3+

7. Metal Folding of Biopolymers PRINCIPLES: Metal-mediated protein structure changes affect transcription Metal-mediated protein structure changes affect translation Metal-induced protein structure changes also activate enzymes Metal-induced DNA structure changes effect anticancer activity ILLUSTRATIONS: Ca 2+, a second messenger and sentinel at the synapse Cisplatin, an anticancer drug

8. Roles of Calcium in Protein Folding and Activation Calcium at the synapse: binds to the C2 domains of synaptotagmin and helps to control neurological signal transduction. Synaptotagmin is the sensor for Ca 2+ influx. Calcium also binds to calmodulin. As Ca 2+ levels in the cell increase, calmodulin senses these changes and binds to its target proteins with K d values of 10 - 100 nM or less. The following are amino acid sequences from activated proteins:

9. Synaptotagmin is a calcium sensor Synaptotagmin III with Mg 2+ C2A-C2B crystal structure Brunger and coworkers 1999

10. How does Ca 2+ activate synaptotagmin? Dimerization via C2 domains? 10-100  M Ca 2+ Phospholipid Binding mM Ca 2+ Binding to protein partner (syntaxin) Change in Conformation? Electrostatic Switch?

11. Ca 2+ C2A C2B 280 340 280 470 FRET C2AC2B

12. Garcia, R. A.; Forde, C. A.; Godwin, H. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5883-5888. Addition of Calcium Results in an Increase in Energy Transfer apo mM Ca 2+

13. Other metal ions that can promote fusion also confer this conformation change, but to a lesser extent than does Ca 2+ Garcia, R. A.; Forde, C. A.; Godwin, H. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5883-5888.

14. Calmodulin - Properties and Structure Properties: 148 amino acids; binds up to four Ca 2+ ions. Primary structure conserved over many species. Binds calcium in pairs of “EF hand” domains. Calcium coordinated by 3 monodentate Asp, 1 bidentate Glu, 1 peptide backbone carbonyl, and 1 water molecule. Seven-coordination favors Ca 2+ over Mg 2+. Ca 2+ K d, 10 -6 to < 10 -7 M Mg 2+ K d, ~ 10 -3 M

15. Structural Changes in Calmodulin upon Binding to a Target Peptide Domain Note how the target peptide is encapsulated following conformational changes in the long central helix

16. A Fluorescent Sensor for Calcium Binding in Cells Can be used to correlate with synaptotagmin as well as calmodulin response to Ca 2+ levels

17. In eukaryotes iron homeostasis is achieved by regulating the concentrations of the transferrin receptor and apoferritin at the translational level. IRP’s are the conformational switch and utilize an Fe 4 S 4 cluster to alter protein structure. A mercury resistance operon in bacteria detoxifies R 2 Hg by lyase and reduction to Hg(0) which volatilizes harmlessly out of the cell. MerR is the sensor which undergoes a conformational change upon Hg binding and activates the genes at the level of transcription. This topic was not covered in class, but please read Chapter 6, Section 6.3. Summary - Points to Remember

18. Zinc fingers form upon metal-binding to the apo peptide domains and activate transcription of thousands of genes. Evidence is accumulating that zinc-binding domains also act as metalloregulators in response to zinc levels in the cell. Synaptotagmin C2 domains are the calcium sensors at the synapse responsible for Ca-dependent signal transduction in helping to achieve synaptic plasticity. Calmodulin alters its structure upon calcium influx into cells and activates many genes at the transcriptional level. Calmodulin allows calcium to serve as a second messenger in response to signals at the cell surface, affording a signal transduction pathway. Summary - Points to Remember, cont’d

19. Bacterial infection Syphilis

23. Factors Associated with Antitumor Drug Development A. Serendipity: > 7000:1 in most screens B. Perseverance: ~ 13 years between laboratory and pharmacy C. Cost: > $750 M D. Moral questions: gene therapy E. The aversion to metal-based pharmaceuticals

24. Cisplatin: From DNA Damage to Cancer Therapy Idea Discovery Understanding & Invention Serendipity Creativity Intelligence

26. Cisplatin: An Extraordinary Anticancer Drug Years after Orchidectomy source: I. Damjanov, Scientific American, 1995 % Survival Cisplatin is highly effective against testicular cancer. It is also used to treat head, neck, ovarian, and several other types of human tumors. Cisplatin cis-diamminedichloroplatinum(II) w/ cisplatin w/o cisplatin