Principles of Bioinorganic Chemistry - 2003

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 • Ca2+, 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. Zn2+ not removed by EDTA. 9 tandem repeats. 7-11 Zn/protein. Y or F – X – C – X2,4 – C – X3 – F – X5 – L – X2 – H – X3,4 – H – X2,6 C C C H H H H H 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. Td 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 Kd value: 2 pM 5nM 2mM 3mM Metal ion: Zn2+ Co2+ Ni2+ Fe3+

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: • Ca2+, 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 Ca2+ influx. Calcium also binds to calmodulin. As Ca2+ levels in the cell increase, calmodulin senses these changes and binds to its target proteins with Kd 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 Mg2+ C2A-C2B crystal structure Brunger and coworkers 1999

10. How does Ca2+ activate synaptotagmin? 10-100 M Ca2+ Phospholipid Binding Dimerization via C2 domains? mM Ca2+ Binding to protein partner (syntaxin) Change in Conformation? Electrostatic Switch?

11. FRET 340 280 470 280 Ca2+ C2A C2B C2B C2A

12. Addition of Calcium Results in an Increase in Energy Transfer apo mM Ca2+ Garcia, R. A.; Forde, C. A.; Godwin, H. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5883-5888.

13. Other metal ions that can promote fusion also confer this conformation change, but to a lesser extent than does Ca2+ 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 Ca2+ 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 Ca2+ over Mg2+. Ca2+ Kd,10-6 to < 10-7 M Mg2+ Kd,~ 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 Ca2+ levels

17. Summary - Points to Remember • 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 Fe4S4 cluster to alter protein structure. • A mercury resistance operon in bacteria detoxifies R2Hg 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.

18. Summary - Points to Remember, cont’d • 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.

19. Syphilis Bacterial infection

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 Understanding & Invention Idea Creativity Serendipity Discovery Intelligence

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