1. How metal ions control protein structure and dynamics (and biomolecular interactions) A: Calmodulin B: Zinc fingers

2. Calcium-binding proteins: Calcium regulates protein structure, dynamics and interactions

3. The EF hand motif Lewit-Bentley, A. & Réty, S. (2000) Curr. Op. Struct. Biol., 10, 637 - 643 Numerous Ca-binding proteins contain pairs of this motif Labels E and F stem from parvalbumin, where this motif was first discovered http://www.agr.nagoya- u.ac.jp/~mcr/Research/EF- hand.html

4. Calmodulin Monomeric, 148 aa, 17 kDa Binds up to 4 Ca 2+ 2 x 2 EF-hands Why specific for Ca 2+ ? log K ≈ 6-7 (Mg 2+ : 3-4) 7 oxygen ligands (hard !): –Not suitable for soft/borderline ions (Cu(I/II), Zn(II)) –Mg 2+ or Fe 3+ too small

5. Calmodulin 1 EF handA pair of EF hands Full-length Ca 4 -calmodulin X-ray: pdb 1cll; Chattopadhyaya, R., Meador, W.E., Means, A.R., Quiocho, F.A. J. Mol. Biol. 228, 1177-1192 (1992) Two domains connected by long  -helix

6. Apo CaM: Disruption of central helix NMR solution structure No fixed orientation of the two domains w.r. to each other Key point: Ca 2+ binding changes protein structure and dynamics NMR structure: pdb 1dmo; M Zhang, T Tanaka, M Ikura: Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nature structural biology. (1995) 2, pp. 758-67

7. Cooperativity Usually, simple binding curves are hyperbolical Cooperativity is frequent in biological systems Binding curves are sigmoidal Means: Binding of the first Ca enhances binding of the second and so on Will see again when discussing Hb http://ead.univ-angers.fr/~jaspard/Page2/ COURS/7RelStructFonction/2Biochimie/5Signalisation/2 Calmoduline/1Calmodulin.htm Bound Ca 2+ non- cooperative binding cooperative binding in CaM

8. Ca-loaded CaM interacts with a plethora of proteins Strong interaction (K d 10-100 nM) with many different proteins: Strange

9. How ? Ca 2+ binding induces the formation of hydrophobic patches on calmodulin surface CaM-binding proteins have a positively charged amphipathic helix (hydrophobic and hydrophilic sides) Interacts with both hydrophobic patches calmodulin “mitts”

10. WE Meador, AR Means, FA Quiocho: Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science. (1992) 257, 1251-5 Less schematic:

11. Summary Binding of Ca 2+ to calmodulin changes protein structure and dynamics This change in properties enables CaM to bind to a plethora of other proteins, but only in the presence of Ca 2+ (and only Ca 2+ ) This is how, by regulation of intracellular Ca 2+ concentrations, the activity of many other proteins can be regulated

12. Bio-Inorganic Chemistry Lecture 6b Zinc fingers: Control of protein structure and biomolecular interactions

13. Zinc proteins Zinc enzymes Zinc transporting proteins Enzymes in which zinc modulates activity Zinc fingers and other proteins with structural zinc Hydrolases others peptidases nucleases Metallo-  -lactamases others

14. Zinc fingers Small protein domains Classical: C 2 H 2 ligand set Tetrahedral coordination Unfolded without zinc DNA binding (only in presence of zinc) N C Transcription factors Often occur in multiples

15. Zif268 binding to DNA M Elrod-Erickson, TE Benson, CO Pabo 3 fingers in one protein Interaction with the major groove

16. DNA recognition: a code ? Corbi, Nicoletta; Libri, Valentina; Onori, Annalisa; Passananti, Claudio. Biochemistry and Cell Biology (2004), 82(4), 428-436.

17. Interactions are mediated by H- bonds in major groove His (+3) Arg (-1) Asp (+2) G G C

18. Artificial zinc fingers Based on unravelling the details of zinc finger DNA recognition Re-design zinc finger proteins for recognition of other DNA sequences Promising for medical applications: e.g. Gene regulation/targeting, antiviral therapy

19. Today: 116 different zinc finger families Zinc fingers are amongst the most populous domains in the human genome

20. The zinc fingers Can have His 2 Cys 2, Cys 3 His, or Cys 4 ligand set Can have 1 or 2 zinc sites Can be classified by the pattern of ligands in the sequence E.g. Pattern for GATA-type zinc fingers: C - x - [DN] - C - x(4,5) - [ST] - x(2) - W - [HR] - [RK] - x(3) - [GN] - x(3,4) - C - N - [AS] - C

21. What all these zinc fingers do Mediate interactions between proteins and other biomolecules (Not only DNA: RNA, lipids, other proteins...) Involved in processes such as –DNA recognition –RNA packaging –Transcriptional activation –Regulation of apoptosis (programmed cell death) –protein folding and assembly –lipid binding Examples 

22. Some principles in DNA-binding zinc fingers Isolated fingers, but more than one required (can be 2- 9, or even more) 2 zinc working together to structure protein Directly bridged by 2 Cys sulfurs Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains, BERT L. VALLEE, JOSEPH E. COLEMAN, AND DAVID S. AULD, PNAS 88, 999-1003 (1991).

23. HIV nucleocapsid zinc fingers Small, basic, 2 zinc fingers Nucleic acid chaperone: protects viral RNA and is important for reverse transcription (RNA as template for DNA synthesis) Drugs targeting the zinc fingers are in development: e.g. 2,2'- dithiobisbenzamides (DIBAs), azadicarbonamide (ADA)) http://jlevinlab.nichd.nih.gov/research.html TL South, MF Summers: Protein science (1993) 2, pp. 3-19 CCHC ligand set

24. Summary Zinc fingers are important for mediation of bio-molecular interactions Cys 2 His 2, Cys 3 His, Cys 4 coordination modes Unfolded without zinc