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- 1. 1 © Ramaiah University of Applied Sciences 1 Lecture No. 12 Zinc protein motifs Course Leaders: Dr. Judy Jays
- 2. 2 © Ramaiah University of Applied Sciences 2 2 Faculty of Pharmacy Lecture No. 12 • At the end of this lecture, student will be able to • Describe the Zinc protein motifs
- 3. 3 © Ramaiah University of Applied Sciences 3 3 Faculty of Pharmacy Zinc finger • A Zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) in order to stabilize the fold • It was originally coined to describe the finger-like appearance of a hypothesized structure from the African clawed frog (Xenopus laevis) transcription factor IIIA. • However, it has been found to encompass a wide variety of differing protein structures in eukaryotic cells. • Xenopus laevis TFIIIA was originally demonstrated to contain zinc and require the metal for function in 1983, the first such reported zinc requirement for a gene regulatory protein followed soon thereafter by the Krüppel factor in Drosophila. It often appears as a metal-binding domain in multi-domain proteins.
- 4. 4 © Ramaiah University of Applied Sciences 4 4 Faculty of Pharmacy Zinc finger • Cartoon representation of the Cys2His2 zinc finger motif, consisting of an α helix and an antiparallel β sheet. The zinc ion (green) is coordinated by two histidine residues and two cysteine residues.
- 5. 5 © Ramaiah University of Applied Sciences 5 5 Faculty of Pharmacy Types • Proteins that contain zinc fingers (zinc finger proteins) are classified into several different structural families • There are a number of types of zinc fingers, each with a unique three-dimensional architecture • A particular zinc finger protein's class is determined by this three- dimensional structure, but it can also be recognized based on the primary structure of the protein or the identity of the ligands coordinating the zinc ion • In spite of the large variety of these proteins, however, the vast majority typically function as interaction modules that bind DNA, RNA, proteins, or other small, useful molecules, and variations in structure serve primarily to alter the binding specificity of a particular protein
- 6. 6 © Ramaiah University of Applied Sciences 6 6 Faculty of Pharmacy Classes • Initially, the term zinc finger was used solely to describe DNA-binding motif found in Xenopus laevis; however, it is now used to refer to any number of structures related by their coordination of a zinc ion. In general, zinc fingers coordinate zinc ions with a combination of cysteine and histidine residues Cys2His2 Two ligands from a knuckle and two more from the c terminus of a helix Gag knuckle Two ligands from a knuckle and two more from a short helix or loop Zinc ribbon Two ligands each from two knuckles Zn2/Cys6 Two ligands from the N terminus of a helix and two more from a loop
- 7. 7 © Ramaiah University of Applied Sciences 7 7 Faculty of Pharmacy Functions Diverse functions • DNA recognition • RNA packaging • Transcriptional activation • Regulation of apoptosis • Protein folding and assembly • Lipid binding
- 8. 8 © Ramaiah University of Applied Sciences 8 8 Faculty of Pharmacy Applications • Zinc-finger proteins that recognize novel DNA sequences are the basis of a powerful technology platform with many uses in drug discovery and therapeutics • Zep, a novel 49 kDa zinc finger protein, was found in the brain of day- 13 mouse embryos and cloned. Zep contains two C2H2-type zinc finger motifs close to the N-terminal region • ZEP-mediated regulation of endogenous genes could make it possible to use genes in a drug discovery application that would otherwise require securing IPR for a corresponding complementary DNA sequence • ZEP transcription factors are made by combining the ZEPs with domains that either activate or repress genes. ZPF TFs are used in drug discovery to regulate genes for target validation, HTS and therapeutics • The most common applications for engineered zinc finger arrays include zinc finger transcription factors and zinc finger nucleases
- 9. 9 © Ramaiah University of Applied Sciences 9 9 Faculty of Pharmacy Zinc-finger TFs • Zinc finger transcription factors are sequence- specific DNA binding proteins that regulate transcription. They possess DNA-binding domains that are formed from repeated Cys2His2 zinc finger motifs • The most common DNA-binding motif in human and multicellular animal TFs is the zinc finger • Two types of zinc finger TFs are discussed here-- C2H2 zinc finger TFs and C4 zinc finger TFs Most TFs that contain C2H2 zinc fingers are monomeric • Its 2 cysteine and 2 histidine residues bind to zinc ions (Zn2), and the a-helix containing the 2 histidines binds to bases in the major groove. • Much less common are TFs containing C4 zinc fingers. Most TFs containing this motif are dimeric. Nuclear receptors, which bind steroid hormones and other compounds, contain this motif. The glucocorticoid receptor is shown in Fig. b. Zinc ions are bound to the DNA recognition helix of this motif, which contacts bases in the major groove.
- 10. 10 © Ramaiah University of Applied Sciences 10 10 Faculty of Pharmacy DNA-Protein Interaction 1.Different protein motifs binding to DNA: Helix-turn-Helix motif; the homeodomain; leucine zipper; helix-loop-helix; zinc finger 2.Dimerization approach 3.Biotechnology to identify protein and DNA sequence interacting each other.
- 11. 11 © Ramaiah University of Applied Sciences 11 ©M. S. Ramaiah University of Applied Sciences 11 Helix-turn-Helix C-terminal binds to major groove, N-terminal helps to position the complex, discovered in Bacteria
- 12. 12 © Ramaiah University of Applied Sciences 12 ©M. S. Ramaiah University of Applied Sciences 12 Zinc Finger Motifs Utilizing a zinc in the center An alpha helix and two beta sheet
- 13. 13 © Ramaiah University of Applied Sciences 13 ©M. S. Ramaiah University of Applied Sciences 13 Three Zinc Finger Motifs forming the recognition site
- 14. 14 © Ramaiah University of Applied Sciences 14 ©M. S. Ramaiah University of Applied Sciences 14 A dimer of the zinc finger domain of the glucocorticoid receptor (belonging to intracellular receptor family) bound to its specific DNA sequence Zinc atoms stabilizing DNA-binding Helix and dimerization interface
- 15. 15 © Ramaiah University of Applied Sciences 15 ©M. S. Ramaiah University of Applied Sciences 15 Beta sheets can also recognize DNA sequence (bacterial met repressor binding to s-adenosyl methionine)
- 16. 16 © Ramaiah University of Applied Sciences 16 ©M. S. Ramaiah University of Applied Sciences 16 Leucine Zipper Dimer Same motif mediating both DNA binding and Protein dimerization (yeast Gcn4 protein)
- 17. 17 © Ramaiah University of Applied Sciences 17 ©M. S. Ramaiah University of Applied Sciences 17 Homodimers and heterodimers can recognize different patterns Helix-loop-Helix (HLH) Motif and its dimer
- 18. 18 © Ramaiah University of Applied Sciences 18 ©M. S. Ramaiah University of Applied Sciences 18 Truncation of HLH tail (DNA binding domain) inhibits binding
- 19. 19 © Ramaiah University of Applied Sciences 19 ©M. S. Ramaiah University of Applied Sciences 19 Six Zinc Finger motifs and their interaction with DNA
- 20. 20 © Ramaiah University of Applied Sciences 20 ©M. S. Ramaiah University of Applied Sciences 20
- 21. 21 © Ramaiah University of Applied Sciences 21 ©M. S. Ramaiah University of Applied Sciences 21
- 22. 22 © Ramaiah University of Applied Sciences 22 22 Faculty of Pharmacy Summary • Helix-turn-Helix, homeodomain, leucine zipper, helix-loop-helix, zinc- finger motif • Homodimer and heterodimer • Techniques to identify gene sequences bound to a known protein (DNA affinity chromatography) or proteins bound to known sequences (gel mobility shift)