Zinc finger domains

1. Zinc finger domains Newcastle 10th January 2008

2. Zinc finger domains • Large superfamily of protein domains • Characterised by 2 anti parallel b sheets and 1 a helix • Structure stabilised by binding of Zinc ion • Zinc binding mediated by specific cysteine (b sheets) and histidine • (a helix) residues N terminal Zn C terminal

3. Role of zinc finger domains • Zinc finger domains make multiple contacts on target molecule • Can bind to DNA, RNA, protein and/or lipids • Binding properties depend on specific type of zinc finger, no of fingers and sequences therein • Versatility in binding results in specialised functions including gene transcription, translation, mRNA trafficking, cytoskeletal organisation and chromatin remodelling. • Over 70 different classes of zinc finger domains recognised

4. Classes of zinc fingers • Differential use of cysteine and histidine residues gives rise to numerous • types of zinc fingers including C2H2, C2HC, C2C2, C3HC4, C4 and C6 C2H2 represents the classic Zinc finger Has consensus sequence CX2-4CX12HX2-6H

5. Sub classes of C2H2 Triple fingered eg Sp1, Zif268, EGR2 • 3 zinc fingers bind in major groove of DNA • Specific bases within fingers and linker sequences interact with specific • bases in target DNA (-1, 2, 3 & 6 of alpha helix)

6. Zif268- DNA binding

7. Sub classes of C2H2 • Multiple adjacent fingers eg WT1, GLI • Separated paired fingers eg TTK, Shn • Each finger may have different binding specificities, thus building an increasing • complexity and specificity of target molecules and thus functional diversity

8. RING domain • Specialised type of Zinc finger • 40-60 residues and two Zinc atoms • Can be of two types; C3HC4 or C3H2C3 • Implicated in diverse biological processes • Usually involved in protein-protein interaction • eg E3 ubiquitin ligase activity

9. KRAB Zinc fingers Approximately 1/3 zinc finger proteins also contain a Kruppel associated boxKRAB) KRAB - potent repressor of transcription (binds to co repressors)

10. Conservation of zinc finger sequences • In typical C2H2 motif 20-44% residues are • structurally important and highly conserved • Evident in • cysteines • histidines • hydrophobic residues(pink) • linker sequences (green) and in • alpha helix (not shown)

11. Conservation of RING domain in BRCA1 across species

12. Role of zinc fingers in disease pathology • High degree of conservation indicates specificity of function • Mutation at specific residues>aberrant functioning>>> disease??? • Examples of disease resulting from mutations within Zinc finger proteins • Breast cancer, hereditary neuropathies, Wilms tumour and myotonic dystrophy type 2

13. Breast Cancer BRCA1 gene - chrom 17 Defects associated with hereditary form of Breast/ovarian cancer BRCA1 protein - contains RING domain (C3HC4 Zinc finger type) Helix 1 C24 RING Motif C64 Helix 3 C-X2-C C-X-H-X2-C-X2-C C-X2-C 39 41 44 47 cys39arg his41arg cys44phe cys47phe cys39ser cys44tyr cys47tyr cys39tyr 61 64 cys61gly cys64arg cys64gly cys64tyr 24 27 C39Y, C61Y and C61G all shown to abolish activity of E3 ubiquitin ligase activity

14. Hereditary neuropathies • EGR2 gene - chrom 10q21 • Defects associated with range of hereditary neuropathies including Dejerrine Sottas disease, congenital hypomyelination and Charcot Marie Tooth disease • EGR1 protein - contains 3 tandem zinc fingers of C2H2 class • DNA binding of mutant constructs to consensus sequence measured R1 Zinc1 Zinc2 Zinc3 R359W S282Y D383Y R409W CMT1 DSS CHN Unable to bind Unable to bind Decreased binding

15. Wilms tumour of kidney Tumour suppressor gene (WT1) - Chrom 11p13 Defects associated with abnormalities of genito-urinary tract Zinc finger domain protein (WT1) - multiple adjacent fingers (4) Mutations identified zinc finger domains> phenotype

16. Myotonic dystrophy type 2 • ZNF9 gene - chrom 3q21 • Defects associated with myotonia, cardiac defects, cataracts • ZNF9 protein – 7 tandem zinc finger domains C2HC type(RNA binding) Ex1 Ex 2-4 (CCTG)n Expansion of CCTG repeat (>100) in intron 1 result in DM2 phenotype Expanded alleles accumulate as RNA foci in nucleus Disease pathology mainly attributed RNA gain of function – binding to RNA binding proteins>>>subsequent dysregulation **Also some evidence for haploinsufficiency of ZFN9 as pathogenic

17. Significance of variants within motifs • eg BRCA1 RING domain • Well characterised sequence • Species alignment - T coffee, Clustal • Grantham scores - Align GV-GD • SIFT • Polyphen • Protein databases (Swiss Prot, Pfam, Prosite, SMART, etc)

18. ClustalW2; BRCA1 Cys61 Cys61 (ring domain residue) http://www.ebi.ac.uk/Tools/clustalw2/index.html

19. Align GV-GD; BRCA1 cys61gly http://agvgd.iarc.fr/agvgd_input.php

20. SIFT; BRCA1 C61G http://blocks.fhcrc.org/sift/SIFT_aligned_seqs_submit.html

21. Polyphen; BRCA1 C61G http://genetics.bwh.harvard.edu/pph/

22. Summary – zinc finger domains • Characterised by 2 anti parallel b sheets and 1 a helix • Structure stabilised by binding of Zinc ion • Mediated by specific cysteine and histidine residues • Differential use of cysteine and histidine residues gives rise to numerous types of zinc fingers including C2H2, C2HC, C2C2, C3HC4, C4 and C6 • Versatility in binding results in specialised functions including gene transcription, translation, mRNA trafficking, cytoskeletal organisation and chromatin remodelling • High degree of conservation – reflects importance of residues in function • Variation at conserved sites>>>> disease