Zinc finger domains
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Zinc finger domains. Newcastle 10 th January 2008. 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

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Zinc finger domains

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Zinc finger domains

Zinc finger domains

Newcastle

10th January 2008


Zinc finger domains1

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


Role of zinc finger domains

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


Classes of zinc fingers

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


Sub classes of c2h2

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)


Zif268 dna binding

Zif268- DNA binding


Sub classes of c2h21

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


Ring domain

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


Krab zinc fingers

KRAB Zinc fingers

Approximately 1/3 zinc finger proteins also contain a Kruppel associated boxKRAB)

KRAB - potent repressor of transcription (binds to co repressors)


Conservation of zinc finger sequences

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)


Conservation of ring domain in brca1 across species

Conservation of RING domain in BRCA1 across species


Role of zinc fingers in disease pathology

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


Breast cancer

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


Hereditary neuropathies

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


Wilms tumour of kidney

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


Myotonic dystrophy type 2

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


Significance of variants within motifs

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)


Clustalw2 brca1 cys61

ClustalW2; BRCA1 Cys61

Cys61 (ring domain residue)

http://www.ebi.ac.uk/Tools/clustalw2/index.html


Align gv gd brca1 cys61gly

Align GV-GD; BRCA1 cys61gly

http://agvgd.iarc.fr/agvgd_input.php


Sift brca1 c61g

SIFT; BRCA1 C61G

http://blocks.fhcrc.org/sift/SIFT_aligned_seqs_submit.html


Polyphen brca1 c61g

Polyphen; BRCA1 C61G

http://genetics.bwh.harvard.edu/pph/


Summary zinc finger domains

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


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