Leucine Zippers

1. Leucine Zippers Louise Slater

2. DNA Binding Proteins • Two fundamental contrasting concepts of sequence-specific DNA binding protein specificity: • Binding specificity results from direct atomic interaction between amino acid side chains and base pairs in minimally distorted B DNA. • Limited number of structural motifs ‘steer’ the appropriate amino acid side chains of a protein in to the grooves of double helical DNA where they can interact directly with base pairs. These motifs are composed primarily of amino acid residues that do not make direct contact with the DNA. These scaffolds dictate appropriate positioning of the interacting protein surface, allowing atomic interaction between amino acid side chains and the specific binding site on the DNA.

3. DNA Binding Proteins • The amino acid sequences of these scaffolds, rather than the surface-contacting sequences that exhibit protein to protein similarity. • Primary sequences and biochemical properties of various DNA binding domains are suggestive of at least four basic structural motifs: • Helix-turn-helix • Common to DNA binding domains of many bacterial repressors and activators • Zinc finger • Common to eukaryotic gene regulatory proteins • RING finger • Leucine zipper

4. Leucine Zippers • First described in 1988 by Landschulz • A third previously undescribed DNA binding motif was found to be common to several DNA binding proteins, 3 nuclear transforming protein and 2 transcriptional regulatory proteins. • Used C/EBP protein to unravel the function of the leucine zipper motif • Protein capable of binding in a sequence specific manner to two cis-regulatory DNA sequences - CCAAT homology and enhancer core homology

5. C/EBP • C/EBP (CCAAT and enhancer binding protein) has a 14 KD DNA binding domain with an abundance of residues with charged side chains BUT no proline. • Proline residues rarely found in α-helices therefore Landschulz proposed a schematic α-helix. When arranged this way a 28 amino acid segment of DNA binding domain exhibited notable amphipathy. • One side predominantly composed of hydrophobic amino acids and the other with charged and uncharged polar side chains.

6. C/EBP Cont… • Periodic repetition of leucine residues at every seventh position over a region of 35 amino acids within the DNA binding domain. • Related protein searches – region of similarity coincides almost perfectly with the proposed α-helix between C/EBP and mouse c-myc. • Also similarity with human c-Myc, n-Myc, L-Myc and the nuclear transforming proteins, Fos and Jun exhibit at least 4 periodic leucine repeats. Jun related to yeast transcription regulatory protein GCN4.

7. α-Helix Stability • Predicted helices would be unusually long with 6-8 helical turns and unstable. • Is stability provided by the amphipathic arrangement of hydrophobic (leucines) and oppositely charged amino acids allowing salt bridge formation? • But why leucine residues exclusively for the hydrophobic region? • Leucine is long, symmetrical and has a bulky tip. This would be able to interdigitate with leucines from a second separate helix, acting as the dimerisation domain. Hydrophobic stability could then be provided by a complementary surface from a separate polypeptide.

8. Sequence Specific DNA Interaction • Leucine zipper of C/EBP not sufficient to confer sequence specific interaction with DNA. • GCN4 and Jun are structurally related and bind essentially identical DNA sequences but are unable to form heterodimers - residues involved in direct contacts to DNA are located elsewhere. • Sequence analysis of C/EBP demonstrated a high proportion of basic residues in a 30 amino acid region immediately adjacent to its leucine zipper. • They proposed that the leucine zipper juxtaposes the basic regions of two polypeptides in a manner suitable for sequence specific recognition of DNA. [They predicted antiparallel dimerisation].

9. Scissors-Grip Model • Model developed for a Y-shaped molecule (Vinson et al, 1989). Stem of the Y = coiled pair of α-helices and bifurcating arms of the Y = linked set of DNA contact surfaces • Conservation of a consensus 16 residue sequence between the basic region and the leucine zipper that starts exactly 7 residues NH2-terminal to the first leucine of the zipper. • Persistence of the coiled coil helical structure would not preserve the amphipathic phase of the zipper. Instead of juxtaposing attractive, hydrophobic residues suitable for a dimerisation interface, the side chains face their symmetrical counterpart and repel one another – disengaging the zipper and causing bifurcation. • DNA binding depends on a 3-D relation between the dimerisation interface and the DNA contact surface.

10. Leucine Zipper

11. Gene Regulation • Mammalian cells have a variety of proteins (Jun, JunB, JunD, fos, fra) that interact with a common sequence – AP-1 site. • Existence of multiple proteins that recognise related sequences increases the precision of flexibility for coordinately and independently regulate genes in a variety of cell types, developmental stages or in response to extra cellular signals • Dimeric nature of the leucine zipper proteins suggest an additional mode of felixibility involving heterodimers between different proteins. • Leucine zippers are usually incorporated in to a helix-loop-helix conformation called the basic helix-loop-helix-leucine zipper (bHLH-Zip)

12. Consequences of Disturbing the Motif • Site directed mutagenesis of Jun and Fos show that single mutations at L1 do not affect dimerisation but do affect DNA binding specificity. Mutations in L2-L5 also prevent dimerisation. • Most reported mutations preventing Fos and Jun binding also prevent DNA binding, indicating that formation of the Fos-Jun heterodimer is required for high affinity DNA binding. • Most consistent abolition of DNA binding occurs following substitution of the basic amino acid cluster with uncharged residues. • Mutations affecting the spacer region also completely abolished DNA binding. Cluster-spacer-cluster arrangement is conserved in many transcription factors.

13. Waardenburg Syndrome Type 2A • WS2 is dominantly inherited disorder characterised by a pigmentation anomaly and hearing impairment due to lack of melanocyte. • Linked to the MITF gene that encodes a transcription factor with a bHLH-Zip motif involved in melanocyte differentiation. • bHLH-Zip motif makes sequence specific DNA contacts with its basic region and the HLH-Zip domain mediates homo- and heterodimeric interactions necessary for DNA binding. • Patients with WS2 found to have mutations in their basic region or truncations leading to lack of HLH-Zip domain.

14. Retinopathies • Neural retina leucine zipper (NRL) is preferentially expressed in rod photoreceptors of the mammalian retina. • It interacts with cone-rod homeobox (CRX) and other transcriptional regulatory proteins to activate the expression of most, if not all, rod photoreceptor genes. • Mutations in NRL are associated with autosomal dominant retinitis pigmentosa and other retinopathies. • Many are suggested to change phosphorylation status and alter NRL-mediated transactivation of rhodopsin promoter.

15. Fanconi Anaemia Group G Gene • Autosomal recessive Fanconi anaemia is genetically highly heterogeneous. • FANCG was the third Fanconi anaemia gene identified (there are probably seven). • Several studies have shown that FANCG is found in cells as a nuclear complex with the FANCA protein. • Majority of Fanconi anaemia mutations are homozygous and result in protein truncation but one group reported a missense variant. • Comparison with the mouse cDNA sequence identified several totally conserved residues. The missense mutation is located directly in a leucine zipper at a conserved leucine residue.

16. Basic region/helix-loop-helix/leucine zipper domain of Myc proto-oncoproteins • Myc is sufficient to drive resting cells into the cell cycle and promote DNA synthesis. Constitutive expression in cells blocks their differentiation therefore initiating and promoting tumour formation - it is over expressed in the majority of human tumours. • Possess a DNA binding domain/dimerisation domain and a transactivation domain (TAD) • DNA binding domain is well understood - composed of three elements, the basic, the helix-loop-helix and the leucine zipper regions (bHLHZip). • Has heterodimerisation partner Max. Myc/Max/Mad network binds to subset of E box recognition sequences. Myc also an interaction partner of BRCA1- inhibits Myc-specific transactivation

17. References • Landschulz et al, 1988. Science. The leucine zipper: ahypothetical structure common to a new class of DNA binding proteins. Jun24;240:1759-64. • Struhl, K. 1989. Helix-turn-helix, zinc-finger, and leucine-zipper motifs for eukaryotic transcriptional regulatory proteins. Trends Bioche Sci. Apr;14(4):137-40. • Busch and Sassone-Corsi, 1990. Dimers, leucine zippers and DNA-binding domains. Trends Genet. Feb;6(2):36-40. • Vinson et al, 1989. Scissors-grip model for DNA recognition by a family of Leucine zipper proteins. Science. Nov 17;246:911-6. • Nobukuni et al, 1996. Analyses of loss-of-function mutations in the MITF gene suggest that haploinsufficiency is a cause of Waardenburg syndrome type 2A. Am. J. Hum. Gene. 59:76-83. • Swain et al, 2007. Mutations associated with retinopathies alter mitogen-activated protein kinase-induced phosphorylation of neural retina leucine-zipper. Molecular vision. 13:1114-20. • Demuth et al, 2000. Spectrum of mutations in the Fanconi anaemia group G gene, FANCG/XRCC9. European journal of human genetics. 8, 861-868. • Luscher and Larsson, 1999. The basic region/helix-loop-helix/leucine zipper domain of Myc proto-oncoproteins: Function and regulation. Oncogene. 18, 2955-2966.