Homology Modeling of the human PAX8 Protein and mechanisms for sequence specific DNA recognition

1. Homology Modeling of the human PAX8 Protein and mechanisms for sequence specific DNA recognition Abhishek Dabral School of Biology, Georgia Institute of Technology

2. What is PAX? The PAX gene family encodes a group of transcription factors that have been conserved through millions of years of evolution and play roles in early development. Pax proteins are transcriptional regulators that have critical roles in mammalian development, the mutations of PAX genes cause profound developmental defects.

4. PAX Organization • All PAX proteins have a paired domain (PD), which spans 128 amino acids near the N-terminus and consists of two helix-turn-helix (HTH) motifs. • Sequence conservation among PAX proteins is highest in the paired domain but can also be extended to a paired-type homeodomain (HD) and to a stretch of residues between paired domain and homeodomain called octapeptide (OP).

5. PAX Structure • PD is composed of amino and carboxy terminal subdomains each of which are made up of 3 alpha helices resembling the HTH (helix-turn-helix) motif found in all HD. • Third helix of PD and HD proteins interacts with the major groove of the DNA. • PDs have the ability to not adopt a fixed structure unless it is bound to DNA, this lends it a great diversity as a protein.

6. PAX Subgroups In mammals, 9 PAX genes have been identified. PAX genes divided into 4 subgroups based on: • Genomic Structure • Sequence Similarity • Conserved Function

7. The PAX Families PAX 8 is the only member of the family expressed in the thyroid tissue. PAX 8 cooperates with TTF1 (Thyroid Transcription Factor 1) to influence thyroid specific gene regulation. Pax8 is extremely important for the correct development of the thyroid gland because inactivation of the Pax8 gene causes absence of follicular cells, and therefore absence of thyroid hormone . PAX 8 co-expresses with Wilms’ tumor gene (WT1) during kidney development suggesting a possible interaction.

8. Splice Variants in PAX 8 Alternative splicing in PAX gene by inclusion or exclusion of exons 7 and/or 8 has produced several known products but the biological significance of the variants is unknown. The human PAX8 gene generates at least five different alternatively spliced transcripts encoding different PAX8 isoforms.

9. Splice Variants in PAX 8 MPHNSIRSGHGGLNQLGGAFVNGRPLPE 1 5’ UTR ATG VVRQRIVDLAHQGVRPCDISRQLRVSHGCVSKILGRYYETGSIRPGVIGGSKPKVATPKVVEKIGDYKRQNPTMFAWEIRDRLL 2 3 LAEGVCDNDTVPSVSSINRIIRTKVQQPFNLPMDSCVATKSLSPGHTLIPSSAVTPPESPQSDSLGSTYSINGLLGIAQPGSDK 5 4 RKMDDSDQDSCRLSIDSQSSSSGPRKHLRTDAFSQHHLEPLECPFERQHYPEAYASPSHTKGEQG LYPLPLLNSTLDDGKATLT 6 PSNTPLGRNLSTHQTYPVVAD PHSPFAIKQETPEVSSSSSTPSSLSSSAFLDLQQVGSGVPPFNAFPHAASVYGQFTGQALLS siRNA 7 8 ? ? GREMVGPTLPGYPPHIPTSGQGSYASSAIAGMVAG SEYSGNAYGHTPYSSYSEAWRFPNSSLLSSPYYYSSTSRPSAPPTTAT ? 9 10 AFDHL 11 UAG = paired domain = intron with de novo CpG island = octapeptide = translocation breakpoints with PPAR-gamma = partial homeodomain = activation domain = intron/exon boundaries = repression domain

10. What questions could a PAX 8 model answer? Better understanding of : Paired Domain-DNA interaction– • Biological function of PD Function of N and C subdomains – • Specific DNA contacts made by them • Do they cooperate with each other, does one affect the function of the other and how? Effects of mutations • Relation to the abnormal phenotype

11. Why Homology modeling? • No solved X- Ray structure for our Target protein ie. PAX 8 Moreover: • X-Ray structure is both time consuming and expensive • Only a small number of proteins can be made to form crystals and crystal is not the protein’s native state.

12. Why Homology modeling? • No solved NMR structure for our Target protein ie. PAX 8 Moreover: • NMR does not work too well for protein complexes. • Very time consuming

13. Steps for Homology Modeling Sequence Database (Genbank) Initial Model Obtain Target Sequence Blastp, CDD Validate Model Get Information about Target Protein WHAT IF, PROCHECK, 3D JIGSAW, Esypred, SWISS Model, FUGUE RAMPAGE BLAST PDB database Template Selection (Crystal Structures) MODELER TOOLBOX Clean PDB files CLUSTALW Create alignment of target with template sequences (Convert aln to ali) MODELER

14. The Template Structure PAX6 5822580|pdb|6PAX|A Chain A, Crystal Structure Of The Human Pax-6 Paired Domain-Dna Complex Reveals A General Model For Pax Protein-Dna Interactions Length = 133 Score = 198 bits (503), Expect = 3e-52 Identities = 92/123 (74%), Positives = 107/123 (86%) Query: 10 HGGLNQLGGAFVNGRPLPEVVRQRIVDLAHQGVRPCDISRQLRVSHGCVSKILGRYYETG 69 H G+NQLGG FVNGRPLP+ RQRIV+LAH G RPCDISR L+VS+GCVSKILGRYY TG Sbjct: 2 HSGVNQLGGVFVNGRPLPDSTRQRIVELAHSGARPCDISRILQVSNGCVSKILGRYYATG 61 Query: 70 SIRPGVIGGSKPKVATPKVVEKIGDYKRQNPTMFAWEIRDRLLAEGVCDNDTVPSVSSIN 129 SIRP IGGSKP+VATP+VV KI YK++ P++FAWEIRDRLL+EGVC ND +PSVSSIN Sbjct: 62 SIRPRAIGGSKPRVATPEVVSKIAQYKQECPSIFAWEIRDRLLSEGVCTNDNIPSVSSIN 121 Query: 130 RII 132 R++ Sbjct: 122 RVL 124

15. Target-Template Alignment

16. DNA Contacts

17. MODEL

18. Hypothetical DNA fit of the model

19. Validation 1. Swiss Model(http:swissmodel.expasy.org) WhatCheck Report generated for your SWISS MODEL request : • No errors in amino acid nomenclature • Improper Dihedral angle distribution OK — The RMS Z-score for all improper dihedrals in the structure is within normal range. • Normal bond angle variability. • A few residues had abnormal backbone torsion angles. • A few pair of atoms had abnormally short interatomic distances. Overall the model conforms to the common refinement constraints

20. Ramachandran plot (http://raven.bioc.cam.ac.uk/rampage.php) Residue [ 43 :ARG] ( 68.15, 44.04) in Allowed region Residue [ 73 :LYS] (-118.37, -75.31) in Allowed region Number of residues in favoured region (~98.0% expected) : 119 ( 98.3%) Number of residues in allowed region ( ~2.0% expected) : 2 ( 1.7%) Number of residues in outlier region : 0 ( 0.0%)

21. Main Chain-Side Chain ContactsSource: MolProbity, an interactive macromolecular structure validation tool provided by the Richardson laboratory, Duke University.

22. Limitations • Could not model the entire protein due to lack of homologous structures and extensive loop region which is tough to model. • The paired box region may undergo some structural changes in the presence of the partial homeodomain (cooperativity in DNA binding). • The DNA contacts made by the model may differ from the template due to presence of other non-identical residues.

23. References 1.

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28. 6. Simon C. Lovell, Ian W. Davis, W. Bryan Arendall III, Paul I. W. de Bakker, J. Michael Word, Michael G. Prisant, Jane S. Richardson, David C. Richardson (2003) Structure validation by C-alpha geometry: phi, psi, and C-beta deviation. Proteins: Structure, Function, and Genetics. 50: 437-450. 7.

29. 8.