1. The FREAKS Session 3.1: Repeats Session 3.2: Biased regions Miguel Andrade Johannes-Gutenberg University of Mainz Andrade@uni-mainz.de of PROTEIN SEQUENCE

2. Definition 14% proteins contains repeats (Marcotte et al, 1999) 1: Single amino acid repeats. 2: Longer imperfect tandem repeats. Assemble in structure.

3. Definition CBRs Perfect repeat: QQQQQQQQQQQ Imperfect: QQQQPQQQQQQ Amino acid type: DDDDDEEEDEDEED Compositionally biased regions (CBRs) High frequency of one or two amino acids in a region. Particular case of low complexity region

4. Conservation => Function Length, amino acid type not necessarily conserved Frequency: 1 in 3 proteins contains a compositionally biased region (Wootton, 1994), ~11% conserved (Sim and Creamer, 2004) Function CBRs

5. Conservation => Function Length, amino acid type not necessarily conserved Functions: Passive: linkers Active: binding, mediate protein interaction, structural integrity (Sim and Creamer, 2004)

6. Structure of CBRs Often variable or flexible: do not easily crystalize

7. 1CJF: profilin bound to polyP

8. 2IF8: Inositol Phosphate Multikinase Ipk2

9. RVSETTTSGSL

10. 2CX5: mitochondrial cytochrome c B subunit N-terminal

11. 2CX5: mitochondrial cytochrome c B subunit N-terminal FFFFIFVFNF

12. Types of CBRs More than 6 aa in length, 1.4% of all, 87% of them in Euk (Faux et al 2005)

13. Types of CBRs (Faux et al 2005) Distribution is not random: Eukaryota: Most common: poly-Q, poly-N, poly-A, poly-S, poly-G Prokaryota: Most common: poly-S, poly-G, poly-A, poly-P Relatively rare: poly-Q, poly-N Very rare or absent in both eukaryota and prokaryota: Poly-I, Poly-M, Poly-W, Poly-C, Poly-Y Toxicity of long stretches of hydrophobic residues.

14. Filtering out CBRs Normally filtered out as low complexity region: they give spurious BLAST hits QQQQQQQQQQ |||||||||| QQQQQQQQQQ 10/10 id IDENTITIES |||||||||| IDENTITIES 10/10 id

15. Filtering out CBRs Normally filtered out as low complexity region: they give spurious BLAST hits QQQQQQQQQQ |||||||||| QQQQQQQQQQ Shuffle: 10/10 id IDENTITIES |||||||||| IDENTITIES 10/10 id

16. Filtering out CBRs Normally filtered out as low complexity region: they give spurious BLAST hits QQQQQQQQQQ |||||||||| QQQQQQQQQQ Shuffle: 10/10 id IDENTITIES | | SIINDIETTE Shuffle: 2/10 id

17. Filtering out CBRs Option for pre-BLAST treatment SEG algorithm: 1) Identify sequence regions with low information content over a sequence window 2) Merge neighbouring regions Eliminates hits against common acidic-, basic- or proline-rich regions (Wootton and Federhen, 1993)

18. Obtain this protein sequence from NCBI. This is a hypothetical protein from Nematocida sp., a microsporidia (spore-forming fungi) that infects the worm Caenorhabditis elegans.protein sequence from NCBI Can you see funny things in this sequence? Go to the NCBI's BLAST web page and go to the "protein blast" optionNCBI's BLAST Search for homologs of the protein Keep the output Do the same search in another NCBI's BLAST window selecting the filter low complexity regions using SEG optionNCBI's BLAST Compare the outputs: Can you identify different hits? Do matches to the same sequence have relevant differences in the E-value? Comment on the relevance of the differences. Exercise 1. Filtering CBRs for BLAST using SEG

19. A particular analysis… AIR9 (1708 aa) Ser rich + basic LRR A9 repeats conserved region Δ1Δ1 Δ15 Δ9Δ9 Δ12 Δ14 Δ10 Δ11 Δ16 Δ3Δ3 Δ2Δ2 Δ6Δ6 Buschmann, et al (2006). Current Biology. Buschmann, et al (2007). Plant Signaling & Behavior Microtubule localization of Δx-GFP

20. …triggers a tool

21. A particular analysis… http://sourceforge.net/projects/biasviz/ Huska, et al. (2007). Bioinformatics …triggers BiasViz

22. http://sourceforge.net/projects/biasviz/ Huska, et al. (2007). Bioinformatics A particular analysis… …triggers BiasViz

23. ADAM15

24. Binds SH3 of endophilin and SH3 PX1 PMID:10531379 Binds SH3 of endophilinI and SH3 PX1 PMID:10531379 Binds SH3 of Fish PMID:12615925 Binds SH3 of Grb2 PMID:11127814 Binds SH3 of Fish PMID:12615925 Binds SH3 of ArgBP1/ABI2 PMID:12463424

25. ADAM19 ADAM9 ADAM11 ADAM20 a b c 0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4

26. Go to the BiasViz2 web pageBiasViz2 Launch BiasViz2 Load this alignment on the step 1 sectionthis alignment Hit the "Go to graphical view" button Try to find combinations of parameters that reveal CBRs Try hydrophobic residues and window size 10. If I tell you this is a transmembrane protein, what is this result telling you? Can you see other biased regions? Exercise 2. Viewing CBRs in an alignment with BiasViz2

27. Function of polyQ Martin Schaefer Human Dog Mouse Opossum Chicken Frog Zebrafish Trout Fugu Stickleback Lancelet Capitella Limpet Nematostella Trichoplax Ciona intestinalis Ciona savignyi D. melanogaster D. mojavensis D. sechellia D. erecta D. yakuba D. grimshawi D. pseudoobscura D. persimilis D. ananassae D. willistoni D. virilis polyQ in Huntingtin Schaefer et al (2012) Nucleic Acids Res.

28. polyQTFslong human non polyQ 1 5 10 50 100 500 1000 partners

29. no polyQ polyQ >14 polyQ 4-14 no polyQ polyQ >14 polyQ 4-14 partners 1 5 10 50 100 500 1000 1 2 5 10 20 50 100 200 500 polyQTFslong human non polyQ 1 5 10 50 100 500 1000 partners polyQnon polyQ TFslong yeast 1 5 10 50 100 500 1000

30. polyQ protein N-terminal C-terminal unbound polyP polyQ disordered coiled coil

31. polyQ protein N-terminal C-terminal unbound polyP polyQ disordered coiled coil polyQ polyP coiled coil protein X bound

32. ATXN1Q82 NT is toxicATXN1Q82 NT aggregates Petrakis et al (2012) PLoS Genetics Spyros Petrakis

33. interactors that change ATXN1Q82 NT toxicity

38. polyQ disordered CC Normal polyQ protein CC partner

39. polyQ disordered CC Normal polyQ protein CC partner

40. polyQ disordered CC Normal polyQ protein non-CC partner polyQ alpha-helix CC partner

41. polyQ disordered CC Normal polyQ protein non-CC partner polyQ alpha-helix CC partner Toxic polyQ protein

42. polyQ disordered CC Normal polyQ protein non-CC partner polyQ alpha-helix CC partner Toxic polyQ protein

43. polyQ disordered CC Normal polyQ protein non-CC partner polyQ alpha-helix CC partner Toxic polyQ protein polyQ beta-aggregates

44. polyQ disordered CC Normal polyQ protein CC partner non-CC partner polyQ alpha-helix Toxic polyQ protein polyQ beta-aggregates polyQ beta-aggregates

45. polyQ disordered CC Normal polyQ protein CC partner non-CC partner polyQ alpha-helix Toxic polyQ protein polyQ beta-aggregates polyQ beta-aggregates

46. polyQ disordered CC Normal polyQ protein CC partner non-CC partner polyQ alpha-helix Toxic polyQ protein polyQ beta-aggregates polyQ increased beta-aggregates

47. polyQ disordered CC Normal polyQ protein CC partner non-CC partner polyQ alpha-helix Toxic polyQ protein polyQ beta-aggregates polyQ increased beta-aggregates

48. polyQ disordered CC Normal polyQ protein CC partner non-CC partner polyQ alpha-helix Toxic polyQ protein polyQ beta-aggregates polyQ increased beta-aggregates

49. polyQ disordered CC Normal polyQ protein CC partner non-CC partner polyQ alpha-helix Toxic polyQ protein polyQ beta-aggregates polyQ increased beta-aggregates

50. BiasViz2

51. Go to the BiasViz2 web pageBiasViz2 Load this alignment of N-terminal huntingtins on the step 1 sectionthis alignment Load this file with secondary structure predicted for the human fragment in the step 2 sectionthis file with secondary structure predicted Load this file with ARD2 predictions for all sequences of the alignmnent in the step 2 section "raw values for each amino acid"this file with ARD2 predictions Hit the "Go to graphical view" button Find the CBRs we have discussed for huntingtin Compare the relative position of the predicted repeats and the predicted secondary structure Exercise 3. All together! View repeats, CBRs, and secondary structure in the N-terminal of huntingtin with BiasViz2