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The FREAKS of PROTEIN SEQUENCE Session 3.1: Repeats Session 3.2: Biased regions Miguel Andrade Johannes-Gutenberg University of Mainz [email protected] Definition 14% proteins contains repeats (Marcotte et al, 1999) 1: Single amino acid repeats. 2: Longer imperfect tandem repeats. Assemble in structure. 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 Function CBRs 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 Conservation => Function Length, amino acid type not necessarily conserved Functions: Passive: linkers Active: binding, mediate protein interaction, structural integrity (Sim and Creamer, 2004) Structure of CBRs Often variable or flexible: do not easily crystalize 1CJF: profilin bound to polyP 2IF8: Inositol Phosphate Multikinase Ipk2 2IF8: Inositol Phosphate Multikinase Ipk2 RVSETTTSGSL 2CX5: mitochondrial cytochrome c B subunit N-terminal FFFFIFVFNF 2CX5: mitochondrial cytochrome c B subunit N-terminal Types of CBRs More than 6 aa in length, 1.4% of all, 87% of them in Euk (Faux et al 2005) Types of CBRs 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. (Faux et al 2005) 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 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 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 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) Exercise 1. Filtering CBRs for BLAST using SEG •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. •Can you see funny things in this sequence? •Go to the NCBI's BLAST web page and go to the "protein blast" option •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 option •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. A particular analysis… AIR9 Ser rich + basic LRR (1708 aa) A9 repeats Δ1 conserved region Δ3 Δ2 Δ15 Δ9 Δ10 Δ6 Δ11 Δ12 Δ14 Δ16 Microtubule localization of Δx-GFP Buschmann, et al (2006). Current Biology. Buschmann, et al (2007). Plant Signaling & Behavior …triggers a tool A particular analysis… …triggers BiasViz http://sourceforge.net/projects/biasviz/ Huska, et al. (2007). Bioinformatics A particular analysis… …triggers BiasViz http://sourceforge.net/projects/biasviz/ Huska, et al. (2007). Bioinformatics ADAM15 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 Fish PMID:12615925 Binds SH3 of ArgBP1/ABI2 PMID:12463424 a b 0.4 0.3 0.4 0.3 0.2 0.2 0.1 0.1 0.0 0.0 0.4 0.3 c ADAM19 ADAM9 0.4 0.3 0.2 0.2 0.1 0.1 0.0 0.0 ADAM11 ADAM20 Exercise 2. Viewing CBRs in an alignment with BiasViz2 •Go to the BiasViz2 web page •Launch BiasViz2 •Load this alignment on the step 1 section •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? Function of polyQ Martin Schaefer polyQ in Huntingtin 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 Schaefer et al (2012) Nucleic Acids Res. 1 5 10 50 100 500 1000 partners human polyQ TFs long non polyQ 5 10 5 10 50 100 500 1000 50 100 500 1000 yeast 1 1 partners human non polyQ polyQ TFs long non polyQ 1 2 5 10 50 100 500 1000 5 10 20 50 100 200 500 TFs long 1 partners polyQ polyQ >14 polyQ 4-14 no polyQ polyQ >14 polyQ 4-14 no polyQ polyQ protein unbound N-terminal coiled coil polyQ disordered polyP C-terminal polyQ protein polyQ protein unbound bound protein X N-terminal coiled coil coiled coil polyQ polyQ disordered polyP polyP C-terminal ATXN1Q82NT is toxic ATXN1Q82NT aggregates Spyros Petrakis Petrakis et al (2012) PLoS Genetics interactors that change ATXN1Q82NT toxicity Normal polyQ protein CC polyQ disordered CC partner Normal polyQ protein CC polyQ disordered CC partner Normal polyQ protein CC polyQ disordered CC partner polyQ alpha-helix non-CC partner Normal polyQ protein Toxic polyQ protein CC polyQ disordered CC partner polyQ alpha-helix non-CC partner Normal polyQ protein Toxic polyQ protein CC polyQ disordered CC partner polyQ alpha-helix non-CC partner Normal polyQ protein Toxic polyQ protein CC polyQ disordered CC partner polyQ alpha-helix non-CC partner polyQ beta-aggregates Normal polyQ protein Toxic polyQ protein CC polyQ disordered polyQ beta-aggregates CC partner polyQ alpha-helix non-CC partner polyQ beta-aggregates Normal polyQ protein Toxic polyQ protein CC polyQ disordered polyQ beta-aggregates CC partner polyQ alpha-helix non-CC partner polyQ beta-aggregates Normal polyQ protein Toxic polyQ protein CC polyQ disordered polyQ beta-aggregates CC partner polyQ alpha-helix non-CC partner polyQ increased beta-aggregates Normal polyQ protein Toxic polyQ protein CC polyQ disordered polyQ beta-aggregates CC partner polyQ alpha-helix non-CC partner polyQ increased beta-aggregates Normal polyQ protein Toxic polyQ protein CC polyQ disordered polyQ beta-aggregates CC partner polyQ alpha-helix non-CC partner polyQ increased beta-aggregates Normal polyQ protein Toxic polyQ protein CC polyQ disordered polyQ beta-aggregates CC partner polyQ alpha-helix non-CC partner polyQ increased beta-aggregates BiasViz2 Exercise 3. All together! View repeats, CBRs, and secondary structure in the N-terminal of huntingtin with BiasViz2 •Go to the BiasViz2 web page •Load this alignment of N-terminal huntingtins on the step 1 section •Load this file with secondary structure predicted for the human fragment in the step 2 section •Load this file with ARD2 predictions for all sequences of the alignmnent in the step 2 section "raw values for each amino acid" •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