Anisimova, Maria

Bliven, Spencer

ZHAW digitalcollection

This work is licensed under a Creative Commons Attribution 3.0 Unported License.Phylogenetics of Tandem Repeats with Circular HMMs: A Case Study on Armadillo Repeat ProteinsSpencer Bliven1,2,*  Maria Anisimova1,21 Institute for Applied Simulation, Zurich University of Applied Sciences, Wädenswil, Switzerland  2 Swiss Institute of Bioinformatics (SIB), Lausanne, SwitzerlandAbstractTandem repeat proteins are characterized by multiple sequential copies of  repeats with significant structural or sequence similarity. Tandem repeats evolve via repeat expansion, duplication and loss, and many protein families exhibit very diverse repeat counts. Identifying the complex relationships between homologous proteins and between individual repeats is a challenging task. Using tools developed in our group, we present a detailed phylogenetic analysis of  the repeats in the Armadillo Repeat Protein (ArmRP) family. The ArmRP family is very diverse, appearing throughout the eukaryotes and having a wide range of  functions. They are well characterized structurally, with ~42 amino acid repeats forming three alpha-helices which assemble into a solenoid structure. ArmRP are exciting candidates for protein design, as they have been shown to bind peptides in a modular manner (Reichen 2016). Phylogenetic analysis of  tandem repeats has several unique challenges. Identifying homologous regions is complicated by the repetitive nature of  the sequence, which can cause register shifts when applying standard alignment tools. We surmount this problem using the Tandem Repeat Annotation Library (TRAL), a tool for accurately identifying repeats using circular profile hidden Markov models (Schaper 2015). After constructing a multiple alignment of  the repeats of  ArmRP representatives, we infer a phylogenetic tree relating the different ArmRP and use it to analyze the conservation and diversification patterns through evolution, based on the information about tandem repeat number, order and their distribution on phylogenies.Gul, I. S., Hulpiau, P., Saeys, Y., & van Roy, F. (2017). Metazoan evolution of the armadillo repeat superfamily. Cellular and Molecular Life Sciences : CMLS, 74(3), 525–541. http://doi.org/10.1007/s00018-016-2319-6 Nguyen, L.-T., Schmidt, H. A., Haeseler, von, A., & Minh, B. Q. (2015). IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Molecular Biology and Evolution, 32(1), 268–274. http://doi.org/10.1093/molbev/msu300 Reichen, C., Hansen, S., Forzani, C., Honegger, A., Fleishman, S. J., Zhou, T., et al. (2016). Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition. Journal of Molecular Biology, 428(22), 4467–4489. http://doi.org/10.1016/j.jmb.2016.09.012 Schaper, E., Korsunsky, A., Pečerska, J., Messina, A., Murri, R., Stockinger, H., et al. (2015). TRAL: tandem repeat annotation library. Bioinformatics, 31(18), 3051–3053. http://doi.org/10.1093/bioinformatics/btv306 Wheeler, T. J., Clements, J., & Finn, R. D. (2014). Skylign: a tool for creating informative, interactive logos representing sequence alignments and profile hidden Markov models. BMC Bioinformatics, 15(1), 7. http://doi.org/10.1186/1471-2105-15-7 EvolutionDetermining the evolution of  tandem repeat proteins is a challenge. The repeats typically have significant homology (for instance, Asn39 is conserved in 51% of  human ArmRP; Leu22 in 67% (Gul, 2017)). This leads to the hypothesis that most tandem repeats evolved through duplication and fusion events. A number of  mechanisms exist for the duplication/fusion of  repeats, both individually and collectively through gene duplication/fusion. Combining this with gene-level speciation and duplications creating paralogs can explain the complex relationships we see among TR families. Tandem Repeat AnnotationOne issue with identifying tandem repeats is that the first and last repeats are often truncated or atypical. With standard sequence alignment methods, this can lead to missing partial repeats and incorrect phase identification: Additionally, misalignments near the repeat boundaries can cause overlapping repeats which must be reconciled. These issues are resolved in the Tandem Repeat Annotation Library (TRAL) (Schaper 2015). The library makes use of  circular profile hidden Markov models (cpHMM) to capture TR profiles.   The cpHMM allows the TR region to start and end at any point in the repeat, making the phase inconsequential. Additionally, it prevents overlaps and provides a rigorous statistical model for dealing with insertions between and within repeats. TRAL contains functionality for constructing a cpHMM from a set of  possibly inconsistent or overlapping seed TR hits.Conclusions & OutlookWe have shown that using cpHMM in TRAL we are able to improve our identification of  ArmRP repeats. It is able to capture the correct phase of  the repeats, as well as handle partial terminal repeats. Using the ArmRP alignment we construct phylogenetic trees of  the repeats. Work continues to reconstruct detailed evolutionary histories for individual repeats. This will allow the identification of  repeat duplication and loss across this complex family. Work is also ongoing to incorporate duplication and fusion events into the model for phylogenetic inference. This would allow reconstruction of  the evolutionary events leading to tandem repeats.Armadillo Repeat ProteinsArmadillo repeat proteins (ArmRP) are composed of  ~40 amino acid repeats forming a alpha solenoid structure. Each repeat contains three alpha helices, with a hydrophobic core and conserved binding residues along the third helix. ArmRP bind extended peptides, which often are disordered prior to binding. Two amino acids binding each repeat. The conserved Asn at position 39 of  the repeat typically forms a hydrogen bond to the peptide backbone. Specificity is conferred by a shallow binding pocket between helix 3 of  adjacent repeats, which binds the peptide side chain. However, many ArmRP show weak specificity and engage multiple partners.Designed ArmRP  YIIIM5AII bound to a (KR)5 peptide [5AEI] 0.250.001.9GANEQTFRH10.470.001.9DNGTSKIC20.800.001.9NESDQRPCYH30.890.001.9EPQDA40.930.001.9QNAGMDY50.950.001.9ILACTFRQ60.990.001.9KRQAEDNH71.000.001.9AYEQFTMW81.000.001.9LVIFM91.000.001.9ILVMCW101.000.001.9EDQRNKH111.000.001.9SLAETQCM120.950.001.9GESNQYH130.980.001.9GALVINCM140.990.001.9LIVFM150.990.001.9PQERDN160.990.001.9KAPTRDH171.000.001.9LIMFY180.980.001.9VLIMFC190.990.001.9QEKNRADHC201.000.001.9LYMQ211.000.001.9LMI221.000.001.9SREQKTNHF231.000.001.9SKHND240.990.001.9ESPDKNRTQH250.990.001.9DNSRQH260.900.001.9EPSFDQC270.960.001.9KEDSNTMH280.990.001.9IVLT291.000.001.9QLVAKTRI301.000.001.9ERKQSFWC311.000.001.9ANESDKQYCH320.990.001.9AVCT330.990.001.9ALVTCFM340.990.001.9GWAQRDKNMC351.000.001.9LAVITCYW361.000.001.9LVIFMW370.980.001.9SRQKGHCW380.970.001.9NSH390.960.001.9LIVMFC400.950.001.9SVTACFIMW410.900.000SRADEKFQY421.6700.83Consensus Arm repeat. (Top) HMM logo based on a multiple alignment of  human Arm proteins by Gul (2017). Image generated with Skylign (Wheeler 2014).  (Left) Repeat structure, showing hydrophobic core (grey), glycine turns (green), positive (blue), negative (red), and amidic (magenta) conserved residues. Side chains in the binding pocket are shown as sticks. M1D1I1.........MNB ED1DN DNIN INCircular TR sequence profile HMM (cpHMM). The alignment starts in any position within the TR with equal probability (blue). States representing alignment matches, insertions and deletions are included as in the HMMER model (black). Additional transitions are added from the end of  a repeat back to the beginning (red) to align multiple repeats. The alignment may end at any position with equal probability (green).0.7KPNA1 10KPNA6 4KPNA5 5KPNA2 1KPNA2 9KPNA6 2KPNA1 2KPNA2 2KPNA1 8KPNA1 1KPNA1 9KPNA5 8KPNA6 7KPNA5 2KPNA1 6KPNA4 3KPNA5 1KPNA4 9KPNA1 7KPNA4 10KPNA5 4KPNA4 1KPNA2 6KPNA6 9KPNA2 3KPNA6 10KPNA2 4KPNA6 3KPNA4 7KPNA6 5KPNA5 7KPNA5 6KPNA4 8KPNA4 2KPNA5 10KPNA2 10KPNA5 9KPNA2 5KPNA1 3KPNA6 8KPNA1 4KPNA1 5KPNA6 1KPNA4 5KPNA2 8KPNA2 7KPNA6 6KPNA4 6KPNA5 3KPNA4 4Phylogenetic tree for human karyopherin (KPNA) proteins. Each protein has a 10 Arm repeats. Each repeat clusters together, indicating that the order of  repeats has not changed since the paralogous expansion. The support for this tree is too weak to draw conclusions about the relationships between repeats. Calculated using IQ-Tree (Nguyen 2015).Canonical phaseDetected phase(Top) Protein with 3.5 repeats with repeat boundaries assigned from literature. (Bottom) Incorrect tandem repeat identification, with non-standard boundaries, a missing partial repeat, and overlapping repeats.Possible evolutionary histories for two 4-repeat proteins. a) TR expansion occurs before speciation, giving identical TR orders in both species. b) Both species undergo independent whole-gene duplications, giving similar subtree patterns in both species. c) Both species undergo independent TR duplications showing different patterns.A4B1 B3 B2A1 A3 A2 B411 21 23 4A4B1 B3 B2A1 A3 A2 B411 2A1 A2B1 B2A4B1 B3B2A1 A3A2 B411 2A1 A2B1 B2Speciationa) b) c)DuplicationKey:TRAL availabilityhttp://elkeschaper.github.io/tral/2.0CAB39L 3KPNA1 10DAAM1 1CTNNB1 10KPNA1 5KPNA6 6JUP 11KPNA5 5JUP 10APC 6FMNL2 5KPNA2 7KPNA5 4USO1 11PKP2 8PKP2 4CTNNB1 5PKP2 7CTNNB1 12UNC45A 2CTNND2 6UNC45A 3KPNA1 6KPNA2 2RQCD1 6DIAPH1 4USO1 9APC 5KPNA4 2JUP 7KPNA4 10KPNA2 6HSPBP1 2RQCD1 4KPNA4 5JUP 8UNC45A 1PKP1 2KPNA6 10USO1 10PKP1 5KPNA6 7HSPBP1 1API5 2APC 7USO1 5CTNND2 7JUP 2PKP1 7PKP2 3UNC45A 7RQCD1 1FMNL2 4USO1 8KPNA4 4CTNNBL1 6KPNA2 4USO1 3API5 3CTNNBL1 5JUP 1CAB39 1UNC45A 8CTNNB1 11PKP2 1APC 2KPNA6 3PKP1 9KPNA2 3CTNND2 2CTNND2 3FMNL2 3KPNA6 1USO1 6PKP1 8USO1 2RQCD1 5KPNA5 6HSPBP1 4JUP 6HSPBP1 3KPNA5 8KPNA2 8CTNNB1 2CTNNBL1 4UNC45A 6JUP 9CAB39L 1CTNNB1 8KPNA1 4CTNNB1 4CAB39 5CTNNBL1 2PKP1 4RQCD1 2KPNA2 9KPNA6 2JUP 12CTNND2 4KPNA4 3JUP 3KPNA5 2PKP2 9KPNA4 9KPNA6 4JUP 5KPNA5 10CAB39L 2CTNNBL1 1UNC45A 5DAAM1 4APC 1KPNA1 1USO1 4KPNA2 5FMNL1 4APC 3KPNA4 6DIAPH1 3PKP2 6KPNA6 8PKP2 2KPNA1 9KPNA5 7CTNNBL1 3PKP1 3KPNA4 8KPNA2 10UNC45A 4DAAM1 5CTNND2 5PKP1 1CTNNB1 7FMNL1 3KPNA1 7PKP2 5JUP 4CTNND2 9CAB39 4DIAPH1 2KPNA6 9CAB39 2KPNA4 1CTNND2 8KPNA2 1CTNNB1 9DIAPH1 5CAB39L 5KPNA1 8CTNNB1 6APC 4KPNA6 5DIAPH1 1USO1 7KPNA1 3KPNA4 7CTNND2 1RQCD1 3CAB39 3PKP1 6KPNA5 1CAB39L 4DAAM1 3KPNA5 3CTNNB1 3KPNA5 9KPNA1 2FMNL1 5USO1 1CTNNB1 1Preliminary tree of  human ArmRP proteins with structural information in the Protein Data Bank. The shown tree does not explicitly model duplication events. Leaves are colored by gene. Calculated using IQ-Tree.Poster presented at SIB Days 2018 (Biel, CH) This research was supported by COST Action BM1405 and COST Switzerland SEFRI project IZCNZ0-174836. ArmRP PrevalencePrevalence of  ArmRP m e m b e r s a c r o s s m e t a z o a n s p e c i e s . ArmRP proteins were detected using TRAL across a 94 metazoan species. Between 14 and 1 7 0 p r o t e i n s w e r e detected containing at least two consecutive repeats.ReferencesSpecies Icons curtesy of the TogoDB (http://togodb.biosciencedbc.jp)

Phylogentics of tandem repeats with circular HMMs : a case study on Armadillo Repeat Proteins

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Phylogentics of tandem repeats with circular HMMs : a case study on Armadillo Repeat Proteins

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