1. Novel vertebrate homologues detected for two families of mechanosensitive channels. HYUN JI KIM and MARK S. P. SANSOM Structural Bioinfomatics and Computational Biochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK *For further information, contact Hyunji Kim Email: hyunji.kim@bioch.ox.ac.ukhyunji.kim@bioch.ox.ac.uk and Mark Sansom Email: mark.sansom@bioch.ox.ac.ukmark.sansom@bioch.ox.ac.uk 1. Genomic scanning of mechanosensitive channels revealed unprecedented vertebrate homologues of Large and Small conductance MS channels. 2. It is speculated that Gly and Aln may serve key residues accounting for mechanosensation observed in families of 2 pore potassium channels, Transient Receptor Potential channels and Large/Small mechanosensitive channels. Phylogenetic Trees with Evolutionary Distance combined. 4 A Vertebrate Homologue of Large- conductance MechanoSensitive Channel 2 A Mammalian Homologue of Small- conductance MechanoSensitive Channel 3 3-D Mapping of Sequence Conservation 5 (1)Biggin PC, Sansom MSP (2003) Mechanosensitive Channels: Stress Relief. Current Biology 13(5): R183-R185. (2) Honore E (2007) The neuronal background K2P channels: focus on TREK1. Nature Reviews Neuroscience 8:251- 261 SBCB website, http://sbcb.bioch.ox.ac.uk. References Conclusions 7 6 Mechanosensitivity is traced down to Gly & Aln. MscL MscS. A) b) C) A)Sequence alignment of MscL family, colour-coded for two conserved residues, Gly and Aln. B)Pairwise comparison of profile Hidden Markov Models: Font in proportion to emission probabilities, whilst letters shown per column correspond to symbols emitted from each state. 80 genomes in total, were screened for homologues of 17 ion channels and related proteins. ‘WU-BLAST’ was employed as search-engine, and ‘blastp’ was executed with 6 various amino acid substitution matrices, for enhanced sensitivity, whereas ‘MSPcrunch’ was plugged in our automation-script for removing spurious hits, thus higher specificity. A Genomic Landscape of Ion Channels 1 TMH1 TMH3 A) Sequence alignment of MscS family, Gly (yellow) and Aln (sky blue), as in MscL-TMH1. B) Pairwise comparison of two profile Hidden Markov Models, each representing MscS- TMH3 (upper) and a consensus mechanosensitive TM helix (lower). The latter was co- extracted from two additional protein families, which are two pore potassium channels (K2P) and transient receptor potential (TRP) channels. A) B) The vertebrate homologues, detected for MscL and MscS, are predicted to encode three TM helices (Pongo and Dr. Cuthbertson’s). Sequence conservation was measured and mapped onto corresponding crystal structures, 2oau (MscL) and 2oar (MscS), in Chimera. Degree of conservation is expressed by the thickness of worm-shaped presentation and also by the strength of blue shades. A)A consensus mechanosensitive motif, detected from 4 protein families, which are MscL, MscS, two pore potassium channel (K2P) and TRP channels. One transmembrane helix (TMH) was extracted from each family, such as TMH1 of MscL, TMH3 of MscS, TMH2 of pore 1 and pore 2 of K2P, and TMH4 of TRP channels. B) Principal Component Analysis showing co-relationship amongst the above 4 protein families. C) A profile Hidden Markov Model was constructed from the assembled alignment on the left, which was subsequently visualised by HMM Logos. Notable are strong signals detected for conserved Gly and Aln, despite a very low overall sequence identity across those ion channel families. MscL TRPV4 NOMPC K2P MscS mouse MscL chick MscLMscS MscL_HAEIN MscL_PSEFL MscL_BACSU MscL_CLOPE MscL_CHICK MscL_STAAW 2oar MscL_MYCTU MscL_SYNY3 MscL_ECOLI MscS_ECO57 2oau MscS_mouse Q51409_BOR Q55882_SYN Q34897_BAC Q05781_MYC MscS_EDWIC MscS_SHIFL Evolutionary distances were calculated from pairwise sequence comparison (using PHYLIP), based on the two alignments presented for MscL and MscS. Identical pairs are lined in the central diagonal direction. Each shade of blue increments 0.5 in the range of 0 to 3.5.