Unbiased search for structure/function correlates in K+ channels - Exploiting viral molecular evolution to study ion channel functionality
Final Report Abstract
The function of a K+-channel is mainly determined by its structure. The relevant complex and synergistic structure/function correlates, which operate in the folded protein, are commonly studied by comparing the function of structurally diverse orthologs. Here we followed a new approach, which provides an un-preceded high variability of K+-channel orthologs. The strategy is based on isolation and functional characterization of viral K+-channels from environmental samples, where Chlorella viruses are abundant, and a subsequent bioinformatics analysis of structures, sequences, and their respective functions. Isolation of new viral K+ channel sequences from environmental samples provided interesting insights into structure and function correlates. We found a functional channel with a monomer size of only 78 amino acids, which presumably is the absolute minimal size for a channel protein. A phylogenetic analysis of new K+ channels from marine viruses established that they are distantly related to channels from fresh water viruses but clearly separated from eukaryotic and prokaryotic K+ channels. This fosters the hypothesis that the viral channels are not the product of molecular piracy from their hosts; they rather form a separate clade. The analysis of structural aspects in these channels has shown that the proteins have maintained a conserved fold with two α-helixes, which span the membrane and a pore α-helix, which hosts the selectivity filter. While the fold in conserved the amino acid composition in these domains is rather unique and not typical of transmembrane domains; indeed established structural prediction algorithms are not able to detect the a-helixes as transmembrane domains. This shows that it is possible to build transmembrane domains by amino acids, which are not commonly found in these domains. The analysis of the new K+ channel proteins was also shedding new light on the mechanism of protein sorting. We found that the same channel protein could be sorted in a sequence specific manner to the secretory pathway or to the mitochondria. The available data suggest that the velocity of protein translation is determining sorting at the level of the nascent protein. These proteins could provide an important model system for understanding protein sorting in cells. In the theoretical part we were able to identify mechanical building principles: the n-terminus turns out to be of uttermost importance for the channel function. We have derived a mechanical model to elucidate in detail the effects of variable n-termini. Furthermore, we were able to prove that the filter region is mechanically uncoupled from the rest of the channel – while stipulated in the experimental literature for some time, we are the first to show that this is an explainable fact, induced by the channel structure alone and not sequence dependent (only implicitly via the fold). We established a new method to quantify evolutionary pressure in sequence sets and showed that it reliable identifies sites of varying or biased molecular evolution. During the sequence preparation phase – namely upon observing artefacts in sequence alignments – we rigorously reviewed frequently source code and found an highimpact error; upon correction, new substitution models outperform older, incorrect ones; we are confident that the new approach will help to improve research in all areas of molecular evolution, protein engineering, and molecular biology. The project succeeded in shedding light on building principles of K+ ion channels – eventually combining theoretical with experimental methods. The project contributed to the profile of the involved researchers within the subsequently approved LOEWE project iNAPO (ionenleitende Nanoporen).
Publications
- Information Theoretic Dissection of the Holobiont - Host-Virus Interaction as an Example. Nova Acta Leopoldina, NF 114, No. 391, 307-315, 2013
Hamacher, K.
- Potassium ion channels: could they have evolved from viruses? Plant Physiol. 162:1215-1224, 2013
Thiel, G., Moroni, A., Blanc, G., Van Etten, J.L.
(See online at https://doi.org/10.1104/pp.113.219360) - Clustering of Giant Virus-DNA Based on Variations in Local Entropy. Viruses 6:2259-2267, 2014
Bose, R., Thiel, G., Hamacher, K.
(See online at https://doi.org/10.3390/v6062259) - Viruses infecting marine picoplancton encode functional potassium ion channels. Virology 467:103-111, 2014
Siotto, F., Martin, C., Rauh, O., Van Etten, J.L., Schroeder, I., Moroni, A., Thiel, G.
(See online at https://doi.org/10.1016/j.virol.2014.05.002) - Visual Analysis of Patterns in Multiple Amino Acid Mutation Graphs Visual Analytics Science and Technology (VAST). IEEE Conference on pp 93-102, 2014
Lenz, O., Keul, F., Bremm, S., Hamacher, K., von Landesberger, T.
(See online at https://doi.org/10.1109/VAST.2014.7042485) - Large dsDNA chloroviruses encode several membrane transport proteins. Virology 479-480:38-45, 2015
Thiel, G., Greiner, T., Dunigan, D.D., Moroni, A., Van Etten, J.L.
(See online at https://doi.org/10.1016/j.virol.2015.02.025) - Tectonics of a K+ channel: the importance of the N-terminus for channel gating. Biochimica et Biophysica Acta – Biomembranes 1848:3197-3204, 2015
Hoffgaard, F., Kast, S.M., Moroni, A., Thiel, G., Hamacher, K.
(See online at https://doi.org/10.1016/j.bbamem.2015.09.015) - Addressing inaccuracies in BLOSUM comptuation improves homology search performance. BMC Bioinformatics, 2016, 17:189
Hess, M., Keul, F., Goesele, M., Hamacher, K.
(See online at https://doi.org/10.1186/s12859-016-1060-3) - Cotranslational intersection between the SRP and GET targeting pathways to the ER of Saccharomyces cerevisiae. Molecular and Cellular Biology, Jun 2016, MCB.00131-16
Zhang, Y. Wölfle, T., Fitzke, E., Thiel, G., Rospert, S.
(See online at https://doi.org/10.1128/MCB.00131-16)
