Multiple sequence alignment of pore modules of K⁺ channel proteins from <i>C. variabilis</i>.

<p>For comparison a K⁺ channel protein CRK from the alga <i>C. reinhardtii</i> is also included. The pore-forming unit begins with the transmembrane domain, prior to the selectivity filter and it finishes at the end of the transmembrane domain after the filter. The locations of transmembrane domains were predicted based on different methods. The selectivity filter sequence is in black; aromatic amino acids upstream of the filter are marked in grey; the transmembrane domains are underlined. Worth noting is the K⁺ channels conserved selectivity filter sequence and an otherwise overall low degree of similarity between the channels.</p

Consensus, unrooted tree obtained by Bayesian estimates of phylogenies for the amino acid and nucleotide sequences, as well as for a protein parsimony approach.

<p>All clades showed a statistical support of 1 (=100%) with reference to the six independent trees computed (Bayesian estimate). The same holds for the statistical support with reference to the 1,000 replicas fed into the protpars program (protein parsimony). The branch length in this tree is arbitrary. The only difference between these is a weaker support in one of the clades (50% support, as indicated by the red star). Note that all phylogenetic approaches resulted in the same tree. Red entries indicate algae channels, while blue entries are viral channels.</p

The consensus sequence of viral K⁺ channel pore is similar to protein LAP from proteobacterium <i>Labrenzia alexandrii DFL-11</i>.

<p>(A) Consensus sequence of viral K⁺ channels. (B) Alignment of K⁺ channel Kcv_ATCV-1 with protein LAP from <i>L. alexandrii</i> DFL-11 (data bank ZP_05113853). Identical amino acids are indicated by (*), conserved or semi-conserved amino acids are indicated by (:) and (.) respectively. Note that the consensus sequence of K⁺ channel selectivity filter (grey box) is only partially conserved in the bacterial protein.</p

Growth phenotype ΔtrkΔtrk2 mutants transformed with different genes.

<p>Yeast cells were transformed with either an empty vector or with genes encoding viral K⁺ channel Kcv, or the protein LAP from <i>L. alexandrii DFL-11</i>. All yeasts were grown on non-selective medium containing either 100 mM K⁺ or lesser amounts. Only yeast transformed with Kcv_PBCV-1 grew on selective medium with low 0.5 mM and 1 mM K⁺ concentrations.</p

Multiple sequence alignment of pore modules of K⁺ channel proteins from <i>E. siliculosus</i>.

<p>The pore-forming unit begins with the transmembrane domain, prior to the selectivity filter and it finishes at the end of the transmembrane domain after the filter. The selectivity filter sequence is in black; aromatic amino acids upstream of the filter are marked in grey; the transmembrane domains are underlined.</p

Multiple sequence alignment of K⁺ channel proteins from different phycodnaviruses.

<p>The genes that code for these proteins, originate from viruses with different host specificities. Kcv_PBCV-1 and Kcv_NY-2A are from viruses that replicate in <i>C. variabilis</i>, Kcv_MT325 and Kcv_CVM-1 from viruses that replicate in <i>M. conductrix</i>, and Kcv_ATCV-1 and Kcv_TN603 from viruses that replicate in <i>C. heliozoae</i>. The channel Kesv is from virus EsV-1, which replicates in <i>E. siliculosus</i>. The selectivity filter sequence is in black; aromatic amino acids upstream of the filter are marked in grey and the transmembrane domains are underlined.</p

Minimal sequence set to test molecular piracy hypothesis. Seven sequences of K⁺ channels are from different phycodnaviruses.

<p>Six of them replicate in specific species of green alga<i>e. C. variabilis</i> is a host for two of these viruses. The seventh phycodnavirus infects <i>E. siliculosus</i>, a brown alga, which is only distantly related to the green algae. The viral channels are compared to putative K⁺ channels from hosts and non-hosts. The host channels include all 7 K⁺ channels from <i>C. variabilis</i> and all 12 K⁺ channels from <i>E. siliculosus</i>. A K⁺ channel sequence from the green alga <i>C. reinhardtii</i>, a non-host of phycodnaviruses and a close relative of <i>Chlorella</i> served as a negative control.</p

Identification of Intrahelical Bifurcated H‑Bonds as a New Type of Gate in K⁺ Channels

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K⁺ channels, Kcv_S and Kcv_NTS. These small viral encoded K⁺ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K⁺ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in Kcv_NTS and 40% in Kcv_S. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in Kcv_S. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels