Membrane phospholipids control gating of the mechanosensitive potassium leak channel TREK1

Tandem pore domain (K2P) potassium channels modulate resting membrane potentials and shape cellular excitability. For the mechanosensitive subfamily of K2Ps, the composition of phospholipids within the bilayer strongly influences channel activity. To examine the molecular details of K2P lipid modulation, we solved cryo-EM structures of the TREK1 K2P channel bound to either the anionic lipid phosphatidic acid (PA) or the zwitterionic lipid phosphatidylethanolamine (PE). At the extracellular face of TREK1, a PA lipid inserts its hydrocarbon tail into a pocket behind the selectivity filter, causing a structural rearrangement that recapitulates mutations and pharmacology known to activate TREK1. At the cytoplasmic face, PA and PE lipids compete to modulate the conformation of the TREK1 TM4 gating helix. Our findings demonstrate two distinct pathways by which anionic lipids enhance TREK1 activity and provide a framework for a model that integrates lipid gating with the effects of other mechanosensitive K2P modulators

Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel

SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the hyperpolarization-activated and cyclic nucleotide-gated channel HCN2 in the family of so-called pacemaker channels. Here, we investigate slow cAMP-induced activation in purified SthK channels using stopped-flow assays, mutagenesis, enzymatic catalysis and inhibition assays revealing that the cis/trans conformation of a conserved proline in the cyclic nucleotide-binding domain determines the activation kinetics of SthK. We propose that SthK exists in two forms: trans Pro300 SthK with high ligand binding affinity and fast activation, and cis Pro300 SthK with low affinity and slow activation. Following channel activation, the cis/trans equilibrium, catalyzed by prolyl isomerases, is shifted towards trans, while steady-state channel activity is unaffected. Our results reveal prolyl isomerization as a regulatory mechanism for SthK, and potentially eukaryotic HCN channels. This mechanism could contribute to electrical rhythmicity in cells

Dimeric structure of the bacterial extracellular foldase PrsA

Secretion of proteins into the membrane-cell wall space is essential for cell wall biosynthesis and pathogenicity in Gram-positive bacteria. Folding and maturation of many secreted proteins depend on a single extracellular foldase, the PrsA protein. PrsA is a 30 kDa protein, lipid-anchored to the outer leaflet of the cell membrane. The crystal structure of Bacillus subtilis PrsA reveals a central catalytic parvulin-type prolyl isomerase domain, which is inserted into a larger composite NC domain formed by the N- and C-terminal regions. This domain architecture resembles, despite a lack of sequence conservation, both trigger factor, a ribosome-binding bacterial chaperone, and SurA, a periplasmic chaperone in Gram-negative bacteria. Two main structural differences are observed in that the N-terminal arm of PrsA is substantially shortened relative to trigger factor and SurA and in that PrsA is found to dimerize in a unique fashion via its NC domain. Dimerization leads to a large, bowl-shaped crevice, which might be involved in vivo in protecting substrate proteins from aggregation. NMR experiments reveal a direct, dynamic interaction of both the parvulin and the NC domain with secretion propeptides, which have been implicated in substrate targeting to PrsA

Molecular Determinants of a Regulatory Prolyl Isomerization in the Signal Adapter Protein c‑CrkII

The cellular CT10 regulator of kinase protein (c-CrkII) transmits signals from oncogenic tyrosine kinases to cellular targets. Nuclear magnetic resonance studies had suggested that in chicken c-CrkII a native state prolyl <i>cis</i>–<i>trans</i> isomerization is involved in signal propagation. Corresponding evidence for the closely related human c-CrkII was not obtained. Here we analyzed the kinetics of folding and substrate binding of the two homologues and found that <i>cis</i>–<i>trans</i> isomerization of Pro238 determines target binding in chicken but not in human c-CrkII. A reciprocal mutational analysis uncovered residues that determine the isomeric state at Pro238 and transmit it to the binding site for downstream target proteins. The transfer of these key residues to human c-CrkII established a regulatory proline switch in this protein, as well. We suggest that Pro238 isomerization extends the lifetime of the signaling-active state of c-CrkII and thereby functions as a long-term molecular storage device

Incorporation of an Unnatural Amino Acid as a Domain-Specific Fluorescence Probe in a Two-Domain Protein

The biophysical analysis of multidomain proteins often is difficult because of overlapping signals from the individual domains. Previously, the fluorescent unnatural amino acid <i>p</i>-cyanophenylalanine has been used to study the folding of small single-domain proteins. Here we extend its use to a two-domain protein to selectively analyze the folding of a specific domain within a multidomain protein

Structural and Functional Characterization of a Novel Family of Cyclophilins, the AquaCyps.

Cyclophilins are ubiquitous cis-trans-prolyl isomerases (PPIases) found in all kingdoms of life. Here, we identify a novel family of cyclophilins, termed AquaCyps, which specifically occurs in marine Alphaproteobacteria, but not in related terrestric species. In addition to a canonical PPIase domain, AquaCyps contain large extensions and insertions. The crystal structures of two representatives from Hirschia baltica, AquaCyp293 and AquaCyp300, reveal the formation of a compact domain, the NIC domain, by the N- and C-terminal extensions together with a central insertion. The NIC domain adopts a novel mixed alpha-helical, beta-sheet fold that is linked to the cyclophilin domain via a conserved disulfide bond. In its overall fold, AquaCyp293 resembles AquaCyp300, but the two proteins utilize distinct sets of active site residues, consistent with differences in their PPIase catalytic properties. While AquaCyp293 is a highly active general PPIase, AquaCyp300 is specific for hydrophobic substrate peptides and exhibits lower overall activity

PPIase activities of AquaCyp293 and AquaCyp300.

<p>(A) Kinetics of <i>cis</i>/<i>trans</i> isomerization of 3 μM Abz-Ala-Ala-Pro-Phe-pNa followed by fluorescence at 416 nm, without enzyme (black), with 8 nM (blue), 12 nM (green), 16 nM (dark blue) and 20 nM (red) AquaCyp293. (B) Refolding kinetics of RCM-T1 in the presence of increasing concentrations of AquaCyp293, 0 nM (black), 10 nM (blue), 300 nM (green) and 750 nM (red). The kinetics of refolding of 0.1 μM RCM-T1 in 0.1 M Tris/HCl pH 8.0; 2 M NaCl were measured at 15°C in the presence of various concentrations of AquaCyp293. (C, D) Catalytic efficiencies of AquaCyp293 (○) and AquaCyp300 (□) for (C) the <i>cis</i>/<i>trans</i> isomerization of Abz-Ala-Ala-Pro-Phe-<i>p</i>NA and (D) the refolding of RCM-T1. The measured proline-limited refolding rate constants <i>k</i>_app are shown as a function of the PPIase concentration. The <i>k</i>_cat/<i>K</i>_m values derived from the slopes are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157070#pone.0157070.t001" target="_blank">Table 1</a>.</p

Dimerization of AquaCyp300.

<p>(A) Surface representation of the dimeric AquaCyp300 crystal structure, one protomer is colored in light the other in dark grey, the N-terminal-, insertion, and C-terminal structural elements in one protomer are colored blue, green and red, respectively, active site residues of both protomers are colored yellow. (B) Sequence conservation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157070#pone.0157070.ref083" target="_blank">83</a>] within the AquaCyp300 family is mapped onto a cartoon representation of AquaCyp300 protomer2. Residues that are highly conserved (e.g. Phe258, Phe259, Phe260) are shown in blue, sequences with lower identity are shown in white and red.</p

Domain structure and conservation of AquaCyp.

<p>The cyclophilin domains are shown in grey, the additonal N-terminal-, insertion, and C-terminal structural elements are colored in blue, green and red, respectively. The indicated amino acids are conserved among cyclophilins. The two invariant cysteine residues in AquaCyp are shown in bold. Extension and insertion regions are indicated above the sequences.</p