Atomistic mechanism of coupling between cytosolic sensor domain and selectivity filter in TREK K2P channels.

The two-pore domain potassium (K2P) channels TREK-1 and TREK-2 link neuronal excitability to a variety of stimuli including mechanical force, lipids, temperature and phosphorylation. This regulation involves the C-terminus as a polymodal stimulus sensor and the selectivity filter (SF) as channel gate. Using crystallographic up- and down-state structures of TREK-2 as a template for full atomistic molecular dynamics (MD) simulations, we reveal that the SF in down-state undergoes inactivation via conformational changes, while the up-state structure maintains a stable and conductive SF. This suggests an atomistic mechanism for the low channel activity previously assigned to the down state, but not evident from the crystal structure. Furthermore, experimentally by using (de-)phosphorylation mimics and chemically attaching lipid tethers to the proximal C-terminus (pCt), we confirm the hypothesis that moving the pCt towards the membrane induces the up-state. Based on MD simulations, we propose two gating pathways by which movement of the pCt controls the stability (i.e., conductivity) of the filter gate. Together, these findings provide atomistic insights into the SF gating mechanism and the physiological regulation of TREK channels by phosphorylation.

The opening dynamics of the lateral gate regulates the activity of rhomboid proteases.

Rhomboid proteases hydrolyze substrate helices within the lipid bilayer to release soluble domains from the membrane. Here, we investigate the mechanism of activity regulation for this unique but wide-spread protein family. In the model rhomboid GlpG, a lateral gate formed by transmembrane helices TM2 and TM5 was previously proposed to allow access of the hydrophobic substrate to the shielded hydrophilic active site. In our study, we modified the gate region and either immobilized the gate by introducing a maleimide-maleimide (M2M) crosslink or weakened the TM2/TM5 interaction network through mutations. We used solid-state nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations, and molecular docking to investigate the resulting effects on structure and dynamics on the atomic level. We find that variants with increased dynamics at TM5 also exhibit enhanced activity, whereas introduction of a crosslink close to the active site strongly reduces activity. Our study therefore establishes a strong link between the opening dynamics of the lateral gate in rhomboid proteases and their enzymatic activity.

Dissecting the activation of insulin degrading enzyme by inositol pyrophosphates and their bisphosphonate analogs.

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are densely phosphorylated eukaryotic messengers, which are involved in numerous cellular processes. To elucidate their signaling functions at the molecular level, non-hydrolyzable bisphosphonate analogs of inositol pyrophosphates, PCP-InsPs, have been instrumental. Here, an efficient synthetic strategy to obtain these analogs in unprecedented quantities is described - relying on the use of combined phosphate ester-phosphoramidite reagents. The PCP-analogs, alongside their natural counterparts, were applied to investigate their regulatory effect on insulin-degrading enzyme (IDE), using a range of biochemical, biophysical and computational methods. A unique interplay between IDE, its substrates and the PP-InsPs was uncovered, in which the PP-InsPs differentially modulated the activity of the enzyme towards short peptide substrates. Aided by molecular docking and molecular dynamics simulations, a flexible binding mode for the InsPs/PP-InsPs was identified at the anion binding site of IDE. Targeting IDE for therapeutic purposes should thus take regulation by endogenous PP-InsP metabolites into account.