Search

The water permeability of aquaporins (AQPs) varies by more than an order of magnitude even though the pore structure, geometry, as well as the channel lining residues are highly conserved. However, channel gating by pH, divalent ions or phosphorylation was only shown for a minority of AQPs. Structural and in silico indications of water flux modulation by flexible side chains of channel lining residues have not been experimentally confirmed yet. Hence, the aquaporin “open state” is still considered to be a continuously open pore with water molecules permeating in a single-file fashion. Using protein mutations outside the selectivity filter in the aqua(glycerol)facilitator GlpF of Escherichia coli we, to the best of our knowledge, for the first time, modulate the position of the highly conserved Arg in the selectivity filter. This in turn enhances or reduces the unitary water permeability of GlpF as shown in silico by molecular dynamics (MD) simulations and in vitro with purified and reconstituted GlpF. This finding suggests that AQP water permeability can indeed be regulated by lipid bilayer asymmetry and the transmembrane potential. Strikingly, our long-term MD simulations reveal that not only the conserved Arg in the selectivity filter, but the position and dynamics of multiple other pore lining residues modulate water passage through GlpF. This finding is expected to trigger a wealth of future investigations on permeability and regulation of AQPs among others with the aim to tune water permeability for biotechnological applications. Fonds zur Förderung der Wissenschaftlichen Forschung P31074 Deutsche Forschungsgemeinschaft EXC2075-390740016 Version of record

G protein-coupled receptors (GPCRs) are the largest class of human membrane proteins that bind extracellular ligands at their orthosteric binding pocket to transmit signals to the cell interior. Ligand binding evokes conformational changes in GPCRs that trigger the binding of intracellular interaction partners (G proteins, G protein kinases, and arrestins), which initiate diverse cellular responses. It has become increasingly evident that the preference of a GPCR for a certain intracellular interaction partner is modulated by a diverse range of factors, e.g., ligands or lipids embedding the transmembrane receptor. Here, by means of molecular dynamics simulations of the β2-adrenergic receptor and β-arrestin2, we study how membrane lipids and receptor phosphorylation regulate GPCR-arrestin complex conformation and dynamics. We find that phosphorylation drives the receptor’s intracellular loop 3 (ICL3) away from a native negatively charged membrane surface to interact with arrestin. If the receptor is embedded in a neutral membrane, the phosphorylated ICL3 attaches to the membrane surface, which widely opens the receptor core. This opening, which is similar to the opening in the G protein-bound state, weakens the binding of arrestin. The loss of binding specificity is manifested by shallower arrestin insertion into the receptor core and higher dynamics of the receptor-arrestin complex. Our results show that receptor phosphorylation and the local membrane composition cooperatively fine-tune GPCR-mediated signal transduction. Moreover, the results suggest that deeper understanding of complex GPCR regulation mechanisms is necessary to discover novel pathways of pharmacological intervention., Frontiers in Cell and Developmental Biology, 9, ISSN:2296-634X

Additional file 1 Tables S1-S6 and Figures S1-S25. Table S1 - Lipid tail names, shortcuts and stereochemistry. Table S2 - Molecular composition of each simulation system. Table S3 - List of performed simulations. Table S4 - Angle parameters for Martini3 tails including cyclopropane bead. Table S5 - Details on water permeability at AA resolution. Table S6 - Details on water permeability at CG resolution. Fig. S1 - Chemical structures of the lipid headgroups in E. coli polar extract membranes. Fig. S2 - Chemical structures of lipid tails included in this study. Fig. S3 - ChemDraw representations of lipid molecules in the Avanti model. Fig. S4 - Angle distributions from AA and CG simulations extracted from the Avanti model of the E. coli polar extract membrane simulations at 310 K. Fig. S5 - The time evolution of the average area per lipid of AA pure lipid bilayers. Fig. S6 - The time evolution of the average area per lipid of CG pure lipid bilayers. Fig. S7 - Area per lipid of each lipid type in pure lipid bilayers at all temperatures and their temperature dependence. Fig. S8 - Statistical analysis of error estimation of APL. Fig. S9 - Ratios of the curved area per lipid and the area per lipid estimated form the box size for all systems at all temperatures. Fig. S10 - Distribution of hydrophobic thickness of 171 transmembrane proteins localized in the inner membrane of E. coli obtained from the OPM database. Fig. S11 - Lipid diffusion in each simulated system for each lipid type including the corresponding activation energies. Fig. S12 - Interdigitation of membrane leaflets. Fig. S13 - Lipid mixing at low (277 K) and high (310 K) temperature visualized by coloring lipids according to their headgroups. Fig. S14 - Lipid tail mixing in individual leaflets at low (277K) and high (310K) temperature visualized by coloring different lipid tail types in different colors. Fig. S15 - Enrichment and depletion of neighboring lipids. Fig. S16 - CL clustering at different temperatures in all three membrane models at CG resolution. Fig. S17 - CL clustering at AA resolution at different temperatures for each lipid mixture. Fig. S18 - Membrane area compressibility of CG membranes. Fig. S19 - Order parameters along the lipid tails in CG simulations at different temperatures for all four membrane models and for the Avanti model including AQP1. Fig. S20 - AQP1 embedded in the E. coli PLE Avanti model at 310 K at AA and CG resolution Fig. S21 - P-P membrane thickness of E. coli PLE Avanti model in pure lipid systems and in 0.5 nm and 1 nm surroundings of AQP1 at different temperatures and at AA and CG resolutions. Fig. S22 - RMSD over time of C α atoms within helices of the AA tetramer documenting the stability of the protein’s quaternary structure in AA simulations. Fig. S23 - RMSD over time of C α atoms within helices of the AA monomer documenting the stability of the protein’s tertiary structure in AA simulations. Fig. S24 - Lipid binding times to CG AQP1 at different temperatures. Fig. S25 - Time evolutions of lipid enrichment in the first solvation layer around CG AQP1.

Frontiers in Immunology, 9, ISSN:1664-3224

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.