Objective The TMEM175/GAK/DGKQ locus is the 3rd strongest risk locus in genome‐wide association studies of Parkinson disease (PD). We aimed to identify the specific disease‐associated variants in this locus, and their potential implications. Methods Full sequencing of TMEM175/GAK/DGKQ followed by genotyping of specific associated variants was performed in PD (n = 1,575) and rapid eye movement sleep behavior disorder (RBD) patients (n = 533) and in controls (n = 1,583). Adjusted regression models and a meta‐analysis were performed. Association between variants and glucocerebrosidase (GCase) activity was analyzed in 715 individuals with available data. Homology modeling, molecular dynamics simulations, and lysosomal localization experiments were performed on TMEM175 variants to determine their potential effects on structure and function. Results Two coding variants, TMEM175 p.M393T (odds ratio [OR] = 1.37, p = 0.0003) and p.Q65P (OR = 0.72, p = 0.005), were associated with PD, and p.M393T was also associated with RBD (OR = 1.59, p = 0.001). TMEM175 p.M393T was associated with reduced GCase activity. Homology modeling and normal mode analysis demonstrated that TMEM175 p.M393T creates a polar side‐chain in the hydrophobic core of the transmembrane, which could destabilize the domain and thus impair either its assembly, maturation, or trafficking. Molecular dynamics simulations demonstrated that the p.Q65P variant may increase stability and ion conductance of the transmembrane protein, and lysosomal localization was not affected by these variants. Interpretation Coding variants in TMEM175 are likely to be responsible for the association in the TMEM175/GAK/DGKQ locus, which could be mediated by affecting GCase activity. ANN NEUROL 2020;87:139–153

Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect formed by hydrophobic mismatch at their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium towards the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the protein's biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties.

ProP is a member of the major facilitator superfamily, a proton-osmolyte symporter, and an osmosensing transporter. ProP proteins share extended cytoplasmic carboxyl terminal domains (CTDs) implicated in osmosensing. The CTDs of the best characterized, group A ProP orthologs, terminate in sequences that form intermolecular, antiparallel a-helical coiled coils (e.g., ProPEc, from Escherichia coli). Group B orthologs lack that feature (e.g., ProPXc, from Xanthomonas campestris). ProPXc was expressed and characterized in E. coli to further elucidate the role of the coiled coil in osmosensing. The activity of ProPXc was a sigmoid function of the osmolality in cells and proteoliposomes. ProPEc and ProPXc attained similar activities at the same expression level in E. coli. ProPEc transports proline and glycine betaine with comparable high affinities at low osmolality. In contrast, proline weakly inhibited high-affinity glycine-betaine uptake via ProPXc. The K M for proline uptake via ProPEc increases dramatically with the osmolality. The K M for glycine-betaine uptake via ProPXc did not. Thus, ProPXc is an osmosensing transporter, and the C-terminal coiled coil is not essential for osmosensing. The role of CTD-membrane interaction in osmosensing was examined further. As for ProPEc, the ProPXc CTD co-sedimented with liposomes comprising E. coli phospholipid. Molecular dynamics simulations illustrated association of the monomeric ProPEc CTD with the membrane surface. Comparison with the available NMR structure for the homodimeric coiled coil formed by the ProPEc-CTD suggested that membrane association and homodimeric coiled-coil formation by that peptide are mutually exclusive. The membrane fluidity in liposomes comprising E. coli phospholipid decreased with increasing osmolality in the range relevant for ProP activation. These data support the proposal that ProP activates as cellular dehydration increases cytoplasmic cation concentration, releasing the CTD from the membrane surface. For group A orthologs, this also favors a-helical coiled-coil formation that stabilizes the transporter in an active form.

Porous coordination networks (PCNs) are promising nanoporous materials in gas separation applications due to their tunable pore sizes, large surface areas, high porosities, and good thermal and mechanical stabilities. In this work, we investigated adsorption-based and membrane-based separation performances of 20 different PCNs for CH4/H2, CO2/H2, CO2/CH4, and CO2/N2 mixtures using molecular simulations. Several PCNs were identified to show higher selectivity than traditional zeolites and polymers in membrane-based CO2 separations. We also developed simple models that can predict adsorption, diffusion, and permeation selectivities of PCNs for CH4/H2 and CO2/H2 mixtures based on the structural properties of materials such as pore volume, surface area, and pore diameter.

Porous coordination networks (PCNs) offer considerable potential for gas separation applications due to their tunable pore sizes, large surface areas, high pore volumes, and good thermal and mechanical stabilities. Although a large number of PCNs have been synthesized to date, the potential performance of PCNs for adsorption-based and/or membrane-based gas separation applications is not known. In this work, we used atomically detailed simulations to predict the performance of PCN materials both in adsorption-based and in membrane-based separations of CH 4 /H 2 , CO 2 /CH 4 , CO 2 /H 2 , and CO 2 /N 2 mixtures. After validating the accuracy of our atomic simulations by comparing simulated adsorption isotherms of CO 2 , CH 4 , H 2 , and N 2 with the available experimental data, we predicted adsorption-based selectivity, working capacity, regenerability, sorbent selection parameter, diffusion-based selectivity, membrane-based selectivity, and gas permeability of various PCNs. Several PCNs were predicted to outperform traditional zeolites and widely studied metal organic frameworks in CO 2 separation processes. PCN-26 was identified as a potential membrane material that can exceed the upper bound established for CO 2 /CH 4 and CO 2 /N 2 separations due to its high CO 2 permeability and selectivity.

Osmosensing transporter ProP detects the increase in cytoplasmic cation concentration associated with osmotically induced cell dehydration and mediates osmolyte uptake into bacteria. ProP is a 12-transmembrane helix protein with an α-helical, cytoplasmic C-terminal domain (CTD) linked to transmembrane helix XII (TM XII). It has been proposed that the CTD helix associates with the anionic membrane surface to lock ProP in an inactive conformation and that the release of the CTD may activate ProP. To investigate this possible activation mechanism, we have built and simulated a structural model in which the CTD was anchored to the membrane by TM XII and the CTD helix was associated with the membrane surface. Molecular dynamics simulations showed specific intrapeptide salt bridges forming when the CTD associated with the membrane. Experiments supported the presence of the salt bridge Lys447–Asp455 and suggested a role for these residues in osmosensing. Simulations performed at different salt concentrations showed weakened CTD–lipid interactions at 0.25 M KCl and gradual stiffening of the membrane with increasing salinity. These results suggest that salt cations may affect CTD release and activate ProP by increasing the order of membrane phospholipids.

The nonpolarizable CHARMM force field is one of the most widely used energy functions for all-atom biomolecular simulations. Chloride is the only halide ion included in the latest version, CHARMM36m, and is used widely in simulation studies, often as an electrolyte ion but also as the biological substrate of transport proteins and enzymes. Here, we find that existing parameters systematically underestimate the interaction of Cl– with proteins and lipids. Accordingly, when examined in solution, little to no Cl–association can be observed with most components of the protein, including backbone, polar side chains and aromatic rings. The strength of the interaction with cationic side chains and with alkali ions is also incongruent with experimental measurements, specifically osmotic coefficients of concentrated solutions. Consistent with these findings, a 4-μs trajectory of the Cl–-specific transport protein CLC-ec1 shows irreversible Cl– dissociation from the so-called Scen binding site, even in a 150 mM NaCl buffer. To correct for these deficiencies, we formulate a series of pair-specific Lennard-Jones parameters that override those resulting from the conventional Lorentz–Berthelot combination rules. These parameters, referred to as NBFIX, are systematically calibrated against available experimental data as well as ab initio geometry optimizations and energy evaluations, for a wide set of binary and ternary Cl– complexes with protein and lipid analogs and alkali cations. Analogously, we also formulate parameter sets for the other three biological halide ions, namely, fluoride, bromide, and iodide. The resulting parameters are used to calculate the potential of mean force defining the interaction of each anion and each of the protein and lipid analogues in bulk water, revealing association free energies in the range of −0.3 to −3.3 kcal/mol, with the F– complexes being the least stable. The NBFIX corrections also preserve the Cl– occupancy of CLC-ec1 in a second 4-μs trajectory. We posit that these optimized molecular-mechanics models provide a more realistic foundation for all-atom simulation studies of processes entailing changes in hydration, recognition, or transport of halide anions.

Over two-thirds of membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins in the membrane remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid an energetic penalty for solvating a thinned-membrane defect caused by their exposed dimerization interfaces. Supporting this theory, we demonstrate that this penalty is drastically reduced with minimal amounts of short-chain lipids, which stabilize the monomeric state by preferentially solvating the defect rather than altering the physical state of the membrane. We thus posit that the energy differentials for local lipid-solvation define membrane-protein association equilibria, and describe a molecular-level physical mechanism for lipid regulation of such processes in biological conditions.