Niklaus Johner

Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H + /Cl − exchanger

Active exchangers dissipate the gradient of one substrate to accumulate nutrients, export xenobiotics and maintain cellular homeostasis. Mechanistic studies have suggested that two fundamental properties are shared by all exchangers: substrate binding is antagonistic, and coupling is maintained by preventing shuttling of the empty transporter. The CLC H+/Cl− exchangers control the homeostasis of cellular compartments in most living organisms, but their transport mechanism remains unclear. We show that substrate binding to CLC-ec1 is synergistic rather than antagonistic: chloride binding induces protonation of a crucial glutamate. The simultaneous binding of H+ and Cl− gives rise to a fully loaded state that is incompatible with conventional transport mechanisms. Mutations in the Cl− transport pathway identically alter the stoichiometries of H+/Cl− exchange and binding. We propose that the thermodynamics of synergistic substrate binding, rather than the kinetics of conformational changes and ion binding, determine the stoichiometry of transport.

Why GPCRs behave differently in cubic and lamellar lipidic mesophases.

Recent successes in the crystallographic determination of structures of transmembrane proteins in the G protein-coupled receptor (GPCR) family have established the lipidic cubic phase (LCP) environment as the medium of choice for growing structure-grade crystals by the method termed “in meso”. The understanding of in meso crystallogenesis is currently at a descriptive level. To enable an eventual quantitative, energy-based description of the nucleation and crystallization mechanism, we have examined the properties of the lipidic cubic phase system and the dynamics of the GPCR rhodopsin reconstituted into the LCP with coarse-grained molecular dynamics simulations with the Martini force-field. Quantifying the differences in the hydrophobic/hydrophilic exposure of the GPCR to lipids in the cubic and lamellar phases, we found that the highly curved geometry of the cubic phase provides more efficient shielding of the protein from unfavorable hydrophobic exposure, which leads to a lesser hydrophobic mismatch and less unfavorable hydrophobic–hydrophilic interactions between the protein and lipid–water interface in the LCP, compared to the lamellar phase. Since hydrophobic mismatch is considered a driving force for oligomerization, the differences in exposure mismatch energies between the LCP and the lamellar structures suggest that the latter provide a more favorable setting in which GPCRs can oligomerize as a prelude to nucleation and crystal growth. These new findings lay the foundation for future investigations of in meso crystallization mechanisms related to the transition from the LCP to the lamellar phase and studies aimed at an improved rational approach for generating structure-quality crystals of membrane proteins.

OpenStructure: an integrated software framework for computational structural biology

Current developments in the computational structural biology framework OpenStructure are presented.

Percolative properties of hard oblate ellipsoids of revolution with a soft shell

We present an in-depth analysis of the geometrical percolation behavior in the continuum of random assemblies of hard oblate ellipsoids of revolution. Simulations were carried out by considering a broad range of aspect ratios, from spheres up to aspect-ratio-100 platelike objects, and with various limiting two-particle interaction distances, from 0.05 times the major axis up to 4.0 times the major axis. We confirm the widely reported trend of a consistent lowering of the hard particle critical volume fraction with increase of the aspect ratio. Moreover, by assimilating the limiting interaction distance to a shell of constant thickness surrounding the ellipsoids, we propose a simple relation based on the total excluded volume of these objects which allows us to estimate the critical concentration from a quantity that is quasi-invariant over a large spectrum of limiting interaction distances. Excluded volume and volume quantities are derived explicitly.

Computational modeling of the N‐terminus of the human dopamine transporter and its interaction with PIP2‐containing membranes

The dopamine transporter (DAT) is a transmembrane protein belonging to the family of neurotransmitter:sodium symporters (NSS). Members of the NSS are responsible for the clearance of neurotransmitters from the synaptic cleft, and for their translocation back into the presynaptic nerve terminal. The DAT contains long intracellular N‐ and C‐terminal domains that are strongly implicated in the transporter function. The N‐terminus (N‐term), in particular, regulates the reverse transport (efflux) of the substrate through DAT. Currently, the molecular mechanisms of the efflux remain elusive in large part due to lack of structural information on the N‐terminal segment. Here we report a computational model of the N‐term of the human DAT (hDAT), obtained through an ab initio structure prediction, in combination with extensive atomistic molecular dynamics (MD) simulations in the context of a lipid membrane. Our analysis reveals that whereas the N‐term is a highly dynamic domain, it contains secondary structure elements that remain stable in the long MD trajectories of interactions with the bilayer (totaling >2.2 μs). Combining MD simulations with continuum mean‐field modeling we found that the N‐term engages with lipid membranes through electrostatic interactions with the charged lipids PIP2 (phosphatidylinositol 4,5‐Biphosphate) or PS (phosphatidylserine) that are present in these bilayers. We identify specific motifs along the N‐term implicated in such interactions and show that differential modes of N‐term/membrane association result in differential positioning of the structured segments on the membrane surface. These results will inform future structure‐based studies that will elucidate the mechanistic role of the N‐term in DAT function. Proteins 2015; 83:952–969.

Electron tunneling in conductor-insulator composites with spherical fillers

We report on our Monte Carlo calculations of the conductivity of monosized and conducting spherical particles dispersed in a homogeneous matrix, with interparticle transport mechanism given by electron tunneling. We show that our numerical results can be reproduced by a simple formula based on a critical path analysis and which gives also a practical way to estimate the characteristic tunneling length ξ in real composites. We find that ξ is about 1 nm for several low structure carbon black polymer composites, in agreement with the expected order of magnitude.

How the Dynamic Properties and Functional Mechanisms of GPCRs Are Modulated by Their Coupling to the Membrane Environment

Experimental observations of the dependence of function and organization of G protein-coupled receptors (GPCRs) on their lipid environment have stimulated new quantitative studies of the coupling between the proteins and the membrane. It is important to develop such a quantitative understanding at the molecular level because the effects of the coupling are seen to be physiologically and clinically significant. Here we review findings that offer insight into how membrane-GPCR coupling is connected to the structural characteristics of the GPCR, from sequence to 3D structural detail, and how this coupling is involved in the actions of ligands on the receptor. The application of a recently developed computational approach designed for quantitative evaluation of membrane remodeling and the energetics of membrane-protein interactions brings to light the importance of the radial asymmetry of the membrane-facing surface of GPCRs in their interaction with the surrounding membrane. As the radial asymmetry creates adjacencies of hydrophobic and polar residues at specific sites of the GPCR, the ability of membrane remodeling to achieve complete hydrophobic matching is limited, and the residual mismatch carries a significant energy cost. The adjacencies are shown to be affected by ligand-induced conformational changes. Thus, functionally important organization of GPCRs in the cell membrane can depend both on ligand-determined properties and on the lipid composition of various membrane regions with different remodeling capacities. That this functionally important reorganization can be driven by oligomerization patterns that reduce the energy cost of the residual mismatch, suggests a new perspective on GPCR dimerization and ligand-GPCR interactions. The relation between the modulatory effects on GPCRs from the binding of specific cell-membrane components, e.g., cholesterol, and those produced by the non-local energetics of hydrophobic mismatch are discussed in this context.

Molecular origins of bending rigidity in lipids with isolated and conjugated double bonds: The effect of cholesterol

We examine the effects of cholesterol (Chol) on the mechanical properties of membranes consisting of 16:0/18:1 POPC lipid and of lipids with conjugated linoleic acid (CLA), cis-9/trans-11 CLA (C9T11) and trans-10/cis-12 CLA (T10C12). Atomistic molecular dynamics (MD) simulations of POPC-Chol and CLA-Chol mixtures at various Chol concentrations are employed within a recently developed and validated computational methodology (Khelashvili et al., 2013) that calculates from MD trajectories the bending rigidity (KC) for these systems. We have found that the addition of 30% Chol stiffens POPC lipid membranes much more significantly (2.3-fold) than it does C9T11 (1.5-fold) or T10C12 (1.75-fold) lipid bilayers. Extensive comparative structural analysis of the simulated mixtures supports a molecular mechanism for the differential effects of cholesterol, whereby the sterol molecules tilt more significantly in CLA membranes where they also insert deeper inside the hydrocarbon core. The observed distinct arrangement of Chol molecules in CLA and POPC bilayers, in turn, is dictated by the interplay between the specific location of the trans double bond in the two CLA lipid isomers and the preferential interaction of the rigid Chol ring with the saturated segments of the lipid tails. The simulations and analysis described in this paper provide novel insights into the specific modes of molecular interaction in bilayers composed of mixtures of Chol and unsaturated lipids that drive emergent macroscopic properties, such as the membranes bending modulus.

Protein and Lipid Interactions Driving Molecular Mechanisms of in meso Crystallization

The recent advances in the in meso crystallization technique for the structural characterization of G-protein coupled receptor (GPCR) proteins have established the usefulness of the lipidic-cubic phases (LCPs) in the field of crystallography of membrane proteins. It is surprising that despite the success of the approach, the molecular mechanisms of the in meso method are still not well understood. Therefore, the approach must rely on extensive screening for a suitable protein construct, for host and additive lipids, and for the appropriate precipitants and temperature. To shed light on the in meso crystallization mechanisms, we used extensive coarse-grained molecular dynamics simulations to study, in molecular detail, LCPs under different conditions (compositions and temperatures relevant to crystallogenesis) and their interactions with different types of GPCR constructs. The results presented show how the modulation of the lattice constant of the LCP (triggered by the addition of precipitant during the in meso assay), or of the host lipid type, can destabilize monomeric proteins in the bilayer of the LCP and thus drive their aggregation into the stacked lamellae, where the residual hydrophobic mismatch between the protein and the membrane can drive the formation of lateral contacts leading to nucleation and crystal growth. Moreover, we demonstrate how particular protein designs (such as transmembrane proteins engineered to contain large polar regions) can promote protein stacking interactions in the third, out-of-plane, dimension. The insights provided by the new aspects of the specific molecular mechanisms responsible for protein–protein interactions inside the cubic phase presented here should be helpful in guiding the rational design of future in meso trials with successful outcomes.

Optimal percolation of disordered segregated composites

We evaluate the percolation threshold values for a realistic model of continuum segregated systems, where random spherical inclusions forbid the percolating objects, modeled by hardcore spherical particles surrounded by penetrable shells, to occupy large regions inside the composite. We find that the percolation threshold is generally a nonmonotonous function of segregation, and that an optimal (i.e., minimum) critical concentration exists well before maximum segregation is reached. We interpret this feature as originating from a competition between reduced available volume effects and enhanced concentrations needed to ensure percolation in the highly segregated regime. The relevance with existing segregated materials is discussed.