Is the Functional Response of a Receptor Determined by the Thermodynamics of Ligand Binding?

For an effective drug, strong binding to the target protein is a prerequisite, but it is not enough. To produce a particular functional response, drugs need to either block the proteins' functions or modulate their activities by changing their conformational equilibrium. The binding free energy of a compound to its target is routinely calculated, but the timescales for the protein conformational changes are prohibitively long to be efficiently modeled via physics-based simulations. Thermodynamic principles suggest that the binding free energies of the ligands with different receptor conformations may infer their efficacy. However, this hypothesis has not been thoroughly validated. We present an actionable protocol and a comprehensive study to show that binding thermodynamics provides a strong predictor of the efficacy of a ligand. We apply the absolute binding free energy perturbation method to ligands bound to active and inactive states of eight G protein-coupled receptors and a nuclear receptor and then compare the resulting binding free energies. We find that carefully designed restraints are often necessary to efficiently model the corresponding conformational ensembles for each state. Our method achieves unprecedented performance in classifying ligands as agonists or antagonists across the various investigated receptors, all of which are important drug targets.

Nanoporous Membranes of Densely Packed Carbon Nanotubes Formed by Lipid-Mediated Self-Assembly.

Nanofiltration technology faces the competing challenges of achieving high fluid flux through uniformly narrow pores of a mechanically and chemically stable filter. Supported dense-packed 2D-crystals of single-walled carbon nanotube (CNT) porins with ∼1 nm wide pores could, in principle, meet these challenges. However, such CNT membranes cannot currently be synthesized at high pore density. Here, we use computer simulations to explore lipid-mediated self-assembly as a route toward densely packed CNT membranes, motivated by the analogy to membrane-protein 2D crystallization. In large-scale coarse-grained molecular dynamics (MD) simulations, we find that CNTs in lipid membranes readily self-assemble into large clusters. Lipids trapped between the CNTs lubricate CNT repacking upon collisions of diffusing clusters, thereby facilitating the formation of large ordered structures. Cluster diffusion follows the Saffman-Delbrück law and its generalization by Hughes, Pailthorpe, and White. On longer time scales, we expect the formation of close-packed CNT structures by depletion of the intervening shared annular lipid shell, depending on the relative strength of CNT-CNT and CNT-lipid interactions. Our simulations identify CNT length, diameter, and end functionalization as major factors for the self-assembly of CNT membranes.

Atypical structural snapshots of human cytomegalovirus GPCR interactions with host G proteins.

Human cytomegalovirus (HCMV) encodes G protein-coupled receptors (GPCRs) US28 and US27, which facilitate viral pathogenesis through engagement of host G proteins. Here we report cryo-electron microscopy structures of US28 and US27 forming nonproductive and productive complexes with Gi and Gq, respectively, exhibiting unusual features with functional implications. The "orphan" GPCR US27 lacks a ligand-binding pocket and has captured a guanosine diphosphate-bound inactive Gi through a tenuous interaction. The docking modes of CX3CL1-US28 and US27 to Gi favor localization to endosome-like curved membranes, where US28 and US27 can function as nonproductive Gi sinks to attenuate host chemokine-dependent Gi signaling. The CX3CL1-US28-Gq/11 complex likely represents a trapped intermediate during productive signaling, providing a view of a transition state in GPCR-G protein coupling for signaling. Our collective results shed new insight into unique G protein-mediated HCMV GPCR structural mechanisms, compared to mammalian GPCR counterparts, for subversion of host immunity.

PDADMAC/PSS Oligoelectrolyte Multilayers: Internal Structure and Hydration Properties at Early Growth Stages from Atomistic Simulations.

We analyze the internal structure and hydration properties of poly(diallyl dimethyl ammonium chloride)/poly(styrene sulfonate sodium salt) oligoelectrolyte multilayers at early stages of their layer-by-layer growth process. Our study is based on large-scale molecular dynamics simulations with atomistic resolution that we presented recently [Sánchez et al., Soft Matter 2019, 15, 9437], in which we produced the first four deposition cycles of a multilayer obtained by alternate exposure of a flat silica substrate to aqueous electrolyte solutions of such polymers at 0.1M of NaCl. In contrast to any previous work, here we perform a local structural analysis that allows us to determine the dependence of the multilayer properties on the distance to the substrate. We prove that the large accumulation of water and ions next to the substrate observed in previous overall measurements actually decreases the degree of intrinsic charge compensation, but this remains as the main mechanism within the interface region. We show that the range of influence of the substrate reaches approximately 3 nm, whereas the structure of the outer region is rather independent from the position. This detailed characterization is essential for the development of accurate mesoscale models able to reach length and time scales of technological interest.

Atomistic simulation of PDADMAC/PSS oligoelectrolyte multilayers: overall comparison of tri- and tetra-layer systems.

By employing large-scale molecular dynamics simulations of atomistically resolved oligoelectrolytes in aqueous solutions, we study in detail the first four layer-by-layer deposition cycles of an oligoelectrolyte multilayer made of poly(diallyl dimethyl ammonium chloride)/poly(styrene sulfonate sodium salt) (PDADMAC/PSS). The multilayers are grown on a silica substrate in 0.1 M NaCl electrolyte solutions and the swollen structures are then subsequently exposed to varying added salt concentration. We investigated the microscopic properties of the films, analyzing in detail the differences between three- and four-layer systems. Our simulations provide insights into the early stages of growth of a multilayer, which are particularly challenging for experimental observations. We found rather strong complexation of the oligoelectrolytes, with fuzzy layering of the film structure. The main charge compensation mechanism is for all cases intrinsic, whereas extrinsic compensation is relatively enhanced for the layer of the last deposition cycle. In addition, we quantified other fundamental observables of these systems, such as the film thickness, water uptake, and overcharge fractions for each deposition layer.

Membrane perforation by the pore-forming toxin pneumolysin.

Pneumolysin (PLY), a major virulence factor of Streptococcus pneumoniae, perforates cholesterol-rich lipid membranes. PLY protomers oligomerize as rings on the membrane and then undergo a structural transition that triggers the formation of membrane pores. Structures of PLY rings in prepore and pore conformations define the beginning and end of this transition, but the detailed mechanism of pore formation remains unclear. With atomistic and coarse-grained molecular dynamics simulations, we resolve key steps during PLY pore formation. Our simulations confirm critical PLY membrane-binding sites identified previously by mutagenesis. The transmembrane ß-hairpins of the PLY pore conformation are stable only for oligomers, forming a curtain-like membrane-spanning ß-sheet. Its hydrophilic inner face draws water into the protein-lipid interface, forcing lipids to recede. For PLY rings, this zone of lipid clearance expands into a cylindrical membrane pore. The lipid plug caught inside the PLY ring can escape by lipid efflux via the lower leaflet. If this path is too slow or blocked, the pore opens by membrane buckling, driven by the line tension acting on the detached rim of the lipid plug. Interestingly, PLY rings are just wide enough for the plug to buckle spontaneously in mammalian membranes. In a survey of electron cryo-microscopy (cryo-EM) and atomic force microscopy images, we identify key intermediates along both the efflux and buckling pathways to pore formation, as seen in the simulations.

Finite-Size-Corrected Rotational Diffusion Coefficients of Membrane Proteins and Carbon Nanotubes from Molecular Dynamics Simulations.

We investigate system-size effects on the rotational diffusion of membrane proteins and other membrane-embedded molecules in molecular dynamics simulations. We find that the rotational diffusion coefficient slows down relative to the infinite-system value by a factor of one minus the ratio of protein and box areas. This correction factor follows from the hydrodynamics of rotational flows under periodic boundary conditions and is rationalized in terms of Taylor-Couette flow. For membrane proteins like transporters, channels, or receptors in typical simulation setups, the protein-covered area tends to be relatively large, requiring a significant finite-size correction. Molecular dynamics simulations of the protein adenine nucleotide translocase (ANT1) and of a carbon nanotube porin in lipid membranes show that the hydrodynamic finite-size correction for rotational diffusion is accurate in standard-use cases. The dependence of the rotational diffusion on box size can be used to determine the membrane viscosity.

Hydrodynamics of Diffusion in Lipid Membrane Simulations.

By performing molecular dynamics simulations with up to 132 million coarse-grained particles in half-micron sized boxes, we show that hydrodynamics quantitatively explains the finite-size effects on diffusion of lipids, proteins, and carbon nanotubes in membranes. The resulting Oseen correction allows us to extract infinite-system diffusion coefficients and membrane surface viscosities from membrane simulations despite the logarithmic divergence of apparent diffusivities with increasing box width. The hydrodynamic theory of diffusion applies also to membranes with asymmetric leaflets and embedded proteins, and to a complex plasma-membrane mimetic.

Molecular dynamics simulations of carbon nanotube porins in lipid bilayers.

Artificial channels made of carbon nanotube (CNT) porins are promising candidates for applications in filtration and molecular delivery devices. Their symmetric shape and high mechanical, chemical, and thermal stability ensure well-defined transport properties, and at the same time make them ideal model systems for more complicated membrane protein pores. As the technology to produce and tune CNTs advances, simulations can aid in the design of customized membrane porins. Here we concentrate on CNTs embedded in lipid membranes. To derive design guidelines, we systematically studied the interaction of CNT porins with their surrounding lipids. For our simulations, we developed an AMBER- and Lipid14-compatible parameterization scheme for CNTs with different chirality and with functional groups attached to their rim, and a flexible coarse-grained description for open-ended CNTs fitting to the MARTINI lipid model. We found that the interaction with lipid acyl chains is independent of the CNT chirality and the chemical details of functional groups at the CNT rims. The latter, however, are important for the interactions with lipid head groups, and for water permeability. The orientation and permeability of the pore are mainly determined by how well its hydrophobicity pattern matches the membrane. By identifying the factors that determine the structure both of isolated CNTs in lipid membranes and of CNT clusters, we set the foundation for a targeted design of CNT-membrane systems.

Carbon Nanotubes Mediate Fusion of Lipid Vesicles.

The fusion of lipid membranes is opposed by high energetic barriers. In living organisms, complex protein machineries carry out this biologically essential process. Here we show that membrane-spanning carbon nanotubes (CNTs) can trigger spontaneous fusion of small lipid vesicles. In coarse-grained molecular dynamics simulations, we find that a CNT bridging between two vesicles locally perturbs their lipid structure. Their outer leaflets merge as the CNT pulls lipids out of the membranes, creating an hourglass-shaped fusion intermediate with still intact inner leaflets. As the CNT moves away from the symmetry axis connecting the vesicle centers, the inner leaflets merge, forming a pore that completes fusion. The distinct mechanism of CNT-mediated membrane fusion may be transferable, providing guidance in the development of fusion agents, e.g., for the targeted delivery of drugs or nucleic acids.

Divergent Diffusion Coefficients in Simulations of Fluids and Lipid Membranes.

We investigate the dependence of single-particle diffusion coefficients on the size and shape of the simulation box in molecular dynamics simulations of fluids and lipid membranes. We find that the diffusion coefficients of lipids and a carbon nanotube embedded in a lipid membrane diverge with the logarithm of the box width. For a neat Lennard-Jones fluid in flat rectangular boxes, diffusion becomes anisotropic, diverging logarithmically in all three directions with increasing box width. In elongated boxes, the diffusion coefficients normal to the long axis diverge linearly with the height-to-width ratio. For both lipid membranes and neat fluids, this behavior is predicted quantitatively by hydrodynamic theory. Mean-square displacements in the neat fluid exhibit intermediate regimes of anomalous diffusion, with t ln t and t(3/2) components in flat and elongated boxes, respectively. For membranes, the large finite-size effects, and the apparent inability to determine a well-defined lipid diffusion coefficient from simulation, rationalize difficulties in comparing simulation results to each other and to those from experiments.

Coarse-grained simulations of polyelectrolyte complexes: MARTINI models for poly(styrene sulfonate) and poly(diallyldimethylammonium).

We present simulations of aqueous polyelectrolyte complexes with new MARTINI models for the charged polymers poly(styrene sulfonate) and poly(diallyldimethylammonium). Our coarse-grained polyelectrolyte models allow us to study large length and long time scales with regard to chemical details and thermodynamic properties. The results are compared to the outcomes of previous atomistic molecular dynamics simulations and verify that electrostatic properties are reproduced by our MARTINI coarse-grained approach with reasonable accuracy. Structural similarity between the atomistic and the coarse-grained results is indicated by a comparison between the pair radial distribution functions and the cumulative number of surrounding particles. Our coarse-grained models are able to quantitatively reproduce previous findings like the correct charge compensation mechanism and a reduced dielectric constant of water. These results can be interpreted as the underlying reason for the stability of polyelectrolyte multilayers and complexes and validate the robustness of the proposed models.