Dynamic Second Harmonic Imaging of Proton Translocation Through Water Needles in Lipid Membranes.

Proton translocation through lipid membranes is a fundamental process in the field of biology. Several theoretical models have been developed and presented over the years to explain the phenomenon, yet the exact mechanism is still not well understood. Here, we show that proton translocation is directly related to membrane potential fluctuations. Using high-throughput wide-field second harmonic (SH) microscopy, we report apparently universal transmembrane potential fluctuations in lipid membrane systems. Molecular simulations and free energy calculations suggest that H+ permeation proceeds predominantly across a thin, membrane-spanning water needle and that the transient transmembrane potential drives H+ ions across the water needle. This mechanism differs from the transport of other cations that require completely open pores for transport and follows naturally from the well-known Grotthuss mechanism for proton transport in bulk water. Furthermore, SH imaging and conductivity measurements reveal that the rate of proton transport depends on the structure of the hydrophobic core of bilayer membranes.

Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements.

Lipid droplet (LD) function relies on proteins partitioning between the endoplasmic reticulum (ER) phospholipid bilayer and the LD monolayer membrane to control cellular adaptation to metabolic changes. It has been proposed that these hairpin proteins integrate into both membranes in a similar monotopic topology, enabling their passive lateral diffusion during LD emergence at the ER. Here, we combine biochemical solvent-accessibility assays, electron paramagnetic resonance spectroscopy and intra-molecular crosslinking experiments with molecular dynamics simulations, and determine distinct intramembrane positionings of the ER/LD protein UBXD8 in ER bilayer and LD monolayer membranes. UBXD8 is deeply inserted into the ER bilayer with a V-shaped topology and adopts an open-shallow conformation in the LD monolayer. Major structural rearrangements are required to enable ER-to-LD partitioning. Free energy calculations suggest that such structural transition is unlikely spontaneous, indicating that ER-to-LD protein partitioning relies on more complex mechanisms than anticipated and providing regulatory means for this trans-organelle protein trafficking.

Molecular Simulations Reveal the Free Energy Landscape and Transition State of Membrane Electroporation.

The formation of pores over lipid membranes by the application of electric fields, termed membrane electroporation, is widely used in biotechnology and medicine to deliver drugs, vaccines, or genes into living cells. Continuum models for describing the free energy landscape of membrane electroporation were proposed decades ago, but they have never been tested against spatially detailed atomistic models. Using molecular dynamics (MD) simulations with a recently proposed reaction coordinate, we computed potentials of mean force of pore nucleation and pore expansion in lipid membranes at various transmembrane potentials. Whereas the free energies of pore expansion are compatible with previous continuum models, the experimentally important free energy barrier of pore nucleation is at variance with established models. The discrepancy originates from different geometries of the transition state; previous continuum models assumed the presence of a membrane-spanning defect throughout the process, whereas, according to the MD simulations, the transition state of pore nucleation is typically passed before a transmembrane defect has formed. A modified continuum model is presented that qualitatively agrees with the MD simulations. Using kinetics of pore opening together with transition state theory, our free energies of pore nucleation are in excellent agreement with previous experimental data.

Phosphopeptide binding to the N-SH2 domain of tyrosine phosphatase SHP2 correlates with the unzipping of its central ß-sheet.

SHP2 is a tyrosine phosphatase that plays a regulatory role in multiple intracellular signaling cascades and is known to be oncogenic in certain contexts. In the absence of effectors, SHP2 adopts an autoinhibited conformation with its N-SH2 domain blocking the active site. Given the key role of N-SH2 in regulating SHP2, this domain has been extensively studied, often by X-ray crystallography. Using a combination of structural analyses and molecular dynamics (MD) simulations we show that the crystallographic environment can significantly influence the structure of the isolated N-SH2 domain, resulting in misleading interpretations. As an orthogonal method to X-ray crystallography, we use a combination of NMR spectroscopy and MD simulations to accurately determine the conformation of apo N-SH2 in solution. In contrast to earlier reports based on crystallographic data, our results indicate that apo N-SH2 in solution primarily adopts a conformation with a fully zipped central ß-sheet, and that partial unzipping of this ß-sheet is promoted by binding of either phosphopeptides or even phosphate/sulfate ions.

Scrutinizing the protein hydration shell from molecular dynamics simulations against consensus small-angle scattering data.

Biological macromolecules in solution are surrounded by a hydration shell, whose structure differs from the structure of bulk solvent. While the importance of the hydration shell for numerous biological functions is widely acknowledged, it remains unknown how the hydration shell is regulated by macromolecular shape and surface composition, mainly because a quantitative probe of the hydration shell structure has been missing. We show that small-angle scattering in solution using X-rays (SAXS) or neutrons (SANS) provide a protein-specific probe of the protein hydration shell that enables quantitative comparison with molecular simulations. Using explicit-solvent SAXS/SANS predictions, we derived the effect of the hydration shell on the radii of gyration Rg of five proteins using 18 combinations of protein force field and water model. By comparing computed Rg values from SAXS relative to SANS in D2O with consensus SAXS/SANS data from a recent worldwide community effort, we found that several but not all force fields yield a hydration shell contrast in remarkable agreement with experiments. The hydration shell contrast captured by Rg values depends strongly on protein charge and geometric shape, thus providing a protein-specific footprint of protein-water interactions and a novel observable for scrutinizing atomistic hydration shell models against experimental data.

Atomistic ensemble of active SHP2 phosphatase.

SHP2 phosphatase plays an important role in regulating several intracellular signaling pathways. Pathogenic mutations of SHP2 cause developmental disorders and are linked to hematological malignancies and cancer. SHP2 comprises two tandemly-arranged SH2 domains, a catalytic PTP domain, and a disordered C-terminal tail. Under physiological, non-stimulating conditions, the catalytic site of PTP is occluded by the N-SH2 domain, so that the basal activity of SHP2 is low. Whereas the autoinhibited structure of SHP2 has been known for two decades, its active, open structure still represents a conundrum. Since the oncogenic mutant SHP2E76K almost completely populates the active, open state, this mutant has been extensively studied as a model for activated SHP2. By molecular dynamics simulations and accurate explicit-solvent SAXS curve predictions, we present the heterogeneous atomistic ensemble of constitutively active SHP2E76K in solution, encompassing a set of conformational arrangements and radii of gyration in agreement with experimental SAXS data.

Molecular Simulations of Liquid Jet Explosions and Shock Waves Induced by X-Ray Free-Electron Lasers.

X-ray free-electron lasers (XFELs) produce x-ray pulses with high brilliance and short pulse duration. These properties enable structural investigations of biomolecular nanocrystals, and they allow one to resolve the dynamics of biomolecules down to the femtosecond timescale. Liquid jets are widely used to deliver samples into the XFEL beam. The impact of the x-ray pulse leads to vaporization and explosion of the liquid jet, while the expanding gas triggers the formation of shock wave trains traveling along the jet, which may affect biomolecular samples before they have been probed. Here, we used molecular dynamics simulations to reveal the structural dynamics of shock waves after an x-ray impact. Analysis of the density and temperature in the jet revealed shock waves that form close to the explosion center, travel along the jet with supersonic velocities, and decay exponentially with an attenuation length proportional to the jet diameter. A trailing shock wave formed after the first shock wave, similar to the shock wave trains in experiments. High shock wave velocities in our simulations are compatible with the phenomenon of "fast sound," as emerging at large sound frequencies. Although using purely classical models in the simulations, the resulting explosion geometry and shock wave dynamics closely resemble experimental findings, and they highlight the importance of atomistic details for modeling shock wave attenuation.

Revealing Allostery in PTPN11 SH2 Domains from MD Simulations.

Src-homology 2 (SH2) domains are protein interaction domains that bind to specific peptide motifs containing phosphotyrosine. SHP2, a tyrosine phosphatase encoded by PTPN11 gene, which has been emerged as positive or negative modulator in multiple signaling pathways, contains two SH2 domains, respectively, called N-SH2 and C-SH2. These domains play a relevant role in regulating SHP2 activity, either by recognizing its binding partners or by blocking its catalytic site. Considering the multiple functions that these domains carry out in SHP2, N-SH2 and C-SH2 represent an interesting case of study. In this chapter, we present a methodology that permits, by means of the principal component analysis (PCA), to study and to rationalize the structures adopted by the SH2 domains, in terms of the conformations of their binding sites. The structures can be distinguished, grouped, classified, and reported in a diagram. This approach permits to identify the accessible conformations of the SH2 domains in different binding conditions and to eventually reveal allosteric interactions. The method further reveals that the conformation dynamics of N-SH2 and C-SH2 strongly differ, which likely reflects their distinct functional roles.

Hydrophobin Bilayer as Water Impermeable Protein Membrane.

One of the most important properties of membranes is their permeability to water and other small molecules. A targeted change in permeability allows the passage of molecules to be controlled. Vesicles made of membranes with low water permeability are preferable for drug delivery, for example, because they are more stable and maintain the drug concentration inside. This study reports on the very low water permeability of pure protein membranes composed of a bilayer of the amphiphilic protein hydrophobin HFBI. Using a droplet interface bilayer setup, we demonstrate that HFBI bilayers are essentially impermeable to water. HFBI bilayers withstand far larger osmotic pressures than lipid membranes. Only by disturbing the packing of the proteins in the HFBI bilayer is a measurable water permeability induced. To investigate possible molecular mechanisms causing the near-zero permeability, we used all-atom molecular dynamics simulations of various HFBI bilayer models. The simulations suggest that the experimental HFBI bilayer permeability is compatible neither with a lateral honeycomb structure, as found for HFBI monolayers, nor with a residual oil layer within the bilayer or with a disordered lateral packing similar to the packing in lipid bilayers. These results suggest that the low permeabilities of HFBI and lipid bilayers rely on different mechanisms. With their extremely low but adaptable permeability and high stability, HFBI membranes could be used as an osmotic pressure-insensitive barrier in situations where lipid membranes fail such as desalination membranes.

Converging PMF Calculations of Antibiotic Permeation across an Outer Membrane Porin with Subkilocalorie per Mole Accuracy.

The emergence of multidrug-resistant pathogens led to a critical need for new antibiotics. A key property of effective antibiotics against Gram-negative bacteria is their ability to permeate through the bacterial outer membrane via transmembrane porin proteins. Molecular dynamics (MD) simulations are, in principle, capable of modeling antibiotic permeation across outer membrane porins (OMPs). However, owing to sampling problems, it has remained challenging to obtain converged potentials of mean force (PMFs) for antibiotic permeation across OMPs. Here, we investigated the convergence of PMFs along a single collective variable aimed at quantifying the permeation of the antibiotic fosmidomycin across the OprO porin. We compared standard umbrella sampling (US) with three advanced flavors of the US technique: (i) Hamiltonian replica exchange with solute tempering in combination with US, (ii) simulated tempering-enhanced US, and (iii) replica-exchange US. To quantify the PMF convergence and to reveal hysteresis problems, we computed several independent sets of US simulations starting from pulling simulations in the outward and inward permeation directions. We find that replica-exchange US in combination with well-chosen restraints is highly successful for obtaining converged PMFs of fosmidomycin permeation through OprO, reaching PMFs converged to subkilocalorie per mole accuracy.

Experiment-guided molecular simulations define a heterogeneous structural ensemble for the PTPN11 tandem SH2 domains.

SHP2 plays an important role in regulating cellular processes, and its pathogenic mutations cause developmental disorders and are linked to cancer. SHP2 is a multidomain protein, comprising two SH2 domains arranged in tandem, a catalytic PTP domain, and a disordered C-terminal tail. SHP2 is activated upon binding two linked phosphopeptides to its SH2 domains, and the peptide orientation and spacing between binding sites are critical for enzymatic activation. For decades, the tandem SH2 has been extensively studied to identify the relative orientation of the two SH2 domains that most effectively binds effectors. So far, neither crystallography nor experiments in solution have provided conclusive results. Using experiment-guided molecular simulations, we determine the heterogeneous structural ensemble of the tandem SH2 in solution in agreement with experimental data from small-angle X-ray scattering and NMR residual dipolar couplings. In the solution ensemble, N-SH2 adopts different orientations and positions relative to C-SH2. We suggest that the intrinsic structural plasticity of the tandem SH2 allows SHP2 to respond to external stimuli and is essential for its functional activity.

Molecular simulations of DEAH-box helicases reveal control of domain flexibility by ligands: RNA, ATP, ADP, and G-patch proteins.

DEAH-box helicases use the energy from ATP hydrolysis to translocate along RNA strands. They are composed of tandem RecA-like domains and a C-terminal domain connected by flexible linkers, and the activity of several DEAH-box helicases is regulated by cofactors called G-patch proteins. We used all-atom molecular dynamics simulations of the helicases Prp43, Prp22, and DHX15 in various liganded states to investigate how RNA, ADP, ATP, or G-patch proteins influence their conformational dynamics. The simulations suggest that apo helicases are highly flexible, whereas binding of RNA renders the helicases more rigid. ATP and ADP control the stability of the RecA1-RecA2 interface, but they have only a smaller effect on domain flexibility in absence of a RecA1-RecA2 interface. Binding of a G-patch protein to DHX15 imposes a more structured conformational ensemble, characterized by more defined relative domain arrangements and by an increased conformational stability of the RNA tunnel. However, the effect of the G-patch protein on domain dynamics is far more subtle as compared to the effects of RNA or ATP/ADP. The simulations characterize DEAH-box helicase as dynamic machines whose conformational ensembles are strongly defined by the presence of RNA, ATP, or ADP and only fine-tuned by the presence of G-patch proteins.

Solvent-Dependent Structural Dynamics in the Ultrafast Photodissociation Reaction of Triiodide Observed with Time-Resolved X-ray Solution Scattering.

Resolving the structural dynamics of bond breaking, bond formation, and solvation is required for a deeper understanding of solution-phase chemical reactions. In this work, we investigate the photodissociation of triiodide in four solvents using femtosecond time-resolved X-ray solution scattering following 400 nm photoexcitation. Structural analysis of the scattering data resolves the solvent-dependent structural evolution during the bond cleavage, internal rearrangements, solvent-cage escape, and bond reformation in real time. The nature and structure of the reaction intermediates during the recombination are determined, elucidating the full mechanism of photodissociation and recombination on ultrafast time scales. We resolve the structure of the precursor state for recombination as a geminate pair. Further, we determine the size of the solvent cages from the refined structures of the radical pair. The observed structural dynamics present a comprehensive picture of the solvent influence on structure and dynamics of dissociation reactions.

Equivalence of Charge Imbalance and External Electric Fields during Free Energy Calculations of Membrane Electroporation.

Electric fields across lipid membranes play important roles in physiology, medicine, and biotechnology, rationalizing the wide interest in modeling transmembrane potentials in molecular dynamics simulations. Transmembrane potentials have been implemented with external electric fields or by imposing charge imbalance between the two water compartments of a stacked double-membrane system. We compare the two methods in the context of membrane electroporation, which involves a large change of membrane structure and capacitance. We show that, given that Ewald electrostatics are defined with tinfoil boundary conditions, the two methods lead to (i) identical potentials of mean force (PMFs) of pore formation and expansion at various potentials, demonstrating that the two methods impose equivalent driving forces for large-scale transitions at membranes, and (ii) to identical polarization of water within thin water wires or open pores, suggesting that the two methods furthermore impose equivalent local electric fields. Without tinfoil boundary conditions, effects from external fields on pore formation are spuriously suppressed or even removed. Together, our study shows that both methods, external fields and charge imbalance, are well suitable for studying large-scale transitions of lipid membranes that involve changes of membrane capacitance. However, using charge imbalance is technically more challenging for maintaining a constant transmembrane potential since it requires updating of the charge imbalance as the membrane capacitance changes.

Continuous millisecond conformational cycle of a DEAH box helicase reveals control of domain motions by atomic-scale transitions.

Helicases are motor enzymes found in every living organism and viruses, where they maintain the stability of the genome and control against false recombination. The DEAH-box helicase Prp43 plays a crucial role in pre-mRNA splicing in unicellular organisms by translocating single-stranded RNA. The molecular mechanisms and conformational transitions of helicases are not understood at the atomic level. We present a complete conformational cycle of RNA translocation by Prp43 in atomic detail based on molecular dynamics simulations. To enable the sampling of such complex transition on the millisecond timescale, we combined two enhanced sampling techniques, namely simulated tempering and adaptive sampling guided by crystallographic data. During RNA translocation, the center-of-mass motions of the RecA-like domains followed the established inchworm model, whereas the domains crawled along the RNA in a caterpillar-like movement, suggesting an inchworm/caterpillar model. However, this crawling required a complex sequence of atomic-scale transitions involving the release of an arginine finger from the ATP pocket, stepping of the hook-loop and hook-turn motifs along the RNA backbone, and several others. These findings highlight that large-scale domain dynamics may be controlled by complex sequences of atomic-scale transitions.

Simulated Tempering-Enhanced Umbrella Sampling Improves Convergence of Free Energy Calculations of Drug Membrane Permeation.

Molecular dynamics simulations have been widely used to study solute permeation across biological membranes. The potential of mean force (PMF) for solute permeation is typically computed using enhanced sampling techniques such as umbrella sampling (US). For bulky drug-like permeants, however, obtaining converged PMFs remains challenging and often requires long simulation times, resulting in an unacceptable computational cost. Here, we augmented US with simulated tempering (ST), an extended-ensemble technique that consists in varying the temperature of the system along a pre-defined temperature ladder. Simulated tempering-enhanced US (STeUS) was employed to improve the convergence of PMF calculations for the permeation of methanol and three common drug molecules. To obtain sufficient sampling of the umbrella histograms, which were computed only from the ground temperature, we modified the simulation time fraction spent at the ground temperature between 1/K and 50%, where K is the number of ST temperature states. We found that STeUS accelerates convergence, when compared to standard US, and that the benefit of STeUS is system-dependent. For bulky molecules, for which standard US poorly converged, the application of ST was highly successful, leading to a more than fivefold accelerated convergence of the PMFs. For the small methanol solute, for which conventional US converges moderately, the application of ST is only beneficial if 50% of the STeUS simulation time is spent at the ground temperature. This study establishes STeUS as an efficient and simple method for PMF calculations, thereby strongly reducing the computational cost of routine high-throughput studies of drug permeability.

Upstream of N-Ras C-terminal cold shock domains mediate poly(A) specificity in a novel RNA recognition mode and bind poly(A) binding protein.

RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition of multiple RBDs remains largely unexplored. Here we use Upstream of N-Ras (Unr), a multidomain RBP, to demonstrate how multiple RBDs orchestrate target specificity. A crystal structure of the three C-terminal RNA binding cold-shock domains (CSD) of Unr bound to a poly(A) sequence exemplifies how recognition goes beyond the classical ππ-stacking in CSDs. Further structural studies reveal several interaction surfaces between the N-terminal and C-terminal part of Unr with the poly(A)-binding protein (pAbp). All interactions are validated by mutational analyses and the high-resolution structures presented here will guide further studies to understand how both proteins act together in cellular processes.

Structure and ensemble refinement against SAXS data: Combining MD simulations with Bayesian inference or with the maximum entropy principle.

Small-angle X-ray scattering (SAXS) is a powerful method for tracking conformational transitions of proteins or soft-matter complexes in solution. However, the interpretation of the experimental data is challenged by the low spatial resolution and the low information content of the data, which lead to a high risk of overinterpreting the data. Here, we illustrate how SAXS data can be integrated into all-atom molecular dynamics (MD) simulation to derive atomic structures or heterogeneous ensembles that are compatible with the data. Besides providing atomistic insight, the MD simulation adds physicochemical information, as encoded in the MD force fields, which greatly reduces the risk of overinterpretation. We present an introduction into the theory of SAXS-driven MD simulations as implemented in GROMACS-SWAXS, a modified version of the GROMACS simulation software. We discuss SAXS-driven parallel-replica ensemble refinement with commitment to the maximum entropy principle as well as a Bayesian formulation of SAXS-driven structure refinement. Practical considerations for running and interpreting the simulations are presented. The methods are freely available via GitLab at https://gitlab.com/cbjh/gromacs-swaxs.

Passive transport of Ca2+ ions through lipid bilayers imaged by widefield second harmonic microscopy.

In biology, release of Ca2+ ions in the cytosol is essential to trigger or control many cell functions. Calcium signaling acutely depends on lipid membrane permeability to Ca2+. For proper understanding of membrane permeability to Ca2+, both membrane hydration and the structure of the hydrophobic core must be taken into account. Here, we vary the hydrophobic core of bilayer membranes and observe different types of behavior in high-throughput wide-field second harmonic imaging. Ca2+ translocation is observed through mono-unsaturated (DOPC:DOPA) membranes, reduced upon the addition of cholesterol, and completely inhibited for branched (DPhPC:DPhPA) and poly-unsaturated (SLPC:SLPA) lipid membranes. We propose, using molecular dynamics simulations, that ion transport occurs through ion-induced transient pores, which requires nonequilibrium membrane restructuring. This results in different rates at different locations and suggests that the hydrophobic structure of lipids plays a much more sophisticated regulating role than previously thought.

Ion-Induced Transient Potential Fluctuations Facilitate Pore Formation and Cation Transport through Lipid Membranes.

Unassisted ion transport through lipid membranes plays a crucial role in many cell functions without which life would not be possible, yet the precise mechanism behind the process remains unknown due to its molecular complexity. Here, we demonstrate a direct link between membrane potential fluctuations and divalent ion transport. High-throughput wide-field non-resonant second harmonic (SH) microscopy of membrane water shows that membrane potential fluctuations are universally found in lipid bilayer systems. Molecular dynamics simulations reveal that such variations in membrane potential reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These transient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu2+, Ca2+, Ba2+, Mg2+) passing through giant unilamellar vesicle (GUV) membranes. Combining the experimental and computational results, we show that permeation through pores formed via an ion-induced electrostatic field is a viable mechanism for unassisted ion transport.