OLIVES: A GoÌ -like Model for Stabilizing Protein Structure via Hydrogen Bonding Native Contacts in the Martini 3 Coarse-Grained Force Field.

Coarse-grained molecular dynamics simulations enable the modeling of increasingly complex systems at millisecond timescales. The transferable coarse-grained force field Martini 3 has shown great promise in modeling a wide range of biochemical processes, yet folded proteins in Martini 3 are not stable without the application of external bias potentials, such as elastic networks or Go̅-like models. We herein develop an algorithm, called OLIVES, which identifies native contacts with hydrogen bond capabilities in coarse-grained proteins and use it to implement a novel Go̅-like model for Martini 3. We show that the protein structure instability originates in part from the lack of hydrogen bond energy in the coarse-grained force field representation. By using realistic hydrogen bond energies obtained from literature ab initio calculations, it is demonstrated that protein stability can be recovered by the reintroduction of a coarse-grained hydrogen bond network and that OLIVES removes the need for secondary structure restraints. OLIVES is validated against known protein complexes and at the same time addresses the open question of whether there is a need for protein quaternary structure bias in Martini 3 simulations. It is shown that OLIVES can reduce the number of bias terms, hereby speeding up Martini 3 simulations of proteins by up to ≈30% on a GPU architecture compared to the established Go̅MARTINI Go̅-like model.

Structural basis of the obligatory exchange mode of human neutral amino acid transporter ASCT2.

ASCT2 is an obligate exchanger of neutral amino acids, contributing to cellular amino acid homeostasis. ASCT2 belongs to the same family (SLC1) as Excitatory Amino Acid Transporters (EAATs) that concentrate glutamate in the cytosol. The mechanism that makes ASCT2 an exchanger rather than a concentrator remains enigmatic. Here, we employ cryo-electron microscopy and molecular dynamics simulations to elucidate the structural basis of the exchange mechanism of ASCT2. We establish that ASCT2 binds three Na+ ions per transported substrate and visits a state that likely acts as checkpoint in preventing Na+ ion leakage, both features shared with EAATs. However, in contrast to EAATs, ASCT2 retains one Na+ ion even under Na+-depleted conditions. We demonstrate that ASCT2 cannot undergo the structural transition in TM7 that is essential for the concentrative transport cycle of EAATs. This structural rigidity and the high-affinity Na+ binding site effectively confine ASCT2 to an exchange mode.

Exploring the Ligand Binding and Conformational Dynamics of the Substrate-Binding Domain 1 of the ABC Transporter GlnPQ.

The adenosine triphosphate (ATP)-binding cassette (ABC) importer GlnPQ from Lactococcus lactis has two sequential covalently linked substrate-binding domains (SBDs), which capture the substrates and deliver them to the translocon. The two SBDs differ in their ligand specificities, binding affinities and the distance to the transmembrane domain; interestingly, both SBDs can bind their ligands simultaneously without affecting each other. In this work, we studied the binding of ligands to both SBDs using X-ray crystallography and molecular dynamics simulations. We report three high-resolution structures of SBD1, namely, the wild-type SBD1 with bound asparagine or arginine, and E184D SBD1 with glutamine bound. Molecular dynamics (MD) simulations provide a detailed insight into the dynamics associated with open-closed transitions of the SBDs.

Capturing chemical reactions inside biomolecular condensates with reactive Martini simulations.

Biomolecular condensates are phase separated systems that play an important role in the spatio-temporal organisation of cells. Their distinct physico-chemical nature offers a unique environment for chemical reactions to occur. The compartmentalisation of chemical reactions is also believed to be central to the development of early life. To demonstrate how molecular dynamics may be used to capture chemical reactions in condensates, here we perform reactive molecular dynamics simulations using the coarse-grained Martini forcefield. We focus on the formation of rings of benzene-1,3-dithiol inside a synthetic peptide-based condensate, and find that the ring size distribution shifts to larger macrocycles compared to when the reaction takes place in an aqueous environment. Moreover, reaction rates are noticeably increased when the peptides simultaneously undergo phase separation, hinting that condensates may act as chaperones in recruiting molecules to reaction hubs.

Building complex membranes with Martini 3.

The Martini model is a popular force field for coarse-grained simulations. Membranes have always been at the center of its development, with the latest version, Martini 3, showing great promise in capturing more and more realistic behavior. In this chapter we provide a step-by-step tutorial on how to construct starting configurations, run initial simulations and perform dedicated analysis for membrane-based systems of increasing complexity, including leaflet asymmetry, curvature gradients and embedding of membrane proteins.

There and back again: bridging meso- and nano-scales to understand lipid vesicle patterning.

We describe a complete methodology to bridge the scales between nanoscale molecular dynamics and (micrometer) mesoscale Monte Carlo simulations in lipid membranes and vesicles undergoing phase separation, in which curving molecular species are furthermore embedded. To go from the molecular to the mesoscale, we notably appeal to physical renormalization arguments enabling us to rigorously infer the mesoscale interaction parameters from its molecular counterpart. We also explain how to deal with the physical timescales at stake at the mesoscale. Simulating the as-obtained mesoscale system enables us to equilibrate the long wavelengths of the vesicles of interest, up to the vesicle size. Conversely, we then backmap from the meso- to the nano-scale, which enables us to equilibrate in turn the short wavelengths down to the molecular length-scales. By applying our approach to the specific situation of patterning a vesicle membrane, we show that macroscopic membranes can thus be equilibrated at all length-scales in achievable computational time offering an original strategy to address the fundamental challenge of timescale in simulations of large bio-membrane systems.

Bartender: Martini 3 Bonded Terms via Quantum Mechanics-Based Molecular Dynamics.

Coarse-grained (CG) molecular dynamics (MD) simulations have grown in applicability over the years. The recently released version of the Martini CG force field (Martini 3) has been successfully applied to simulate many processes, including protein-ligand binding. However, the current ligand parametrization scheme is manual and requires an a priori reference all-atom (AA) simulation for benchmarking. For systems with suboptimal AA parameters, which are often unknown, this translates into a CG model that does not reproduce the true dynamical behavior of the underlying molecule. Here, we present Bartender, a quantum mechanics (QM)/MD-based parametrization tool written in Go. Bartender harnesses the power of QM simulations and produces reasonable bonded terms for Martini 3 CG models of small molecules in an efficient and user-friendly manner. For small, ring-like molecules, Bartender generates models whose properties are indistinguishable from the human-made models. For more complex, drug-like ligands, it is able to fit functional forms beyond simple harmonic dihedrals and thus better captures their dynamical behavior. Bartender has the power to both increase the efficiency and the accuracy of Martini 3-based high-throughput applications by producing numerically stable and physically realistic CG models.

Modeling membranes in situ.

Molecular dynamics simulations of cellular membranes have come a long way-from simple model lipid bilayers to multicomponent systems capturing the crowded and complex nature of real cell membranes. In this opinionated minireview, we discuss the current challenge to simulate the dynamics of membranes in their native environment, in situ, with the prospect of reaching the level of whole cells and cell organelles using an integrative modeling framework.

Probing the stability and interdomain interactions in the ABC transporter OpuA using single-molecule optical tweezers.

Transmembrane transporter proteins are essential for maintaining cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the wealth of detailed structural and functional data available for these systems, our understanding of their dynamics and, consequently, how they function is generally limited. We introduce an innovative approach that enables us to directly measure the dynamics and stability of interdomain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally relevant motions and interdomain interactions of individual transmembrane transporter proteins in real time in a lipid bilayer.

Vesicle protrusion induced by antimicrobial peptides suggests common carpet mechanism for short antimicrobial peptides.

Short-cationic alpha-helical antimicrobial peptides (SCHAMPs) are promising candidates to combat the growing global threat of antimicrobial resistance. They are short-sequenced, selective against bacteria, and have rapid action by destroying membranes. A full understanding of their mechanism of action will provide key information to design more potent and selective SCHAMPs. Molecular Dynamics (MD) simulations are invaluable tools that provide detailed insights into the peptide-membrane interaction at the atomic- and meso-scale level. We use atomistic and coarse-grained MD to look into the exact steps that four promising SCHAMPs-BP100, Decoralin, Neurokinin-1, and Temporin L-take when they interact with membranes. Following experimental set-ups, we explored the effects of SCHAMPs on anionic membranes and vesicles at multiple peptide concentrations. Our results showed all four peptides shared similar binding steps, initially binding to the membrane through electrostatic interactions and then flipping on their axes, dehydrating, and inserting their hydrophobic moieties into the membrane core. At higher concentrations, fully alpha-helical peptides induced membrane budding and protrusions. Our results suggest the carpet mode of action is fit for the description of SCHAMPs lysis activity and discuss the importance of large hydrophobic residues in SCHAMPs design and activity.

Shocker─A Molecular Dynamics Protocol and Tool for Accelerating and Analyzing the Effects of Osmotic Shocks.

The process of osmosis, a fundamental phenomenon in life, drives water through a semipermeable membrane in response to a solute concentration gradient across this membrane. In vitro, osmotic shocks are often used to drive shape changes in lipid vesicles, for instance, to study fission events in the context of artificial cells. While experimental techniques provide a macroscopic picture of large-scale membrane remodeling processes, molecular dynamics (MD) simulations are a powerful tool to study membrane deformations at the molecular level. However, simulating an osmotic shock is a time-consuming process due to slow water diffusion across the membrane, making it practically impossible to examine its effects in classic MD simulations. In this article, we present Shocker, a Python-based MD tool for simulating the effects of an osmotic shock by selecting and relocating water particles across a membrane over the course of several pumping cycles. Although this method is primarily aimed at efficiently simulating volume changes in vesicles, it can also handle membrane tubes and double bilayer systems. Additionally, Shocker is force field-independent and compatible with both coarse-grained and all-atom systems. We demonstrate that our tool is applicable to simulate both hypertonic and hypotonic osmotic shocks for a range of vesicular and bilamellar setups, including complex multicomponent systems containing membrane proteins or crowded internal solutions.

Identification of inhibitors targeting the energy-coupling factor (ECF) transporters.

The energy-coupling factor (ECF) transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. The central role of vitamin transport in the metabolism of bacteria and absence from humans make the ECF transporters an attractive target for inhibition with selective chemical probes. Here, we report on the identification of a promising class of inhibitors of the ECF transporters. We used coarse-grained molecular dynamics simulations on Lactobacillus delbrueckii ECF-FolT2 and ECF-PanT to profile the binding mode and mechanism of inhibition of this novel chemotype. The results corroborate the postulated mechanism of transport and pave the way for further drug-discovery efforts.

CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs.

For the successful generative engineering of functional artificial cells, a convenient and controllable means of delivering membrane proteins into membrane lipid bilayers is necessary. Here we report a delivery system that achieves this by employing membrane protein-carrying nanodiscs and the calcium-dependent fusion of phosphatidylserine lipid membranes. We show that lipid nanodiscs can fuse a transported lipid bilayer with the lipid bilayers of small unilamellar vesicles (SUVs) or giant unilamellar vesicles (GUVs) while avoiding recipient vesicles aggregation. This is triggered by a simple, transient increase in calcium concentration, which results in efficient and rapid fusion in a one-pot reaction. Furthermore, nanodiscs can be loaded with membrane proteins that can be delivered into target SUV or GUV membranes in a detergent-independent fashion while retaining their functionality. Nanodiscs have a proven ability to carry a wide range of membrane proteins, control their oligomeric state, and are highly adaptable. Given this, our approach may be the basis for the development of useful tools that will allow bespoke delivery of membrane proteins to protocells, equipping them with the cell-like ability to exchange material across outer/subcellular membranes.

Pragmatic Coarse-Graining of Proteins: Models and Applications.

The molecular details involved in the folding, dynamics, organization, and interaction of proteins with other molecules are often difficult to assess by experimental techniques. Consequently, computational models play an ever-increasing role in the field. However, biological processes involving large-scale protein assemblies or long time scale dynamics are still computationally expensive to study in atomistic detail. For these applications, employing coarse-grained (CG) modeling approaches has become a key strategy. In this Review, we provide an overview of what we call pragmatic CG protein models, which are strategies combining, at least in part, a physics-based implementation and a top-down experimental approach to their parametrization. In particular, we focus on CG models in which most protein residues are represented by at least two beads, allowing these models to retain some degree of chemical specificity. A description of the main modern pragmatic protein CG models is provided, including a review of the most recent applications and an outlook on future perspectives in the field.

Watching Molecular Nanotubes Self-Assemble in Real Time.

Molecular self-assembly is a fundamental process in nature that can be used to develop novel functional materials for medical and engineering applications. However, their complex mechanisms make the short-lived stages of self-assembly processes extremely hard to reveal. In this article, we track the self-assembly process of a benchmark system, double-walled molecular nanotubes, whose structure is similar to that found in biological and synthetic systems. We selectively dissolved the outer wall of the double-walled system and used the inner wall as a template for the self-reassembly of the outer wall. The reassembly kinetics were followed in real time using a combination of microfluidics, spectroscopy, cryogenic transmission electron microscopy, molecular dynamics simulations, and exciton modeling. We found that the outer wall self-assembles through a transient disordered patchwork structure: first, several patches of different orientations are formed, and only on a longer time scale will the patches interact with each other and assume their final preferred global orientation. The understanding of patch formation and patch reorientation marks a crucial step toward steering self-assembly processes and subsequent material engineering.

Martini 3 Coarse-Grained Force Field for Cholesterol.

Cholesterol plays a crucial role in biomembranes by regulating various properties, such as fluidity, rigidity, permeability, and organization of lipid bilayers. The latest version of the Martini model, Martini 3, offers significant improvements in interaction balance, molecular packing, and inclusion of new bead types and sizes. However, the release of the new model resulted in the need to reparameterize many core molecules, including cholesterol. Here, we describe the development and validation of a Martini 3 cholesterol model, addressing issues related to its bonded setup, shape, volume, and hydrophobicity. The proposed model mitigates some limitations of its Martini 2 predecessor while maintaining or improving the overall behavior.

Multiscale simulations reveal TDP-43 molecular-level interactions driving condensation.

The RNA-binding protein TDP-43 is associated with mRNA processing and transport from the nucleus to the cytoplasm. TDP-43 localizes in the nucleus as well as accumulating in cytoplasmic condensates such as stress granules. Aggregation and formation of amyloid-like fibrils of cytoplasmic TDP-43 are hallmarks of numerous neurodegenerative diseases, most strikingly present in >90% of amyotrophic lateral sclerosis (ALS) patients. If excessive accumulation of cytoplasmic TDP-43 causes, or is caused by, neurodegeneration is presently not known. In this work, we use molecular dynamics simulations at multiple resolutions to explore TDP-43 self- and cross-interaction dynamics. A full-length molecular model of TDP-43, all 414 amino acids, was constructed from select structures of the protein functional domains (N-terminal domain, and two RNA recognition motifs, RRM1 and RRM2) and modeling of disordered connecting loops and the low complexity glycine-rich C-terminus domain. All-atom CHARMM36m simulations of single TDP-43 proteins served as guides to construct a coarse-grained Martini 3 model of TDP-43. The Martini model and a coarser implicit solvent C⍺ model, optimized for disordered proteins, were subsequently used to probe TDP-43 interactions; self-interactions from single-chain full-length TDP-43 simulations, cross-interactions from simulations with two proteins and simulations with assemblies of dozens to hundreds of proteins. Our findings illustrate the utility of different modeling scales for accessing TDP-43 molecular-level interactions and suggest that TDP-43 has numerous interaction preferences or patterns, exhibiting an overall strong, but dynamic, association and driving the formation of biomolecular condensates.

Membrane manipulation by free fatty acids improves microbial plant polyphenol synthesis.

Microbial synthesis of nutraceutically and pharmaceutically interesting plant polyphenols represents a more environmentally friendly alternative to chemical synthesis or plant extraction. However, most polyphenols are cytotoxic for microorganisms as they are believed to negatively affect cell integrity and transport processes. To increase the production performance of engineered cell factories, strategies have to be developed to mitigate these detrimental effects. Here, we examine the accumulation of the stilbenoid resveratrol in the cell membrane and cell wall during its production using Corynebacterium glutamicum and uncover the membrane rigidifying effect of this stilbenoid experimentally and with molecular dynamics simulations. A screen of free fatty acid supplements identifies palmitelaidic acid and linoleic acid as suitable additives to attenuate resveratrol's cytotoxic effects resulting in a three-fold higher product titer. This cost-effective approach to counteract membrane-damaging effects of product accumulation is transferable to the microbial production of other polyphenols and may represent an engineering target for other membrane-active bioproducts.

Seeing the Invisibles: Detection of Peptide Enantiomers, Diastereomers, and Isobaric Ring Formation in Lanthipeptides Using Nanopores.

Mass spectrometry (MS) is widely used in proteomic analysis but cannot differentiate between molecules with the same mass-to-charge ratio. Nanopore technology might provide an alternative method for the rapid and cost-effective analysis and sequencing of proteins. In this study, we demonstrate that nanopore currents can distinguish between diastereomeric and enantiomeric differences in l- and d-peptides, not observed by conventional MS analysis, down to individual d-amino acids in small opioid peptides. Molecular dynamics simulations suggest that similar to chiral chromatography the resolution likely arises from multiple chiral interactions during peptide transport across the nanopore. Additionally, we used nanopore recordings to rapidly assess 4- and 11-amino acid ring formation in lanthipeptides, a process used in the synthesis of pharmaceutical peptides. The cyclization step requires distinguishing between constitutional isomers, which have identical MS signals and typically involve numerous tedious experiments to confirm. Hence, nanopore technology offers new possibilities for the rapid and cost-effective analysis of peptides, including those that cannot be easily differentiated by mass spectrometry.