Computational microbiology of bacteria: Advancements in molecular dynamics simulations.

Microbiology is traditionally considered within the context of wet laboratory methodologies. Computational techniques have a great potential to contribute to microbiology. Here, we describe our loose definition of "computational microbiology" and provide a short survey focused on molecular dynamics simulations of bacterial systems that fall within this definition. It is our contention that increased compositional complexity and realistic levels of molecular crowding within simulated systems are key for bridging the divide between experimental and computational microbiology.

Role of lipid nanodomains for inhibitory FcγRIIb function.

The inhibitory Fcγ receptor FcγRIIb is involved in immune regulation and is known to localize to specific regions of the plasma membrane called lipid rafts. Previous studies suggested a link between the altered lateral receptor localization within the plasma membrane and the functional impairment of the FcγRIIb-I232T variant that is associated with systemic lupus erythematosus. Here, we conducted microsecond all-atom molecular dynamics simulations and IgG binding assays to investigate the lipid nano-environment of FcγRIIb monomers and of the FcγRIIb-I232T mutant within a plasma membrane model, the orientation of the FcγRIIb ectodomain, and its accessibility to IgG ligands. In contrast to previously proposed models, our simulations indicated that FcγRIIb does not favor a cholesterol- or a sphingolipid-enriched lipid environment. Interestingly, cholesterol was depleted for all studied FcγRIIb variants within a 2-3 nm environment of the receptor, counteracting the usage of raft terminology for models on receptor functionality. Instead, the receptor interacts with lipids that have poly-unsaturated fatty acyl chains and with (poly-) anionic lipids within the cytosolic membrane leaflet. We also found that FcγRIIb monomers adopt a conformation that is not suitable for binding to its IgG ligand, consistent with a lack of detectable binding of monomeric IgG in experiments on primary immune cells. However, our results propose that multivalent IgG complexes might stabilize FcγRIIb in a binding-competent conformation. We suggest differences in receptor complex formation within the membrane as a plausible cause of the altered membrane localization or clustering and the altered suppressive function of the FcγRIIb-I232T variant.

IDH3γ functions as a redox switch regulating mitochondrial energy metabolism and contractility in the heart.

Redox signaling and cardiac function are tightly linked. However, it is largely unknown which protein targets are affected by hydrogen peroxide (H2O2) in cardiomyocytes that underly impaired inotropic effects during oxidative stress. Here, we combine a chemogenetic mouse model (HyPer-DAO mice) and a redox-proteomics approach to identify redox sensitive proteins. Using the HyPer-DAO mice, we demonstrate that increased endogenous production of H2O2 in cardiomyocytes leads to a reversible impairment of cardiac contractility in vivo. Notably, we identify the γ-subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch, linking its modification to altered mitochondrial metabolism. Using microsecond molecular dynamics simulations and experiments using cysteine-gene-edited cells reveal that IDH3γ Cys148 and 284 are critically involved in the H2O2-dependent regulation of IDH3 activity. Our findings provide an unexpected mechanism by which mitochondrial metabolism can be modulated through redox signaling processes.

Structural basis for host recognition and superinfection exclusion by bacteriophage T5.

A key but poorly understood stage of the bacteriophage life cycle is the binding of phage receptor-binding proteins (RBPs) to receptors on the host cell surface, leading to injection of the phage genome and, for lytic phages, host cell lysis. To prevent secondary infection by the same or a closely related phage and nonproductive phage adsorption to lysed cell fragments, superinfection exclusion (SE) proteins can prevent the binding of RBPs via modulation of the host receptor structure in ways that are also unclear. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the phage T5 outer membrane (OM) receptor FhuA in complex with the T5 RBP pb5, and the crystal structure of FhuA complexed to the OM SE lipoprotein Llp. Pb5 inserts four loops deeply into the extracellular lumen of FhuA and contacts the plug but does not cause any conformational changes in the receptor, supporting the view that DNA translocation does not occur through the lumen of OM channels. The FhuA-Llp structure reveals that Llp is periplasmic and binds to a nonnative conformation of the plug of FhuA, causing the inward folding of two extracellular loops via "reverse" allostery. The inward-folded loops of FhuA overlap with the pb5 binding site, explaining how Llp binding to FhuA abolishes further infection of Escherichia coli by phage T5 and suggesting a mechanism for SE via the jamming of TonB-dependent transporters by small phage lipoproteins.

Spontaneous Membrane Nanodomain Formation in the Absence or Presence of the Neurotransmitter Serotonin.

Detailed knowledge on the formation of biomembrane domains, their structure, composition, and physical characteristics is scarce. Despite its frequently discussed importance in signaling, e.g., in obtaining localized non-homogeneous receptor compositions in the plasma membrane, the nanometer size as well as the dynamic and transient nature of domains impede their experimental characterization. In turn, atomistic molecular dynamics (MD) simulations combine both, high spatial and high temporal resolution. Here, using microsecond atomistic MD simulations, we characterize the spontaneous and unbiased formation of nano-domains in a plasma membrane model containing phosphatidylcholine (POPC), palmitoyl-sphingomyelin (PSM), and cholesterol (Chol) in the presence or absence of the neurotransmitter serotonin at different temperatures. In the ternary mixture, highly ordered and highly disordered domains of similar composition coexist at 303 K. The distinction of domains by lipid acyl chain order gets lost at lower temperatures of 298 and 294 K, suggesting a phase transition at ambient temperature. By comparison of domain ordering and composition, we demonstrate how the domain-specific binding of the neurotransmitter serotonin results in a modified domain lipid composition and a substantial downward shift of the phase transition temperature. Our simulations thus suggest a novel mode of action of neurotransmitters possibly of importance in neuronal signal transmission.

Serotonin Alters the Phase Equilibrium of a Ternary Mixture of Phospholipids and Cholesterol.

Unsaturated and saturated phospholipids tend to laterally segregate, especially in the presence of cholesterol. Small molecules such as neurotransmitters, toxins, drugs etc. possibly modulate this lateral segregation. The small aromatic neurotransmitter serotonin (5-HT) has been found to bind to membranes. We studied the lipid structure and packing of a ternary membrane mixture consisting of palmitoyl-oleoyl-phosphatidylcholine, palmitoyl-sphingomyelin, and cholesterol at a molar ratio of 4/4/2 in the absence and in the presence of 5-HT, using a combination of solid-state 2H NMR, atomic force microscopy, and atomistic molecular dynamics (MD) simulations. Both NMR and MD report formation of a liquid ordered (L o ) and a liquid disordered (L d ) phase coexistence with small domains. Lipid exchange between the domains was fast such that single component 2H NMR spectra are detected over a wide temperature range. A drastic restructuring of the domains was induced when 5-HT is added to the membranes at a 9 mol% concentration relative to the lipids. 2H NMR spectra of all components of the mixture showed two prominent contributions indicative of molecules of the same kind residing both in the disordered and the ordered phase. Compared to the data in the absence of 5-HT, the lipid chain order in the disordered phase was further decreased in the presence of 5-HT. Likewise, addition of serotonin increased lipid chain order within the ordered phase. These characteristic lipid chain order changes were confirmed by MD simulations. The 5-HT-induced larger difference in lipid chain order results in more pronounced differences in the hydrophobic thickness of the individual membrane domains. The correspondingly enlarged hydrophobic mismatch between ordered and disordered phases is assumed to increase the line tension at the domain boundary, which drives the system into formation of larger size domains. These results not only demonstrate that small membrane binding molecules such as neurotransmitters have a profound impact on essential membrane properties. It also suggests a mechanism by which the interaction of small molecules with membranes can influence the function of membrane proteins and non-cognate receptors. Altered membrane properties may modify lateral sorting of membrane protein, membrane protein conformation, and thus influence their function as suspected for neurotransmitters, local anesthetics, and other small drug molecules.

Modelling lipid systems in fluid with Lattice Boltzmann Molecular Dynamics simulations and hydrodynamics.

In this work we present the coupling between Dry Martini, an efficient implicit solvent coarse-grained model for lipids, and the Lattice Boltzmann Molecular Dynamics (LBMD) simulation technique in order to include naturally hydrodynamic interactions in implicit solvent simulations of lipid systems. After validating the implementation of the model, we explored several systems where the action of a perturbing fluid plays an important role. Namely, we investigated the role of an external shear flow on the dynamics of a vesicle, the dynamics of substrate release under shear, and inquired the dynamics of proteins and substrates confined inside the core of a vesicle. Our methodology enables future exploration of a large variety of biological entities and processes involving lipid systems at the mesoscopic scale where hydrodynamics plays an essential role, e.g. by modulating the migration of proteins in the proximity of membranes, the dynamics of vesicle-based drug delivery systems, or, more generally, the behaviour of proteins in cellular compartments.

Structural dataset from microsecond-long simulations of yeast mitofusin Fzo1 in the context of membrane docking.

In this work we present a novel set of possible auto-oligomerisation states of yeast protein Fzo1 in the context of membrane docking. The dataset reports atomistic models and trajectories derived from a molecular dynamics study of the yeast mitofusin Fzo1, residues 101-855. The initial modelling was followed by coarse-grained molecular dynamics simulation to evaluate the stability and the dynamics of each structural model in a solvated membrane environment. Simulations were run for 1 µs and collected with GROMACS v5.0.4 using the martini v2.1 force field. For each structural model, the dataset comprises the production phase under semi-isotropic condition at 1 bar, 310 K and 150 mn NaCl. The integration step is 20 fs and coordinates have been saved every 1 ns. Each trajectory is associated with a ready-available visualization state for the VMD software. These structural detailed informations are a ready-available platform to plan integrative studies on the mitofusin Fzo1 and will aid the community to further elucidate the mitochondrial tethering process during membrane fusion. This dataset is based on the publication "Physics-based oligomeric models of the yeast mitofusin Fzo1 at the molecular scale in the context of membrane docking." (Brandner and De Vecchis et al., 2019)".

A Molecular Perspective on Mitochondrial Membrane Fusion: From the Key Players to Oligomerization and Tethering of Mitofusin.

Mitochondria are dynamic organelles characterized by an ultrastructural organization which is essential in maintaining their quality control and ensuring functional efficiency. The complex mitochondrial network is the result of the two ongoing forces of fusion and fission of inner and outer membranes. Understanding the functional details of mitochondrial dynamics is physiologically relevant as perturbations of this delicate equilibrium have critical consequences and involved in several neurological disorders. Molecular actors involved in this process are large GTPases from the dynamin-related protein family. They catalyze nucleotide-dependent membrane remodeling and are widely conserved from bacteria to higher eukaryotes. Although structural characterization of different family members has contributed in understanding molecular mechanisms of mitochondrial dynamics in more detail, the complete structure of some members as well as the precise assembly of functional oligomers remains largely unknown. As increasing structural data become available, the domain modularity across the dynamin superfamily emerged as a foundation for transfering the knowledge towards less characterized members. In this review, we will first provide an overview of the main actors involved in mitochondrial dynamics. We then discuss recent example of computational methodologies for the study of mitofusin oligomers, and present how the usage of integrative modeling in conjunction with biochemical data can be an asset in progressing the still challenging field of membrane dynamics.

Physics-based oligomeric models of the yeast mitofusin Fzo1 at the molecular scale in the context of membrane docking.

Tethering and homotypic fusion of mitochondrial outer membranes is mediated by large GTPases of the dynamin-related proteins family called the mitofusins. The yeast mitofusin Fzo1 forms high molecular weight complexes and its assembly during membrane fusion likely involves the formation of high order complexes. Consistent with this possibility, mitofusins form oligomers in both cis (on the same lipid bilayer) and trans to mediate membrane attachment and fusion. Here, we utilize our recent Fzo1 model to investigate and discuss the formation of cis and trans mitofusin oligomers. We have built three distinct cis-assembly Fzo1 models that gave rise to three distinct trans-oligomeric models of mitofusin constructs. Each model involves two main components of mitofusin oligomerization: the GTPase and the trunk domains. The oligomeric models proposed in this study were further assessed for stability and dynamics in a membrane environment using a coarse-grained molecular dynamics (MD) simulation approach. A narrow opening 'head-to-head' cis-oligomerization (via the GTPase domain) followed by the antiparallel 'back-to-back' trans-associations (via the trunk domain) appears to be in agreement with all of the available experimental data. More broadly, this study opens new possibilities to start exploring cis and trans conformations for Fzo1 and mitofusins in general.

Exploring DNA dynamics within oligonucleosomes with coarse-grained simulations: SIRAH force field extension for protein-DNA complexes.

Describing the regulation of chromatin segments by protein recognition events constitute a major goal in biology and biotechnology. Despite astonishing experimental developments, achieving nearly atomistic spatial/temporal resolution on such macromolecular systems remains a big challenge owing to the intrinsic flexibility of large biological assemblies. Although computer simulations have become a reliable complement to experimental techniques, computational cost limits their routine applications to relatively small systems. However, the development of accurate and cost-effective coarse-grained (CG) models helps to bridge the gap between molecular dynamics simulations and biologically relevant scales. Performing an exhaustive search on a set of well-resolved crystallographic protein-DNA complexes, we introduced improvements on the CG SIRAH force field to describe protein-DNA interfaces. Modifications were validated against a set of non redundant structures and applied to the simulation of the longest DNA segment in complex with proteins that has been crystallized to date, i.e. a tetranucleosome. Multimicrosecond simulation of this small chromatin segment evidences a large mobility of the external DNA filaments, which is consistent with results from FRET experiments in solution. Moreover, we found that the sub-microsecond dynamics of DNA is strongly modulated by the quaternary structure, partially overcoming the intrinsic dynamics dictated by the primary structure.

SIRAH: a structurally unbiased coarse-grained force field for proteins with aqueous solvation and long-range electrostatics.

Modeling of macromolecular structures and interactions represents an important challenge for computational biology, involving different time and length scales. However, this task can be facilitated through the use of coarse-grained (CG) models, which reduce the number of degrees of freedom and allow efficient exploration of complex conformational spaces. This article presents a new CG protein model named SIRAH, developed to work with explicit solvent and to capture sequence, temperature, and ionic strength effects in a topologically unbiased manner. SIRAH is implemented in GROMACS, and interactions are calculated using a standard pairwise Hamiltonian for classical molecular dynamics simulations. We present a set of simulations that test the capability of SIRAH to produce a qualitatively correct solvation on different amino acids, hydrophilic/hydrophobic interactions, and long-range electrostatic recognition leading to spontaneous association of unstructured peptides and stable structures of single polypeptides and protein-protein complexes.