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.

Staphylococcus aureus functional amyloids catalyze degradation of ß-lactam antibiotics.

Antibiotic resistance of bacteria is considered one of the most alarming developments in modern medicine. While varied pathways for bacteria acquiring antibiotic resistance have been identified, there still are open questions concerning the mechanisms underlying resistance. Here, we show that alpha phenol-soluble modulins (PSMαs), functional bacterial amyloids secreted by Staphylococcus aureus, catalyze hydrolysis of ß-lactams, a prominent class of antibiotic compounds. Specifically, we show that PSMα2 and, particularly, PSMα3 catalyze hydrolysis of the amide-like bond of the four membered ß-lactam ring of nitrocefin, an antibiotic ß-lactam surrogate. Examination of the catalytic activities of several PSMα3 variants allowed mapping of the active sites on the amyloid fibrils' surface, specifically underscoring the key roles of the cross-α fibril organization, and the combined electrostatic and nucleophilic functions of the lysine arrays. Molecular dynamics simulations further illuminate the structural features of ß-lactam association upon the fibril surface. Complementary experimental data underscore the generality of the functional amyloid-mediated catalytic phenomenon, demonstrating hydrolysis of clinically employed ß-lactams by PSMα3 fibrils, and illustrating antibiotic degradation in actual S. aureus biofilms and live bacteria environments. Overall, this study unveils functional amyloids as catalytic agents inducing degradation of ß-lactam antibiotics, underlying possible antibiotic resistance mechanisms associated with bacterial biofilms.

Elevated concentrations cause upright alpha-synuclein conformation at lipid interfaces.

The amyloid aggregation of α-synuclein (αS), related to Parkinson's disease, can be catalyzed by lipid membranes. Despite the importance of lipid surfaces, the 3D-structure and orientation of lipid-bound αS is still not known in detail. Here, we report interface-specific vibrational sum-frequency generation (VSFG) experiments that reveal how monomeric αS binds to an anionic lipid interface over a large range of αS-lipid ratios. To interpret the experimental data, we present a frame-selection method ("ViscaSelect") in which out-of-equilibrium molecular dynamics simulations are used to generate structural hypotheses that are compared to experimental amide-I spectra via excitonic spectral calculations. At low and physiological αS concentrations, we derive flat-lying helical structures as previously reported. However, at elevated and potentially disease-related concentrations, a transition to interface-protruding αS structures occurs. Such an upright conformation promotes lateral interactions between αS monomers and may explain how lipid membranes catalyze the formation of αS amyloids at elevated protein concentrations.

Automatic Optimization of Lipid Models in the Martini Force Field Using SwarmCG.

After two decades of continued development of the Martini coarse-grained force field (CG FF), further refinment of the already rather accurate Martini lipid models has become a demanding task that could benefit from integrative data-driven methods. Automatic approaches are increasingly used in the development of accurate molecular models, but they typically make use of specifically designed interaction potentials that transfer poorly to molecular systems or conditions different than those used for model calibration. As a proof of concept, here, we employ SwarmCG, an automatic multiobjective optimization approach facilitating the development of lipid force fields, to refine specifically the bonded interaction parameters in building blocks of lipid models within the framework of the general Martini CG FF. As targets of the optimization procedure, we employ both experimental observables (top-down references: area per lipid and bilayer thickness) and all-atom molecular dynamics simulations (bottom-up reference), which respectively inform on the supra-molecular structure of the lipid bilayer systems and on their submolecular dynamics. In our training sets, we simulate at different temperatures in the liquid and gel phases up to 11 homogeneous lamellar bilayers composed of phosphatidylcholine lipids spanning various tail lengths and degrees of (un)saturation. We explore different CG representations of the molecules and evaluate improvements a posteriori using additional simulation temperatures and a portion of the phase diagram of a DOPC/DPPC mixture. Successfully optimizing up to ∼80 model parameters within still limited computational budgets, we show that this protocol allows the obtainment of improved transferable Martini lipid models. In particular, the results of this study demonstrate how a fine-tuning of the representation and parameters of the models may improve their accuracy and how automatic approaches, such as SwarmCG, may be very useful to this end.

Predicting molecular properties of α-synuclein using force fields for intrinsically disordered proteins.

Independent force field validation is an essential practice to keep track of developments and for performing meaningful Molecular Dynamics simulations. In this work, atomistic force fields for intrinsically disordered proteins (IDP) are tested by simulating the archetypical IDP α-synuclein in solution for 2.5 µs. Four combinations of protein and water force fields were tested: ff19SB/OPC, ff19SB/TIP4P-D, ff03CMAP/TIP4P-D, and a99SB-disp/TIP4P-disp, with four independent repeat simulations for each combination. We compare our simulations to the results of a 73 µs simulation using the a99SB-disp/TIP4P-disp combination, provided by D. E. Shaw Research. From the trajectories, we predict a range of experimental observations of α-synuclein and compare them to literature data. This includes protein radius of gyration and hydration, intramolecular distances, NMR chemical shifts, and 3 J-couplings. Both ff19SB/TIP4P-D and a99SB-disp/TIP4P-disp produce extended conformational ensembles of α-synuclein that agree well with experimental radius of gyration and intramolecular distances while a99SB-disp/TIP4P-disp reproduces a balanced α-synuclein secondary structure content. It was found that ff19SB/OPC and ff03CMAP/TIP4P-D produce overly compact conformational ensembles and show discrepancies in the secondary structure content compared to the experimental data.

Effect of palmitoylation on the dimer formation of the human dopamine transporter.

The human dopamine transporter (hDAT) is one in three members of the monoamine transporter family (MAT). hDAT is essential for regulating the dopamine concentration in the synaptic cleft through dopamine reuptake into the presynaptic neuron; thereby controlling hDAT dopamine signaling. Dysfunction of the transporter is linked to several psychiatric disorders. hDAT and the other MATs have been shown to form oligomers in the plasma membrane, but only limited data exists on which dimeric and higher order oligomeric states are accessible and energetically favorable. In this work, we present several probable dimer conformations using computational coarse-grained self-assembly simulations and assess the relative stability of the different dimer conformations using umbrella sampling replica exchange molecular dynamics. Overall, the dimer conformations primarily involve TM9 and/or TM11 and/or TM12 at the interface. Furthermore, we show that a palmitoyl group (palm) attached to hDAT on TM12 modifies the free energy of separation for interfaces involving TM12, suggesting that S-palmitoylation may change the relative abundance of dimers involving TM12 in a biological context. Finally, a comparison of the identified interfaces of hDAT and palmitoylated hDAT to the human serotonin transporter interfaces and the leucine transporter interface, suggests similar dimer conformations across these protein family.