Making Structural Sense of Dimerization Interfaces of Delta Opioid Receptor Homodimers†

ABSTRACT: Opioid receptors, like other members of theG protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate. We have recently calculated binding affinities from free energy estimates to predict transient association between mouse delta opioid receptor (DOR) protomers at a symmetric interface involving the fourth transmembrane (TM4) helix (herein termed “4 ” dimer). Here we present disulfide cross-linking experiments with DOR constructs with cysteines substituted at the extracellular ends of TM4 or TM5 that confirm the formation of DOR complexes involving these helices. Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities. Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed “4/5 ” dimer) in an explicit lipid-water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer. Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, wit

Structures of monomeric and oligomeric forms of the Toxoplasma gondiiperforin-like protein 1

Toxoplasma and Plasmodium are the parasitic agents of toxoplasmosis and malaria, respectively, and use perforin-like proteins (PLPs) to invade host organisms and complete their life cycles. The Toxoplasma gondii PLP1 (TgPLP1) is required for efficient exit from parasitophorous vacuoles in which proliferation occurs. We report structures of the membrane attack complex/perforin (MACPF) and Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains of TgPLP1. The MACPF domain forms hexameric assemblies, with ring and helix geometries, and the APCβ domain has a novel β-prism fold joined to the MACPF domain by a short linker. Molecular dynamics simulations suggest that the helical MACPF oligomer preserves a biologically important interface, whereas the APCβ domain binds preferentially through a hydrophobic loop to membrane phosphatidylethanolamine, enhanced by the additional presence of inositol phosphate lipids. This mode of membrane binding is supported by site-directed mutagenesis data from a liposome-based assay. Together, these structural and biophysical findings provide insights into the molecular mechanism of membrane targeting by TgPLP1

Functionalized surfaces with tailored wettability determine Influenza A infectivity

Surfaces contaminated with pathogenic microorganisms contribute to their transmission and spreading. The development of 'active surfaces' that can reduce or eliminate this contamination necessitates a detailed understanding of the molecular mechanisms of interactions between the surfaces and the microorganisms. Few studies have shown that, among the different surface characteristics, the wetting properties play an important role in reducing virus infectivity. Here, we systematically tailored the wetting characteristics of flat and nanostructured glass surfaces by functionalizing them with alkyl- and fluoro-silanes. We studied the effects of these functionalized surfaces on the infectivity of Influenza A viruses using a number of experimental and computational methods including real-time fluorescence microscopy and molecular dynamics simulations. Overall, we show that surfaces that are simultaneously hydrophobic and oleophilic are more efficient in deactivating enveloped viruses. Our results suggest that the deactivation mechanism likely involves disruption of the viral membrane upon its contact with the alkyl chains. Moreover, enhancing these specific wetting characteristics by surface nanostructuring led to an increased deactivation of viruses. These combined features make these substrates highly promising for applications in hospitals and similar infrastructures where antiviral surfaces can be crucial

Development of IKATP Ion Channel Blockers Targeting Sulfonylurea Resistant Mutant KIR6.2 Based Channels for Treating DEND Syndrome

Introduction: DEND syndrome is a rare channelopathy characterized by a combination of developmental delay, epilepsy and severe neonatal diabetes. Gain of function mutations in the KCNJ11 gene, encoding the KIR6.2 subunit of the IKATP potassium channel, stand at the basis of most forms of DEND syndrome. In a previous search for existing drugs with the potential of targeting Cantú Syndrome, also resulting from increased IKATP, we found a set of candidate drugs that may also possess the potential to target DEND syndrome. In the current work, we combined Molecular Modelling including Molecular Dynamics simulations, with single cell patch clamp electrophysiology, in order to test the effect of selected drug candidates on the KIR6.2 WT and DEND mutant channels. Methods: Molecular dynamics simulations were performed to investigate potential drug binding sites. To conduct in vitro studies, KIR6.2 Q52R and L164P mutants were constructed. Inside/out patch clamp electrophysiology on transiently transfected HEK293T cells was performed for establishing drug-channel inhibition relationships. Results: Molecular Dynamics simulations provided insight in potential channel interaction and shed light on possible mechanisms of action of the tested drug candidates. Effective IKIR6.2/SUR2a inhibition was obtained with the pore-blocker betaxolol (IC50 values 27-37 μM). Levobetaxolol effectively inhibited WT and L164P (IC50 values 22 μM) and Q52R (IC50 55 μM) channels. Of the SUR binding prostaglandin series, travoprost was found to be the best blocker of WT and L164P channels (IC50 2-3 μM), while Q52R inhibition was 15-20% at 10 μM. Conclusion: Our combination of MD and inside-out electrophysiology provides the rationale for drug mediated IKATP inhibition, and will be the basis for 1) screening of additional existing drugs for repurposing to address DEND syndrome, and 2) rationalized medicinal chemistry to improve IKATP inhibitor efficacy and specificity

Multiscale simulation of DNA

DNA is not only among the most important molecules in life, but a meeting point for biology, physics and chemistry, being studied by numerous techniques. Theoretical methods can help in gaining a detailed understanding of DNA structure and function, but their practical use is hampered by the multiscale nature of this molecule. In this regard, the study of DNA covers a broad range of different topics, from sub-Angstrom details of the electronic distributions of nucleobases, to the mechanical properties of millimeter-long chromatin fibers. Some of the biological processes involving DNA occur in femtoseconds, while others require years. In this review, we describe the most recent theoretical methods that have been considered to study DNA, from the electron to the chromosome, enriching our knowledge on this fascinating molecule

Material design using Martini:Accelerating discovery through coarse-grained simulations

Advanced materials are a fundamental aspect of our modern everyday life. From batteries to solar cells and vaccines, the development of new functional materials is key in addressing societal challenges of the 21st century, yet the development process is often slow and expensive. Computational studies can accelerate this process but to fully realize their potential, they require infrastructure in the form of software, protocols, and benchmarks of these protocols. The thesis at hand starts with an in-depth review of the current usage of the Martini coarse-grained (CG) simulation technique in the field of material science. Subsequently, it lays the groundwork to realize the next stage of the rational design of complex heterogeneous materials utilizing Martini CG simulations. In particular, two newly developed software tools are introduced to facilitate high-throughput workflows. The open-source Vermouth python library and Polyply software suite efficiently automate the simulation setup of complex materials and sharing of simulation input parameters. Subsequently, two libraries of simulation parameters for carbohydrates and synthetic polymers are presented and benchmarked. Finally, a proof-of-concept method is presented to incorporate pH effects in Martini simulations. This protocol allows to study the response of materials to changes in pH, which is a common mechanism to functionalize materials. Taken together these developments enable the simulation of highly complex materials and offer a comprehensive collection of Martini (bio)-polymer parameters

Untersuchung der Wirkung von Anästhetika auf Membranen und der Adsorption von Proteinen an Festkörperoberflächen mittels Moleküldynamik-Simulationen

In this thesis, the mechanisms underlying anesthesia and the adsorption of proteins on solid surfaces have been studied using the method of molecular dynamics simulations. It is generally assumed that biological membranes are the site of anesthetic action. However, there is no consensus whether anesthetics act directly by binding to membrane proteins, thereby inhibiting their function, or indirectly by modulating the physical properties of the lipid part of the membrane. In the simulations presented here, distinct changes of lipid bilayer properties in response to the presence of alkanols, a group of anesthetics, have been observed. An anesthetic-induced shift of the equilibrium between different membrane protein conformations, modeled by simple geometric shapes, has been found. In simulations with the ion channel gramicidin A embedded in a lipid bilayer, alkanols distributed inhomogeneously in the bilayer, with almost no alkanol molecules residing in close vicinity to the gramicidin. These results provide evidence for an indirect mode of anesthetic action. Spontaneous protein adsorption on solid-liquid interfaces is the first step in the formation of biofilms. Here, a coarse-grained molecular dynamics scheme has been applied to study this complex process at high resolution, but still reaching the necessary time and length scales. Changes in protein structure and dynamics after adsorption and preferred orientations of proteins on the surface were observed.In dieser Arbeit wurde die Wirkungsweise von Anästhetika und die Adsorption von Proteinen an Festkörperoberflächen mittels Moleküldynamik-Simulationen untersucht. Es wird allgemein angenommen, dass Anästhetika auf biologische Membranen wirken. Umstritten ist jedoch, ob Anästhetika direkt an Membranproteine binden und damit deren Funktion hemmen, oder ob sie indirekt wirken, indem sie die physikalischen Eigenschaften der Lipiddoppelschicht der Membran verändern. Solche indirekten Effekte wurden in den hier vorgestellten Simulationen bei Anwesenheit von Alkanolen, einer Gruppe von Anästhetika, beobachtet. Gleichzeitig wurde eine durch Anästhetika verursachte Verschiebung des Gleichgewichts zwischen unterschiedlichen, vereinfacht dargestellten Proteinkonformationen gefunden. Simulationen eines in einer Lipiddoppelschicht eingebetteten Ionenkanals zeigten eine sehr geringe Konzentration von Alkanolen in unmittelbarer Nähe des Kanals. Diese Ergebnisse deuten auf eine indirekte Wirkungsweise von Anästhetika hin. Spontane Adsorption von Proteinen an fest-flüssig Grenzflächen ist der erste Schritt bei der Bildung von Biofilmen. Um diesen Prozess der Proteinadsorption mit hoher Auflösung auf ausreichend langen Zeit- und Längenskalen zu untersuchen, wurde ein "coarse-grained" Moleküldynamik-Schema verwendet. Es wurden Veränderungen in der Proteinstruktur und -dynamik und bevorzugte Ausrichtungen der Proteine auf der Oberfläche beobachtet

The Martini Model in Materials Science

The Martini model, a coarse-grained force field initially developed with biomolecular simulations in mind, has found an increasing number of applications in the field of soft materials science. The model's underlying building block principle does not pose restrictions on its application beyond biomolecular systems. Here, the main applications to date of the Martini model in materials science are highlighted, and a perspective for the future developments in this field is given, particularly in light of recent developments such as the new version of the model, Martini 3

Probing the lipid environment of the G-protein coupled receptor Metabotropic glutamate receptor 2

The Metabotropic glutamate receptor 2 (mGluR2) belongs to the family of G-protein coupled receptors, a specific class of transmembrane proteins involved in cellular signaling. The functionality of such transmembrane proteins has been identified to largely depend on their microenvironment, namely the lipid bilayer surrounding them. However, the regulation of the receptors by their lipid microenvironment remains poorly understood. In particular, it remains unclear how specific protein-lipid interactions may modulate the function of mGluR2. In the last years, general motifs for non-covalent cholesterol and sphingolipid interaction within helical domains of transmembrane proteins have been described. In these motifs, both tryptophan and tyrosine residues have been found to play a decisive role. For that reason, an alanine mutagenesis screening, targeting tryptophan and tyrosine residues at the transmembrane surface, was carried out in the search of specific sphingolipid or cholesterol interaction sites for mGluR2. For the different Y→A and W→A variants, surface biotinylation and co-immunoprecipitation showed that neither trafficking nor dimerization were disturbed by substitution of these aromatic residues. In contrast, cellular photo-crosslinking assays demonstrated that cholesterol binding was compromised if one tyrosine residue located at the helix five or another at the helix six was replaced. Thus, these experiments suggested these two helices to contain specific cholesterol binding sites. To get a better molecular insight into these specific protein-lipid interactions, lipid binding to the transmembrane domain of mGluR2 was investigated in molecular dynamics (MD) simulation. The molecular dynamics simulations in GROMACS were performed in collaboration with the Max Planck tandem group of Dr. Camilo Aponte-Santamaría. All-atom and coarse-grained MD simulations of the mGluR2 transmembrane domain confirmed the experimental observation, by revealing a highly-localized density of cholesterol near these residues in helices five and six, which smeared out when they were changed to alanine in silico. The simulations also revealed flexibility of the protein structure at the exoplasmic end of helix six which changed upon introduction of point mutations. Overall, the work combining functional assays and MD simulations demonstrated the existence of specific cholesterol binding sites in mGluR2. It will be highly interesting to investigate the functional implications of this newly-found specific protein–cholesterol interaction on the activity and conformation of the receptor