Jan K. Marzinek

South-east Asian Zika virus strain linked to cluster of cases in Singapore, August 2016.

Zika virus (ZIKV) is an ongoing global public health emergency with 70 countries and territories reporting evidence of ZIKV transmission since 2015. On 27 August 2016, Singapore reported its first case of local ZIKV transmission and identified an ongoing cluster. Here, we report the genome sequences of ZIKV strains from two cases and find through phylogenetic analysis that these strains form an earlier branch distinct from the recent large outbreak in the Americas.

Characterizing the Conformational Landscape of Flavivirus Fusion Peptides via Simulation and Experiment

Conformational changes in the envelope proteins of flaviviruses help to expose the highly conserved fusion peptide (FP), a region which is critical to membrane fusion and host cell infection, and which represents a significant target for antiviral drugs and antibodies. In principle, extended timescale atomic-resolution simulations may be used to characterize the dynamics of such peptides. However, the resultant accuracy is critically dependent upon both the underlying force field and sufficient conformational sampling. In the present study, we report a comprehensive comparison of three simulation methods and four force fields comprising a total of more than 40 μs of sampling. Additionally, we describe the conformational landscape of the FP fold across all flavivirus family members. All investigated methods sampled conformations close to available X-ray structures, but exhibited differently populated ensembles. The best force field / sampling combination was sufficiently accurate to predict that the solvated peptide fold is less ordered than in the crystallographic state, which was subsequently confirmed via circular dichroism and spectrofluorometric measurements. Finally, the conformational landscape of a mutant incapable of membrane fusion was significantly shallower than wild-type variants, suggesting that dynamics should be considered when therapeutically targeting FP epitopes.

Free energy predictions of ligand binding to an α-helix using steered molecular dynamics and umbrella sampling simulations.

Free energy prediction of ligand binding to macromolecules using explicit solvent molecular dynamics (MD) simulations is computationally very expensive. Recently, we reported a linear correlation between the binding free energy obtained via umbrella sampling (US) versus the rupture force from steered molecular dynamics (SMD) simulations for epigallocatechin-3-gallate (EGCG) binding to α-helical-rich keratin. This linear correlation suggests a potential route for fast free energy predictions using SMD alone. In this work, the generality of the linear correlation is further tested for several ligands interacting with the α-helical motif of keratin. These molecules have significantly varying properties, i.e., octanol/water partition coefficient (log P), and/or overall charges (oleic acid, catechin, Fe(2+), citric acid, hydrogen citrate, dihydrogen citrate, and citrate). Using the constant loading rate of our previous study of the keratin-EGCG system, we observe that the linear correlation for keratin-EGCG can be extended to other uncharged molecules where interactions are governed by hydrogen bonds and/or a combination of hydrogen bonds and hydrophobic forces. For molecules where interactions with the keratin helix are governed primarily by electrostatics between charged molecules, a second, alternative linear correlation model is derived. While further investigations are needed to expand the molecular space and build a fully predictive model, the current approach represents a promising methodology for fast free energy predictions based on short SMD simulations (requiring picoseconds to nanoseconds of sampling) for defined biomolecular systems.

A Study on Fe2+ – α-Helical-Rich Keratin Complex Formation Using Isothermal Titration Calorimetry and Molecular Dynamics Simulation

Iron binding to protein is common in biological processes of dioxygen transport, electron transfer as well as in stabilizing drug-protein complexes. α-Helix is the most prevalent secondary structure of proteins. In this study, Fe(2+) binding to α-helix has been studied by isothermal titration calorimetry (ITC) and explicitly solvated molecular dynamics (MD) simulation. Ferrous gluconate and α-helix-rich keratin are used for the ITC study and the results revealed followed one set of identical sites binding model. The MD simulations further revealed that only the acidic side-chain functional groups and η(2) (O,O) coordination modes are involved in the binding of Fe(2+) to α-helix. The ITC results also showed that the binding of ferrous gluconate to keratin was entropy driven and the higher the temperature, the stronger the binding free energy. The favorable entropy of Fe(2+) binding to keratin was attributed to the displacement of water molecules on the α-helix surface, and was confirmed via MD simulations. The most stable coordination states of Fe(2+) and α-helix were identified via simulation: Fe(2+) stacks between two glutamic acid side chain carboxylate groups, displacing water molecules. The binding free energies calculated using MD simulation and the theoretical values were in excellent agreement with the ITC results.

Multiscale molecular dynamics simulation approaches to the structure and dynamics of viruses

Viral pathogens are a significant source of human morbidity and mortality, and have a major impact on societies and economies around the world. One of the challenges inherent in targeting these pathogens with drugs is the tight integration of the viral life cycle with the hosts cellular machinery. However, the reliance of the virus on the host cell replication machinery is also an opportunity for therapeutic targeting, as successful entry- and exit-inhibitors have demonstrated. An understanding of the extracellular and intracellular structure and dynamics of the virion - as well as of the entry and exit pathways in host and vector cells - is therefore crucial to the advancement of novel antivirals. In recent years, advances in computing architecture and algorithms have begun to allow us to use simulations to study the structure and dynamics of viral ultrastructures at various stages of their life cycle in atomistic or near-atomistic detail. In this review, we outline specific challenges and solutions that have emerged to allow for structurally detailed modelling of viruses in silico. We focus on the history and state of the art of atomistic and coarse-grained approaches to simulate the dynamics of the large, macromolecular structures associated with viral infection, and on their usefulness in explaining and expanding upon experimental data. We discuss the types of interactions that need to be modeled to describe major components of the virus particle and advances in modelling techniques that allow for the treatment of these systems, highlighting recent key simulation studies.

The architecture of the OmpC-MlaA complex sheds light on the maintenance of outer membrane lipid asymmetry in Escherichia coli

A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults, including antibiotics and bile salts. In Escherichia coli, the complex comprising osmoporin OmpC and the OM lipoprotein MlaA is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How this complex performs this function is unknown. Here, we defined the molecular architecture of the OmpC–MlaA complex to gain insights into its role in PL transport. Using in vivo photo-cross-linking and molecular dynamics simulations, we established that MlaA interacts extensively with OmpC and is located entirely within the lipid bilayer. In addition, MlaA forms a hydrophilic channel, likely enabling PL translocation across the OM. We further showed that flexibility in a hairpin loop adjacent to the channel is critical in modulating MlaA activity. Finally, we demonstrated that OmpC plays a functional role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC–MlaA complex transports PLs across the OM and has important implications for future antibacterial drug development.

Infectivity of dengue virus serotypes 1 and 2 is correlated to E protein intrinsic dynamics but not to envelope conformations

Dengue is a mosquito-borne virus with dire health and economic impact. Dengue is responsible for an estimated ~390 million infections per year, with Dengue 2 (DENV2) being the most virulent strain among the four serotypes. Interestingly, it is also for strains of this serotype that temperature-dependent large scale morphological changes, termed as “breathing”, have been observed. Although, the structure of these morphologies has been solved to 3.5 Å resolution, the dynamics of the viral envelope are unknown. Here, we combine fluorescence and mass spectrometry and molecular dynamics simulations to provide insights into DENV2 structural dynamics in comparison to DENV1. We observe hitherto unseen conformational changes and structural dynamics of the DENV2 envelope that are influenced by both temperature and divalent cations. Our results show that for DENV2 and DENV1 the intrinsic dynamics but not the specific morphologies are correlated to viral infectivity. Graphical Abstract/ cover Figure

Single-molecule studies of flavivirus envelope dynamics: Experiment and computation

Flaviviruses are simple enveloped viruses exhibiting complex structural and functional heterogeneities. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology approaches. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. Here, we review different single-molecule sensitive fluorescence methods that are employed to investigate flaviviruses. In particular, we review how (i) time-resolved Förster resonance energy transfer (trFRET) was applied to probe dengue envelope conformations; (ii) FRET-fluorescence correlation spectroscopy to investigate dengue envelope intrinsic dynamics and (iii) single particle tracking to follow the path of dengue viruses in cells. We also discuss how such methods may be supported by molecular dynamics (MD) simulations over a range of spatio-temporal scales, to provide complementary data on the structure and dynamics of flaviviral systems. We describe recent improvements in multiscale MD approaches that allowed the simulation of dengue particle envelopes in near-atomic resolution. We hope this review is an incentive for setting up and applying similar single-molecule studies and combine them with MD simulations to investigate structural dynamics of entire flavivirus particles over the nanosecond-to-millisecond time-scale and follow viruses during infection in cells over milliseconds to minutes.

A Funneled Conformational Landscape Governs Flavivirus Fusion Peptide Interaction with Lipid Membranes

During host cell infection by flaviviruses such as dengue and Zika, acidic pH within the endosome triggers a conformational change in the envelope protein on the outer surface of the virion. This results in exposure of the ∼15 residue fusion peptide (FP) region, freeing it to induce fusion between the viral and endosomal membranes. A better understanding of the conformational dynamics of the FP in the presence of membranes, and the basis for its selectivity for anionic lipid species present within the endosome, would facilitate its therapeutic targeting with antiviral drugs and antibodies. In this work, multiscale modeling, simulations, and free energy calculations (including a total of ∼75 μs of atomic-resolution sampling), combined with imaging total internal reflection fluorescence correlation spectroscopy experiments, were employed to investigate the mechanisms of interaction of FP variants with lipid bilayers. Wild-type FPs (in the presence or absence of a fluorescein isothiocyanate tag) were shown to possess a funneled conformational landscape governing their exit from solvent and penetration into the lipid phase and to exhibit an electrostatically favored >2-fold affinity for membranes containing anionic species over purely zwitterionic ones. Conversely, the landscape was abolished in a nonfunctional point mutant, leading to a 2-fold drop in host membrane affinity. Collectively, our data reveal how the highly conserved flavivirus FP has evolved to funnel its conformational space toward a maximally fusogenic state anchored within the endosomal membrane. Therapeutically targeting the accessible ensemble of FP conformations may represent a new, rational strategy for blocking viral infection.

Partial Intrinsic Disorder Governs the Dengue Capsid Protein Conformational Ensemble

The 11 kDa, positively charged dengue capsid protein (C protein) exists stably as a homodimer and colocalizes with the viral genome within mature viral particles. Its core is composed of four alpha helices encompassing a small hydrophobic patch that may interact with lipids, but approximately 20% of the protein at the N-terminus is intrinsically disordered, making it challenging to elucidate its conformational landscape. Here, we combine small-angle X-ray scattering (SAXS), amide hydrogen-deuterium exchange mass spectrometry (HDXMS), and atomic-resolution molecular dynamics (MD) simulations to probe the dynamics of dengue C proteins. We show that the use of MD force fields (FFs) optimized for intrinsically disordered proteins (IDPs) is necessary to capture their conformational landscape and validate the computationally generated ensembles with reference to SAXS and HDXMS data. Representative ensembles of the C protein dimer are characterized by alternating, clamp-like exposure and occlusion of the internal hydrophobic patch, as well as by residual helical structure at the disordered N-terminus previously identified as a potential source of autoinhibition. Such dynamics are likely to determine the multifunctionality of the C protein during the flavivirus life cycle and hence impact the design of novel antiviral compounds.