Probing effects of the SARS-CoV-2 E protein on membrane curvature and intracellular calcium.

SARS-CoV-2 contains four structural proteins in its genome. These proteins aid in the assembly and budding of new virions at the ER-Golgi intermediate compartment (ERGIC). Current fundamental research efforts largely focus on one of these proteins - the spike (S) protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other structural proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length envelope (E) protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.

Identification of electroporation sites in the complex lipid organization of the plasma membrane.

The plasma membrane of a biological cell is a complex assembly of lipids and membrane proteins, which tightly regulate transmembrane transport. When a cell is exposed to strong electric field, the membrane integrity becomes transiently disrupted by formation of transmembrane pores. This phenomenon termed electroporation is already utilized in many rapidly developing applications in medicine including gene therapy, cancer treatment, and treatment of cardiac arrhythmias. However, the molecular mechanisms of electroporation are not yet sufficiently well understood; in particular, it is unclear where exactly pores form in the complex organization of the plasma membrane. In this study, we combine coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis to identify how formation of pores depends on the local lipid organization. We show that pores do not form homogeneously across the membrane, but colocalize with domains that have specific features, the most important being high density of polyunsaturated lipids. We further show that knowing the lipid organization is sufficient to reliably predict poration sites with machine learning. Additionally, by analysing poration kinetics with Bayesian survival analysis we show that poration does not depend solely on local lipid arrangement, but also on membrane mechanical properties and the polarity of the electric field. Finally, we discuss how the combination of atomistic and coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis can guide the design of future experiments and help us to develop an accurate description of plasma membrane electroporation on the whole-cell level. Achieving this will allow us to shift the optimization of electroporation applications from blind trial-and-error approaches to mechanistic-driven design.

Allosteric Effect of Nanobody Binding on Ligand-Specific Active States of the ß2 Adrenergic Receptor.

Nanobody binding stabilizes G-protein-coupled receptors (GPCR) in a fully active state and modulates their affinity for bound ligands. However, the atomic-level basis for this allosteric regulation remains elusive. Here, we investigate the conformational changes induced by the binding of a nanobody (Nb80) on the active-like ß2 adrenergic receptor (ß2AR) via enhanced sampling molecular dynamics simulations. Dimensionality reduction analysis shows that Nb80 stabilizes structural features of the ß2AR with an ∼14 Å outward movement of transmembrane helix 6 and a close proximity of transmembrane (TM) helices 5 and 7, and favors the fully active-like conformation of the receptor, independent of ligand binding, in contrast to the conditions under which no intracellular binding partner is bound, in which case the receptor is only stabilized in an intermediate-active state. This activation is supported by the residues located at hotspots located on TMs 5, 6, and 7, as shown by supervised machine learning methods. Besides, ligand-specific subtle differences in the conformations assumed by intracellular loop 2 and extracellular loop 2 are captured from the trajectories of various ligand-bound receptors in the presence of Nb80. Dynamic network analysis further reveals that Nb80 binding triggers tighter and stronger local communication networks between the Nb80 and the ligand-binding sites, primarily involving residues around ICL2 and the intracellular end of TM3, TM5, TM6, as well as ECL2, ECL3, and the extracellular ends of TM6 and TM7. In particular, we identify unique allosteric signal transmission mechanisms between the Nb80-binding site and the extracellular domains in conformations modulated by a full agonist, BI167107, and a G-protein-biased partial agonist, salmeterol, involving mainly TM1 and TM2, and TM5, respectively. Altogether, our results provide insights into the effect of intracellular binding partners on the GPCR activation mechanism, which should be taken into account in structure-based drug discovery.

Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel.

As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

Combining EXAFS and Computer Simulations to Refine the Structural Description of Actinyls in Water.

EXAFS spectroscopy is one of the most used techniques to solve the structure of actinoid solutions. In this work a systematic analysis of the EXAFS spectra of four actinyl cations, [UO2]2+, [NpO2]2+, [NpO2]+ and [PuO2]2+ has been carried out by comparing experimental results with theoretical spectra. These were obtained by averaging individual contributions from snapshots taken from classical Molecular Dynamics simulations which employed a recently developed [AnO2]2+/+ -H2O force field based on the hydrated ion model using a quantum-mechanical (B3LYP) potential energy surface. Analysis of the complex EXAFS signal shows that both An-Oyl and An-OW single scattering paths as well as multiple scattering ones involving [AnO2]+/2+ molecular cation and first-shell water molecules are mixed up all together to produce a very complex signal. Simulated EXAFS from the B3LYP force field are in reasonable agreement for some of the cases studied, although the k= 6-8 Å-1 region is hard to be reproduced theoretically. Except uranyl, all studied actinyls are open-shell electron configurations, therefore it has been investigated how simulated EXAFS spectra are affected by minute changes of An-O bond distances produced by the inclusion of static and dynamic electron correlation in the quantum mechanical calculations. A [NpO2]+-H2O force field based on a NEVPT2 potential energy surface has been developed. The small structural changes incorporated by the electron correlation on the actinyl aqua ion geometry, typically smaller than 0.07 Å, leads to improve the simulated spectrum with respect to that obtained from the B3LYP force field. For the other open-shell actinyls, [NpO2]2+ and [PuO2]2+, a simplified strategy has been adopted to improve the simulated EXAFS spectrum. It is computed taking as reference structure the NEVPT2 optimized geometry and including the DW factors of their corresponding MD simulations employing the B3LYP force field. A better agreement between the experimental and the simulated EXAFS spectra is found, confirming the a priori guess that the inclusion of dynamic and static correlation refine the structural description of the open-shell actinyl aqua ions.

Network analysis reveals how lipids and other cofactors influence membrane protein allostery.

Many membrane proteins are modulated by external stimuli, such as small molecule binding or change in pH, transmembrane voltage, or temperature. This modulation typically occurs at sites that are structurally distant from the functional site. Revealing the communication, known as allostery, between these two sites is key to understanding the mechanistic details of these proteins. Residue interaction networks of isolated proteins are commonly used to this end. Membrane proteins, however, are embedded in a lipid bilayer, which may contribute to allosteric communication. The fast diffusion of lipids hinders direct use of standard residue interaction networks. Here, we present an extension that includes cofactors such as lipids and small molecules in the network. The novel framework is applied to three membrane proteins: a voltage-gated ion channel (KCNQ1), a G-protein coupled receptor (GPCR-ß2 adrenergic receptor), and a pH-gated ion channel (KcsA). Through systematic analysis of the obtained networks and their components, we demonstrate the importance of lipids for membrane protein allostery. Finally, we reveal how small molecules may stabilize different protein states by allosterically coupling and decoupling the protein from the membrane.

A general study of actinyl hydration by molecular dynamics simulations using ab initio force fields.

A set of new ab initio force fields for aqueous [AnO2]2+/+ (An = Np(vi,v), Pu(vi), Am(vi)) has been developed using the Hydrated Ion (HI) model methodology previously used for [UO2]2+. Except for the non-electrostatic contribution of the HI-bulk water interaction, the interaction potentials are individually parameterized. Translational diffusion coefficients, hydration enthalpies, and vibrational normal mode frequencies were calculated from the MD simulations. Physico-chemical properties satisfactorily agree with experiments validating the robustness of the force field strategy. The solvation dynamics and structure for all hexavalent actinoids are extremely similar and resemble our previous analysis of the uranyl cation. This supports the idea of using the uranyl cation as a reference for the study of other minor actinyls. The comparison between the NpO2 2+ and NpO2 + hydration only provides significant differences in first and second shell distances and second-shell mean residence times. We propose a single general view of the [AnO2]2+/+ hydration structure: aqueous actinyls are amphiphilic anisotropic solutes which are equatorially conventional spherically symmetric cations capped at the poles by clathrate-like water structures.

Extracting the Americyl Hydration from an Americium Cationic Mixture in Solution: A Combined X-ray Absorption Spectroscopy and Molecular Dynamics Study.

Am(VI) solution chemistry differs from that of lighter actinoids, as U, Pu, and Np, where the actinyl [AnO2]2+ is the most stable form and plays an important role in nuclear fuel technology. The behavior of americium in solution shows the trend to stabilize lower oxidation states, mainly Am(III). Riddle and co-workers recently reported the EXAFS and first XANES spectra of an americium-containing aqueous solution where the americyl species is detected in a mixture. We have developed Am3+-H2O and [AmO2]2+-H2O intermolecular potentials based on quantum-mechanical calculations to carry out classical MD simulations of these two cations in water. Structural information extracted from the statistical trajectories has been used to simulate EXAFS and XANES spectra of both solutions. For the Am3+ case the theoretical-experimental agreement for both EXAFS and XANES spectra is satisfactory. This is not the case for the [AmO2]2+ aqueous solutions. However, when an aqueous solution mixture of both cationic forms in a 55/45 [AmO2]2+/Am3+ ratio is considered, the theoretical-experimental agreement is recovered. EXAFS and XANES spectra which would correspond to a pure [AmO2]2+ aqueous solution are proposed. In the XANES case, the main features characterizing the simulated spectrum are consistent with those previously found in the experimental XANES spectra of stable [UO2]2+ and [PuO2]2+ in water.

A hydrated ion model of [UO2]2+ in water: Structure, dynamics, and spectroscopy from classical molecular dynamics.

A new ab initio interaction potential based on the hydrated ion concept has been developed to obtain the structure, energetics, and dynamics of the hydration of uranyl in aqueous solution. It is the first force field that explicitly parameterizes the interaction of the uranyl hydrate with bulk water molecules to accurately define the second-shell behavior. The [UO2(H2O)5]2+ presents a first hydration shell U-O average distance of 2.46 Å and a second hydration shell peak at 4.61 Å corresponding to 22 molecules using a coordination number definition based on a multisite solute cavity. The second shell solvent molecules have longer mean residence times than those corresponding to the divalent monatomic cations. The axial regions are relatively de-populated, lacking direct hydrogen bonding to apical oxygens. Angle-solved radial distribution functions as well as the spatial distribution functions show a strong anisotropy in the ion hydration. The [UO2(H2O)5]2+ solvent structure may be regarded as a combination of a conventional second hydration shell in the equatorial and bridge regions, and a clathrate-like low density region in the axial region. Translational diffusion coefficient, hydration enthalpy, power spectra of the main vibrational modes, and the EXAFS spectrum simulated from molecular dynamics trajectories agree fairly well with the experiment.