Evelyne Deplazes

Widespread convergence in toxin resistance by predictable molecular evolution

Significance Convergence has strong bearing on the fundamental debate about whether evolution is stochastic and unpredictable or subject to constraints. Here we show that, in certain circumstances, evolution can be highly predictable. We demonstrate that several lineages of insects, amphibians, reptiles, and mammals have utilized the same molecular solution, via the process of convergence, to evolve resistance to toxic cardiac glycosides produced defensively by plants and bufonid toads. The repeatability of this process across the animal kingdom demonstrates that evolution can be constrained to proceed along highly predictable pathways at molecular and functional levels. Our study has important implications for conservation biology by providing a predictive framework for assessing the vulnerability of native fauna to the introduction of invasive toxic toads. The question about whether evolution is unpredictable and stochastic or intermittently constrained along predictable pathways is the subject of a fundamental debate in biology, in which understanding convergent evolution plays a central role. At the molecular level, documented examples of convergence are rare and limited to occurring within specific taxonomic groups. Here we provide evidence of constrained convergent molecular evolution across the metazoan tree of life. We show that resistance to toxic cardiac glycosides produced by plants and bufonid toads is mediated by similar molecular changes to the sodium-potassium-pump (Na+/K+-ATPase) in insects, amphibians, reptiles, and mammals. In toad-feeding reptiles, resistance is conferred by two point mutations that have evolved convergently on four occasions, whereas evidence of a molecular reversal back to the susceptible state in varanid lizards migrating to toad-free areas suggests that toxin resistance is maladaptive in the absence of selection. Importantly, resistance in all taxa is mediated by replacements of 2 of the 12 amino acids comprising the Na+/K+-ATPase H1–H2 extracellular domain that constitutes a core part of the cardiac glycoside binding site. We provide mechanistic insight into the basis of resistance by showing that these alterations perturb the interaction between the cardiac glycoside bufalin and the Na+/K+-ATPase. Thus, similar selection pressures have resulted in convergent evolution of the same molecular solution across the breadth of the animal kingdom, demonstrating how a scarcity of possible solutions to a selective challenge can lead to highly predictable evolutionary responses.

Structural investigation of MscL gating using experimental data and coarse grained MD simulations.

The mechanosensitive channel of large conductance (MscL) has become a model system in which to understand mechanosensation, a process involved in osmoregulation and many other physiological functions. While a high resolution closed state structure is available, details of the open structure and the gating mechanism remain unknown. In this study we combine coarse grained simulations with restraints from EPR and FRET experiments to study the structural changes involved in gating with much greater level of conformational sampling than has previously been possible. We generated a set of plausible open pore structures that agree well with existing open pore structures and gating models. Most interestingly, we found that membrane thinning induces a kink in the upper part of TM1 that causes an outward motion of the periplasmic loop away from the pore centre. This previously unobserved structural change might present a new mechanism of tension sensing and might be related to a functional role in osmoregulation.

Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes is a Prerequisite for its Inhibition of Human Voltage-gated Sodium Channel NaV1.7

ProTx-II is a disulfide-rich peptide toxin from tarantula venom able to inhibit the human voltage-gated sodium channel 1.7 (hNaV1.7), a channel reported to be involved in nociception, and thus it might have potential as a pain therapeutic. ProTx-II acts by binding to the membrane-embedded voltage sensor domain of hNaV1.7, but the precise peptide channel-binding site and the importance of membrane binding on the inhibitory activity of ProTx-II remain unknown. In this study, we examined the structure and membrane-binding properties of ProTx-II and several analogues using NMR spectroscopy, surface plasmon resonance, fluorescence spectroscopy, and molecular dynamics simulations. Our results show a direct correlation between ProTx-II membrane binding affinity and its potency as an hNaV1.7 channel inhibitor. The data support a model whereby a hydrophobic patch on the ProTx-II surface anchors the molecule at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. This is the first study to demonstrate that binding of ProTx-II to the lipid membrane is directly linked to its potency as an hNaV1.7 channel inhibitor.

Bacterial Mechanosensitive Channels: Models for Studying Mechanosensory Transduction

SIGNIFICANCE Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis. RECENT ADVANCES As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction. CRITICAL ISSUES In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis. FUTURE DIRECTIONS A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology.

Testing the use of molecular dynamics to simulate fluorophore motions and FRET

Fluorescence resonance energy transfer (FRET) is commonly used to determine the proximity of fluorophores, but usually many assumptions are required to gain a quantitative relationship between the likelihood of energy transfer and fluorophore separation. Molecular Dynamics (MD) simulations provide one way of checking these assumptions, but before using simulations to study complex systems it is important to make sure that they can correctly model the motions of fluorophores and the likely FRET efficiency in a simple system. Here we simulate a well characterised situation of independent fluorophores in solution so that we can compare the predictions with expected values. Our simulations reproduce the experimental fluorescence anisotropy of Alexafluor488 and predict that of AlexaFluor568. At the ensemble level we are able to reproduce the expected isotropic and dynamic motion of the fluorophores as well as the FRET efficiency of the system. At the level of single donor-acceptor pairs, however, very long simulations are required to adequately sample the translational motion of the fluorophores and more surprisingly also the rotational motion. Our studies demonstrate how MD simulations can be used in more complex systems to check if the dynamic orientation averaging regime applies, if the fluorophores have isotropic orientational motion, to calculate the likely values of the orientation factor κ(2) and to determine the FRET efficiency of the system in both dynamic and static orientational averaging regimes. We also show that it is possible in some situations to create system specific relationships between FRET efficiency and fluorophore separation that can be used to interpret experimental data and find any correlations between κ(2) and separation that may influence distance measurements.

Molecular dynamics and functional studies define a hot spot of crystal contacts essential for PcTx1 inhibition of acid-sensing ion channel 1a

The spider‐venom peptide PcTx1 is the most potent and selective inhibitor of acid‐sensing ion channel (ASIC) 1a. It has centrally acting analgesic activity and is neuroprotective in rodent models of ischaemic stroke. Understanding the molecular details of the PcTx1 : ASIC1a interaction should facilitate development of therapeutically useful ASIC1a modulators. Previously, we showed that several key pharmacophore residues of PcTx1 reside in a dynamic β‐hairpin loop; conclusions confirmed by recent crystal structures of the complex formed between PcTx1 and chicken ASIC1 (cASIC1). Numerous peptide : channel contacts were observed in these crystal structures, but it remains unclear which of these are functionally important.

Membrane-binding properties of gating modifier and pore-blocking toxins: Membrane interaction is not a prerequisite for modification of channel gating.

Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that bind to the extracellular side of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. In the case of gating modifiers, it has been debated whether the peptide must partition into the membrane to reach its binding site. In this study, we used surface plasmon resonance, fluorescence spectroscopy and molecular dynamics to directly compare the lipid-binding properties of two gating modifiers (μ-TRTX-Hd1a and ProTx-I) and two pore blockers (ShK and KIIIA). Only ProTx-I was found to bind to model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we characterised the surface of ProTx-I that mediates its interaction with neutral and anionic phospholipid membranes and show that it preferentially interacts with anionic lipids.

Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNaV1.7.

The human voltage-gated sodium channel sub-type 1.7 (hNaV1.7) is emerging as an attractive target for the development of potent and sub-type selective novel analgesics with increased potency and fewer side effects than existing therapeutics. HwTx-IV, a spider derived peptide toxin, inhibits hNaV1.7 with high potency and is therefore of great interest as an analgesic lead. In the current study we examined whether engineering a HwTx-IV analogue with increased ability to bind to lipid membranes would improve its inhibitory potency at hNaV1.7. This hypothesis was explored by comparing HwTx-IV and two analogues [E1PyrE]HwTx-IV (mHwTx-IV) and [E1G,E4G,F6W,Y30W]HwTx-IV (gHwTx-IV) on their membrane-binding affinity and hNaV1.7 inhibitory potency using a range of biophysical techniques including computational analysis, NMR spectroscopy, surface plasmon resonance, and fluorescence spectroscopy. HwTx-IV and mHwTx-IV exhibited weak affinity for lipid membranes, whereas gHwTx-IV showed improved affinity for the model membranes studied. In addition, activity assays using SH-SY5Y neuroblastoma cells expressing hNaV1.7 showed that gHwTx-IV has increased activity at hNaV1.7 compared to HwTx-IV. Based on these results we hypothesize that an increase in the affinity of HwTx-IV for lipid membranes is accompanied by improved inhibitory potency at hNaV1.7 and that increasing the affinity of gating modifier toxins to lipid bilayers is a strategy that may be useful for improving their potency at hNaV1.7.

ExiFRET: flexible tool for understanding FRET in complex geometries

Fluorescence resonance energy transfer (FRET) can be utilized to gain low-resolution structural information by reporting on the proximity of molecules or measuring inter- and intramolecular distances. This method exploits the fact that the probability of the energy transfer is related to the separation between the fluorescent molecules. This relationship is well described for a single pair of fluorophores but is complicated in systems containing more than two fluorophores. Here, we present a Monte Carlo calculation scheme that has been implemented through a user-friendly web-based program called ExiFRET that can be used to determine the FRET efficiency in a wide range of fluorophore arrangements. ExiFRET is useful to model FRET for individual fluorophores randomly distributed in two or three dimensions, fluorophores linked in pairs or arranged in regular geometries with or without predefined stoichiometries. ExiFRET can model both uniform distributions and fluorophores that are aggregated in clusters. We demonstrate how this tool can be employed to understand the effect of labeling efficiency on FRET efficiency, estimate relative contributions of inter- and intramolecular FRET, investigate the structure of multimeric proteins, stoichiometries, and oligomers, and to aid experiments studying the aggregation of lipids and proteins in membrane environments. We also present an extension that can be used to study instances in which fluorophores have constrained orientations.

Comparing the Ability of Enhanced Sampling Molecular Dynamics Methods To Reproduce the Behavior of Fluorescent Labels on Proteins

Adequately sampling the large number of conformations accessible to proteins and other macromolecules is one of the central challenges in molecular dynamics (MD) simulations; this activity can be difficult, even for relatively simple systems. An example where this problem arises is in the simulation of dye-labeled proteins, which are now being widely used in the design and interpretation of Förster resonance energy transfer (FRET) experiments. In this study, MD simulations are used to characterize the motion of two commonly used FRET dyes attached to an immobilized chain of polyproline. Even in this simple system, the dyes exhibit complex behavior that is a mixture of fast and slow motions. Consequently, very long MD simulations are required to sufficiently sample the entire range of dye motion. Here, we compare the ability of enhanced sampling methods to reproduce the behavior of fluorescent labels on proteins. In particular, we compared Accelerated Molecular Dynamics (AMD), metadynamics, Replica Exchange Molecular Dynamics (REMD), and High Temperature Molecular Dynamics (HTMD) to equilibrium MD simulations. We find that, in our system, all of these methods improve the sampling of the dye motion, but the most significant improvement is achieved using REMD.