Visualization of the mechanosensitive ion channel MscS under membrane tension.

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes1-3. However, how exactly they sense mechanical force remains under investigation4. The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels4-8, but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states9-11. Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tension. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformation of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensation. Pore lipids are necessary to prevent ion conduction in the closed state. Gatekeeper lipids stabilize the closed conformation and dissociate with membrane tension, allowing the channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tension, allowing the channel to transition to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensation4,11.

Structural basis of Focal Adhesion Kinase activation on lipid membranes.

Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions.

The role of flow in the self-assembly of dragline spider silk proteins.

Hydrodynamic flow in the spider duct induces conformational changes in dragline spider silk proteins (spidroins) and drives their assembly, but the underlying physical mechanisms are still elusive. Here we address this challenging multiscale problem with a complementary strategy of atomistic and coarse-grained molecular dynamics simulations with uniform flow. The conformational changes at the molecular level were analyzed for single-tethered spider silk peptides. Uniform flow leads to coiled-to-stretch transitions and pushes alanine residues into ß sheet and poly-proline II conformations. Coarse-grained simulations of the assembly process of multiple semi-flexible block copolymers using multi-particle collision dynamics reveal that the spidroins aggregate faster but into low-order assemblies when they are less extended. At medium-to-large peptide extensions (50%-80%), assembly slows down and becomes reversible with frequent association and dissociation events, whereas spidroin alignment increases and alanine repeats form ordered regions. Our work highlights the role of flow in guiding silk self-assembly into tough fibers by enhancing alignment and kinetic reversibility, a mechanism likely relevant also for other proteins whose function depends on hydrodynamic flow.

A ß-barrel for oil transport through lipid membranes: Dynamic NMR structures of AlkL.

The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters. Dynamic lateral exit sites large enough to accommodate substrates such as carvone or octane occur through restructuring of a barrel extension formed by the extracellular loops.

Mechanical force can enhance c-Src kinase activity by impairing autoinhibition.

Cellular mechanosensing is pivotal for virtually all biological processes, and many molecular mechano-sensors and their way of function are being uncovered. In this work, we suggest that c-Src kinase acts as a direct mechano-sensor. c-Src is responsible for, among others, cell proliferation, and shows increased activity in stretched cells. In its native state, c-Src has little basal activity, because its kinase domain binds to an SH2 and SH3 domain. However, it is known that c-Src can bind to p130Cas, through which force can be transmitted to the membrane. Using molecular dynamics simulations, we show that force acting between the membrane-bound N-terminus of the SH3 domain and p130Cas induces partial SH3 unfolding, thereby impeding rebinding of the kinase domain onto SH2/SH3 and effectively enhancing kinase activity. Forces involved in this process are slightly lower or similar to the forces required to pull out c-Src from the membrane through the myristoyl linker, and key interactions involved in this anchoring are salt bridges between negative lipids and nearby basic residues in c-Src. Thus, c-Src appears to be a candidate for an intriguing mechanosensing mechanism of impaired kinase inhibition, which can be potentially tuned by membrane composition and other environmental factors.

The plakin domain of C. elegans VAB-10/plectin acts as a hub in a mechanotransduction pathway to promote morphogenesis.

Mechanical forces can elicit a mechanotransduction response through junction-associated proteins. In contrast to the wealth of knowledge available for focal adhesions and adherens junctions, much less is known about mechanotransduction at hemidesmosomes. Here, we focus on the C. elegans plectin homolog VAB-10A, the only evolutionary conserved hemidesmosome component. In C. elegans, muscle contractions induce a mechanotransduction pathway in the epidermis through hemidesmosomes. We used CRISPR to precisely remove spectrin repeats (SRs) or a partially hidden Src homology 3 (SH3) domain within the VAB-10 plakin domain. Deleting the SH3 or SR8 domains in combination with mutations affecting mechanotransduction, or just the part of SR5 shielding the SH3 domain, induced embryonic elongation arrest because hemidesmosomes collapse. Notably, recruitment of GIT-1, the first mechanotransduction player, requires the SR5 domain and the hemidesmosome transmembrane receptor LET-805. Furthermore, molecular dynamics simulations confirmed that forces acting on VAB-10 could make the central SH3 domain, otherwise in contact with SR4, available for interaction. Collectively, our data strongly indicate that the plakin domain plays a central role in mechanotransduction and raise the possibility that VAB-10/plectin might act as a mechanosensor.

Structural and mechanistic insights into mechanoactivation of focal adhesion kinase.

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the low-force range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.

Lipid-protein forces predict conformational changes in a mechanosensitive channel.

The mechanosensitive TREK-2 potassium channel, a member of the K2P family, has essential physiological roles and is, therefore, a pharmaceutical target. A combination of experimental and computational studies have established that of the two known conformations, "up" and "down", membrane tension directly favors the "up" state, which displays a higher conductance. However, these studies did not reveal the exact mechanism by which the membrane affects the channel conformation. In this work, we show that changes in protein-lipid interaction patterns suffice in predicting this conformational change, and pinpoint potentially important residues involved in this phenomenon.

How ARVC-Related Mutations Destabilize Desmoplakin: An MD Study.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial heart disease linked to mutations in several desmosomal proteins, but the specific effects of these mutations on the molecular level are poorly understood. Among the many documented ARVC-related genetic variants, a striking hotspot of nine mutations has been identified in the plakin domain of desmoplakin. This hotspot can be found at the meeting point of three different subdomains of desmoplakin: two spectrin repeats and a Src homology 3 domain. We set out to understand the effect of these mutations. We determine, using molecular dynamics simulations, how these mutations affect the mechanics of this interface, performing two different classes of simulations. First, we sample the dynamics of the plakin domain, in particular the tendency of the interdomain hinge to buckle, and then we apply an external force onto the constructs and determine the force necessary to break them. We find that surface-exposed mutations are not affecting the dynamics to a very large degree but that most buried mutations make the junction more flexible and decrease the rupture forces observed. Our data suggest that buried ARVC mutations destabilize desmoplakin and thereby impair desmosome integrity under tension.

CONAN: A Tool to Decode Dynamical Information from Molecular Interaction Maps.

The analysis of contacts is a powerful tool to understand biomolecular function in a series of contexts, from the investigation of dynamical behavior at equilibrium to the study of nonequilibrium dynamics in which the system moves between multiple states. We thus propose a tool called CONtact ANalysis (CONAN) that, from molecular dynamics (MD) trajectories, analyzes interresidue contacts, creates videos of time-resolved contact maps, and performs correlation, principal component, and cluster analysis, revealing how specific contacts relate to functionally relevant states sampled by MD. We present how CONAN can identify features describing the dynamics of ubiquitin both at equilibrium and during mechanical unfolding. Additionally, we show the analysis of MD trajectories of an α-synuclein mutant peptide that undergoes an α-ß conformational transition that can be easily monitored using CONAN, which identifies the multiple states that the peptide explores along its conformational dynamics. The high versatility and ease of use of the software make CONAN a tool that can significantly facilitate the understanding of the complex dynamical behavior of proteins or other biomolecules. CONAN and its documentation are freely available for download on GitHub.

Understanding Conformational Dynamics of Complex Lipid Mixtures Relevant to Biology.

This is a perspective article entitled "Frontiers in computational biophysics: understanding conformational dynamics of complex lipid mixtures relevant to biology" which is following a CECAM meeting with the same name.

Wavefunction in density functional theory embedding for excited states: which wavefunctions, which densities?

We present a detailed analysis of our recently proposed wavefunction in density functional theory method to include differential polarization effects through state-specific embedding potentials. We study methylenecyclopropene and acrolein in water by using several wavefunction approaches to validate the supermolecular reference and to assess their response to embedding. We find that quantum Monte Carlo, complete-active space second-order perturbation theory, and coupled cluster methods give very consistent solvatochromic shifts and a similar response to embedding. Our scheme corrects the excitation energies produced with a frozen environment, but the values are often overshot. To ameliorate the problem, one needs to use wavefunction densities to polarize the environment. The choice of the exchange-correlation functional in the construction of the potential has little effect on the excitation, whereas the approximate kinetic-energy functional appears to be the largest source of error.

Advances in molecular simulations of protein mechanical properties and function.

Single-molecule force spectroscopy and classical molecular dynamics are natural allies. Recent advances in both experiments and simulations have increasingly facilitated a direct comparison of SMFS and MD data, most importantly by closing the gap between time scales, which has been traditionally at least 5 orders of magnitudes wide. In this review, we will explore these advances chiefly on the computational side. We focus on protein dynamics under force and highlight recent studies that showcase how lower loading rates and more statistics help to better interpret previous experiments and to also motivate new ones. At the same time, steadily increasing system sizes are used to mimic more closely the mechanical environment in the biological context. We showcase some of these advances on atomistic and coarse-grained scale, from asymmetric membrane tension to larger (multidomain/multimeric) protein assemblies under force.

Mechanoradicals in tensed tendon collagen as a source of oxidative stress.

As established nearly a century ago, mechanoradicals originate from homolytic bond scission in polymers. The existence, nature and biological relevance of mechanoradicals in proteins, instead, are unknown. We here show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species, essential biological signaling molecules. Electron-paramagnetic resonance (EPR) spectroscopy of stretched rat tail tendon, atomistic molecular dynamics simulations and quantum-chemical calculations show that the radicals form by bond scission in the direct vicinity of crosslinks in collagen. Radicals migrate to adjacent clusters of aromatic residues and stabilize on oxidized tyrosyl radicals, giving rise to a distinct EPR spectrum consistent with a stable dihydroxyphenylalanine (DOPA) radical. The protein mechanoradicals, as a yet undiscovered source of oxidative stress, finally convert into hydrogen peroxide. Our study suggests collagen I to have evolved as a radical sponge against mechano-oxidative damage and proposes a mechanism for exercise-induced oxidative stress and redox-mediated pathophysiological processes.

How Fast Is Too Fast in Force-Probe Molecular Dynamics Simulations?

While molecular dynamics (MD) simulations are routinely used to interpret atomic force microscopy (AFM) experiments of protein unfolding, computational cost in MD simulations still mostly imposes a large difference in loading rates and time scales in this comparison. Loading rate dependencies of unfolding forces and mechanisms have been studied in depth in experiments, simulations, and theory. One potential additional implication of the larger MD pulling velocity that remains to be assessed is that regions of the proteins that are close to the point of force application will be under force earlier or under more force than more shielded regions, resulting in a bias of the protein unfolding sequence which is likely marginal at the slower AFM velocities. We here, for the first time, quantify the parameters of this bias using a model system of four tandem spectrin repeats (SRs) linked with long, flexible poly-glycine linkers. We subject the system to seven different pulling velocities ranging from 0.01 to 10 m/s and find that for the fastest velocities, down to 1 m/s, the outer domains preferentially unfold; in fact, at 10 m/s, this happened in 100 cases out of 100. On the basis of these data, and also through analyzing the amount of partial unfolding in the beginning of the simulations, we show that the bias is equivalent to an effective signal propagation of 5-100 m/s, which is about 2 orders of magnitude slower than the expected speed of sound. Our results can help in identifying and removing this bias from future simulations.

Stability of Biological Membranes upon Mechanical Indentation.

Mechanical perturbations are ubiquitous in living cells, and many biological functions are dependent on the mechanical response of lipid membranes. Recent force-spectroscopy studies have captured the stepwise fracture of stacks of bilayers, avoiding substrate effects. However, the effect of stacking bilayers, as well as the exact molecular mechanism of the fracture process, is unknown. Here, we use atomistic and coarse-grained force-clamp molecular dynamics simulation to assess the effects of mechanical indentation on stacked and single bilayers. Our simulations show that the rupture process obeys the laws of force-activated barrier crossing, and stacking multiple membranes stabilizes them. The rupture times follow a log-normal distribution which allows the interpretation of membrane rupture as a pore-growth process. Indenter hydrophobicity determines the type of pore formation, the preferred dwelling region, and the resistance of the bilayer against rupture. Our results provide a better understanding of the nanomechanics underlying the plastic rupture of lipid membranes.

The mechano-sensing role of the unique SH3 insertion in plakin domains revealed by Molecular Dynamics simulations.

The plakin family of proteins, important actors in cross-linking force-bearing structures in the cell, contain a curious SH3 domain insertion in their chain of spectrin repeats (SRs). While SH3 domains are known to mediate protein-protein interactions, here, its canonical binding site is autoinhibited by the preceding SR. Under force, however, this SH3 domain could be released, and possibly launch a signaling cascade. We performed large-scale force-probe molecular dynamics simulations, across two orders of magnitude of loading rates, to test this hypothesis, on two prominent members of the plakin family: desmoplakin and plectin, obligate proteins at desmosomes and hemidesmosomes, respectively. Our simulations show that force unravels the SRs and abolishes the autoinhibition of the SH3 domain, an event well separated from the unfolding of this domain. The SH3 domain is free and fully functional for a significant portion of the unfolding trajectories. The rupture forces required for the two proteins significantly decrease when the SH3 domain is removed, which implies that the SH3 domain also stabilizes this junction. Our results persist across all simulations, and support a force-sensing as well as a stabilizing role of the unique SH3 insertion, putting forward this protein family as a new class of mechano-sensors.

Introducing QMC/MMpol: Quantum Monte Carlo in Polarizable Force Fields for Excited States.

We present for the first time a quantum mechanics/molecular mechanics scheme which combines quantum Monte Carlo with the reaction field of classical polarizable dipoles (QMC/MMpol). In our approach, the optimal dipoles are self-consistently generated at the variational Monte Carlo level and then used to include environmental effects in diffusion Monte Carlo. We investigate the performance of this hybrid model in describing the vertical excitation energies of prototypical small molecules solvated in water, namely, methylenecyclopropene and s-trans acrolein. Two polarization regimes are explored where either the dipoles are optimized with respect to the ground-state solute density (polGS) or different sets of dipoles are separately brought to equilibrium with the states involved in the electronic transition (polSS). By comparing with reference supermolecular calculations where both solute and solvent are treated quantum mechanically, we find that the inclusion of the response of the environment to the excitation of the solute leads to superior results than the use of a frozen environment (point charges or polGS), in particular, when the solute-solvent coupling is dominated by electrostatic effects which are well recovered in the polSS condition. QMC/MMpol represents therefore a robust scheme to treat important environmental effects beyond static point charges, combining the accuracy of QMC with the simplicity of a classical approach.

Chromophore-protein coupling beyond nonpolarizable models: understanding absorption in green fluorescent protein.

The nature of the coupling of the photoexcited chromophore with the environment in a prototypical system like green fluorescent protein (GFP) is to date not understood, and its description still defies state-of-the-art multiscale approaches. To identify which theoretical framework of the chromophore-protein complex can realistically capture its essence, we employ here a variety of electronic-structure methods, namely, time-dependent density functional theory (TD-DFT), multireference perturbation theory (NEVPT2 and CASPT2), and quantum Monte Carlo (QMC) in combination with static point charges (QM/MM), DFT embedding (QM/DFT), and classical polarizable embedding through induced dipoles (QM/MMpol). Since structural modifications can significantly affect the photophysics of GFP, we also account for thermal fluctuations through extensive molecular dynamics simulations. We find that a treatment of the protein through static point charges leads to significantly blue-shifted excitation energies and that including thermal fluctuations does not cure the coarseness of the MM description. While TDDFT calculations on large cluster models indicate the need of a responsive protein, this response is not simply electrostatic: An improved description of the protein in the ground state or in response to the excitation of the chromophore via ground-state or state-specific DFT and MMpol embedding does not significantly modify the results obtained with static point charges. Through the use of QM/MMpol in a linear response formulation, a different picture in fact emerges in which the main environment response to the chromophore excitation is the one coupling the transition density and the corresponding induced dipoles. Such interaction leads to significant red-shifts and a satisfactory agreement with full QM cluster calculations at the same level of theory. Our findings demonstrate that, ultimately, faithfully capturing the effects of the environment in GFP requires a quantum treatment of large photoexcited regions but that a QM/classical model can be a useful approximation when extended beyond the electrostatic-only formulation.

State-Specific Embedding Potentials for Excitation-Energy Calculations.

Embedding potentials are frequently used to describe the effect of an environment on the electronic structure of molecules in larger systems, including their excited states. If such excitations are accompanied by significant rearrangements in the electron density of the embedded molecule, large differential polarization effects may take place, which in turn can require state-specific embedding potentials for an accurate theoretical description. We outline here how to extend wave function in density functional theory (WF/DFT) methods to compute the excitation energies of a molecule in a responsive environment through the use of state-specific density-based embedding potentials constructed within a modified subsystem DFT approach. We evaluate the general expression of the ground- and excited-state energy difference of the total system both with the use of state-independent and state-dependent embedding potentials and propose some practical recipes to construct the approximate excited-state DFT density of the active part used to polarize the environment. We illustrate these concepts with the state-independent and state-dependent WF/DFT computation of the excitation energies of p-nitroaniline, acrolein, methylenecyclopropene, and p-nitrophenolate in various solvents.