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 β 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.

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.

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes 1 – 3 . However, how exactly they sense mechanical force remains under investigation 4 . The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels 4 – 8 , but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states 9 – 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 mechanosensation 4 , 11 .

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.

When channels permeable to both ions and water are subjected to osmotic pressure, a small but measurable voltage (on the millivolt scale) called streaming potential is produced. This happens due to a flux coupling between water and the ions. The magnitude of the streaming potential can be used to infer the strength of this coupling, in particular, the ratio of fluxes between water and ions. This ratio can then be linked to the permeation mechanism of the given channel. In the past, streaming potentials have been used to deduce the structure of the channel gramicidin A [1] and have suggested that for certain ion channels like KcsA, water can co-permeate with ions [2, 3]. For potassium channels, ratios of 1-2 waters/ion were measured, implying a mechanism involving alternating water and potassium molecules in the selectivity filter. This finding is seemingly in contrast to other experimental and simulation data [3]. In this work, we set out to reconcile this apparent contradiction by applying both an osmotic pressure (modeled by hydrostatic pressure) and an external potential and changing the contributions of these two driving forces. By large-scale molecular dynamics simulations (more than 100 µs per system), we recover a small but significant coupling between water and ion fluxes for water-ion co-permeating channels and observe no such effect for the case of waterless permeation, confirming the traditional interpretation of streaming potentials. By a detailed analysis of our data, we gain deeper insight into condition-dependent conduction mechanisms of ion channels and identify the pressure-induced conformational changes in ion channels responsible for this. [1] P. A. Rosenberg et al, J. Gen. Physiol 1978 [2] M. Iwamoto et al, J. Neurosci. 2011 [3] C. Alcayaga et al, Biophys. J. 1989 [4] C. Öster et al, Sci. Adv. 2019

Focal Adhesions (FA) are large, multi-protein complexes connecting the cytoskeleton to the extracellular matrix. Their adhesive functionality is tightly regulated by mechanical stress. A key component of FA-associated mechanosensing is vinculin. It consists of a globular head, a proline-rich neck region, and a rod-like tail domain that contains binding sites for many other cytoplasmic proteins. Vinculin can assume either a closed ("inactive") or open (“active”) conformation. The underlying activation mechanism, however, remains yet to be fully understood. Here we employ molecular dynamics (MD) simulation to demonstrate that vinculin activation is greatly facilitated by the binding of vinculin on talin's vinculin binding site. Steered MD simulations reveal that the force required for vinculin activation is drastically reduced, by more than 50%, upon formation of the vinculin-talin complex. To explore this observation further, we use dynamic network fluctuation analysis showing how force propagation through vinculin changes upon complex formation. Interestingly, after talin dissociation, vinculin returns to its native conformation on a submicrosecond time scale, with 60% of its native contacts restored. Our results suggest a rapid dynamic equilibrium between 'tight' and 'loosened' inactive vinculin, which depends on talin and determines the level of mechanical stress required for activation. Our study has important implications for our understanding of mechano-sensing mechanisms at FAs.

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.

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.

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,FAKadopts 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 howFAKassociates with lipid membranes and how membrane interactions unlockFAKautoinhibition to promote activation. Intriguingly, initial binding ofFAKto 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 promoteFAKoligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential forFAKautophosphorylation and resulting cellular activity including cancer cell invasion, while stableFAKoligomerization 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 ofFAKthat is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediatedFAKactivation and signaling at focal adhesions.

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.

Molecular Dynamics (MD) simulations are routinely used to interpret single-molecule atomic force microscopy (AFM) experiments. Most previous studies have focused on proteins with a relatively simple topology and their dissociation or unfolding. When it comes to more complex proteins, the interpretation of force-extension curves can be challenging. We perform the direct comparison of the experimental and simulated unfolding pathways of focal adhesion kinase (FAK), a protein that, due to its complex topology, can unfold in numerous ways. FAK is a membrane-associated enzyme at focal adhesions and its kinase domain is inhibited by a so-called FERM domain. The two domains disassociate under force (Zhou et al 2015), suggesting a mechanosensing role. We examine FAK unfolding in both atomistic MD simulations, involving 1.5M atoms and 40μs of simulation time, and AFM experiments, using an engineered FAK construct to apply force through the membrane-binding basic patch of the FERM domain. We experimentally identify the previously predicted activation step in the first stage of the unfoldings. The further pathway depends on experimental conditions: in the presence of ATP, the kinase domain is stabilised and therefore the FERM domain unfolds first, while without ATP, the opposite is the case. In the corresponding simulations, in the presence of ATP, the FERM domain indeed preferentially unfolds before the kinase, and this tendency is stronger at lower pulling velocities and lower spring constants. From the combined data of MD simulations and AFM experiments, we find that FAK can extend to at least 50nm with the kinase domain still functional and the membrane-binding basic patch still in its original conformation. The thus found “safety margin” of force-activated kinase activity supplies further insight into the physiological role of FAK in force-bearing adhesion complexes of the cell.

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.

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.

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.

AbstractMechanical forces control many cellular processes by eliciting a mechanotransduction response in target cells. The initial steps of mechanotransduction at hemidesmosomes remain undefined in contrast to focal adhesions and adherens junctions. Here, we focus on theC. elegansplectin homolog VAB-10A, the only evolutionary conserved hemidesmosome component. InC. elegans, muscle contractions induce a mechanotransduction pathway in the epidermis through hemidesmosomes. We used CRISPR to precisely remove spectrin repeats (SR) 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 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 can render the central SH3 domain, otherwise in contact with SR4, available for interaction. Collectively, our data strongly argue that the plakin domain plays a central role in mechanotransduction and raise the possibility that VAB-10/plectin might act as a mechanosensor.Summary statementCRISPR-derived deletions reveal the roles of three spectrin repeats and an atypical SH3 domain from the plakin domain of the VAB-10 hemidesmosome component in mechanotransduction duringC. elegansmorphogenesis

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.

Mechanical forces control many cellular processes by eliciting mechanotransduction upon changes in tension (mechanosensing). Whereas mechanosensors acting at focal adhesion and adherens junction are known, they remain undefined for hemidesmosomes. Here, we focus on the plectin homolog VAB-10A, the only evolutionary conserved hemidesmosome component. In , muscles contractions induce a mechanotransduction pathway in the epidermis involving hemidesmosomes. We used CRISPR to precisely remove spectrin repeats (SR) or a partially hidden Src-homology-3 (SH3) domain within the VAB-10 plakin domain. Deleting the SH3 domain in combination with mutations affecting mechanotransduction, or just 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. Interestingly, Molecular Dynamics simulations confirmed that forces acting on VAB-10 relieve the inhibition of the central SH3 domain by the adjacent spectrin repeats. Collectively, our data strongly argue that we identified a hemidesmosome mechanosensor and that the SH3 domain, together with its shielding spectrin repeat, play a key role in mechanosensing.

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.

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.

Biological tissues are subjected to vastly varying levels of mechanical load. Protein regulation by force-induced conformational changes has been established as a crucial mode of mechanosensing. However, protein-based biological materials like skin, cartilage, and tendon can experience excessive stresses all the way up to tissue rupture. Whether and how covalent bond scission in stressed protein materials could contribute to mechanosensing remains unknown. We addressed this question for collagen, the most abundant protein in humans, the major force-bearing component of essentially all tissues, and a heavily crosslinked biopolymer. Using combined rheology and electron paramagnetic resonance (EPR) experiments, we show that physiological levels of stress on collagen fibrils from rat tail tendon lead to the formation of mechano-radicals by covalent bond rupture. Radical formation increases with the level of applied stress and occurs already much before fiber rupture. We identify specific bonds in the lysine-based crosslinks as points of stress concentration in tensed fibers through classical molecular dynamics simulations. Using high-level quantum calculations, we establish the chemical nature of the scission to be homolytic. The radicals subsequently undergo rapid reactions with water, yielding reactive oxygen species. Taken together, our study proposes a new mode of coupling between mechanical and oxidative stress in collagen-based tissues as a missing link between mechanical load and biological processes, from pain sensation to inflammation.