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Binding of the spike protein of SARS-CoV-2 to the human angiotensin-converting enzyme 2 (ACE2) receptor triggers translocation of the virus into cells. Both the ACE2 receptor and the spike protein are heavily glycosylated, including at sites near their binding interface. We built fully glycosylated models of the ACE2 receptor bound to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Using atomistic molecular dynamics (MD) simulations, we found that the glycosylation of the human ACE2 receptor contributes substantially to the binding of the virus. Interestingly, the glycans at two glycosylation sites, N90 and N322, have opposite effects on spike protein binding. The glycan at the N90 site partly covers the binding interface of the spike RBD. Therefore, this glycan can interfere with the binding of the spike protein and protect against docking of the virus to the cell. By contrast, the glycan at the N322 site interacts tightly with the RBD of the ACE2-bound spike protein and strengthens the complex. Remarkably, the N322 glycan binds to a conserved region of the spike protein identified previously as a cryptic epitope for a neutralizing antibody. By mapping the glycan binding sites, our MD simulations aid in the targeted development of neutralizing antibodies and SARS-CoV-2 fusion inhibitors.

We study lipid translocation (“flip-flop”) between the leaflets of planar lipid bilayers with artificial intelligence (AI) guided transition path sampling (TPS). Rare flip-flops compete with biological machineries that actively establish asymmetric lipid compositions. By initializing molecular dynamics simulations near transition states, AI for molecular mechanism discovery (AIMMD) captures lipid flip-flop without biasing the dynamics. Four distinct mechanisms of flip-flop emerge, as encoded in neural networks trained on the fly to predict the commitment probability (or “committor”) for a lipid to proceed to one or the other leaflet. Whereas coarse-grained DMPC lipids “tunnel” through the hydrophobic bilayer, unaided by water, atomistic DMPC lipids cross the membrane through spontaneously formed water nanopores. In an atomistic plasma membrane mimetic, cholesterol tunnels unaided by water, whereas PLPC lipids exploit both transient water threads and nanodroplets to cross a locally thinned membrane, as seen also in an atomistic bilayer of DSPC lipids. Remarkably, in the high (~660) dimensional feature space of the deep neural networks in AIMMD, the reaction coordinate becomes effectively linear, in line with Cover’s theorem and consistent with the idea of dominant reaction tubes. Biological membranes are tightly sealed. Here, Post and Hummer capture rare lipid flip-flop events and the formation of water nanopores across lipid bilayers with AI-guided simulations. The mechanism is encoded in close-to-linear neural networks.

We develop a detailed description of protein translational and rotational diffusion in concentrated solution on the basis of allatom molecular dynamics simulations in explicit solvent. Our systems contain up to 540 fully flexible proteins with 3.6 million atoms. In concentrated protein solutions (100 mg/mL and higher), the proteins ubiquitin and lysozyme, as well as the protein domains third IgG-binding domain of protein G and villin headpiece, diffuse not as isolated particles, but as members of transient clusters between which they constantly exchange. A dynamic cluster model nearly quantitatively explains the increase in viscosity and the decrease in protein diffusivity with protein volume fraction, which both exceed the predictions from widely used colloid models. The Stokes–Einstein relations for translational and rotational diffusion remain valid, but the effective hydrodynamic radius grows linearly with protein volume fraction. This increase follows the observed increase in cluster size and explains the more dramatic slowdown of protein rotation compared with translation. Baxter’s sticky-sphere model of colloidal suspensions captures the concentration dependence of cluster size, viscosity, and rotational and translational diffusion. The consistency between simulations and experiments for a diverse set of soluble globular proteins indicates that the cluster model applies broadly to concentrated protein solutions, with equilibrium dissociation constants for nonspecific protein–protein binding in the Kd ≈ 10-mM regime.

The recent availability of long equilibrium simulations of protein folding in atomistic detail for more than 10 proteins allows us to identify the key interactions driving folding. We find that the collective fraction of native amino acid contacts, Q , captures remarkably well the transition states for all the proteins with a folding free energy barrier. Going beyond this global picture, we devise two different measures to quantify the importance of individual interresidue contacts in the folding mechanism: (i) the log-ratio of lifetimes of contacts during folding transition paths and in the unfolded state and (ii) a Bayesian measure of how predictive the formation of each contact is for being on a transition path. Both of these measures indicate that native, or near-native, contacts are important for determining mechanism, as might be expected. More remarkably, however, we found that for almost all the proteins, with the designed protein α ₃D being a notable exception, nonnative contacts play no significant part in determining folding mechanisms.

Channelrhodopsin-2 (ChR2) is a light-sensitive ion channel widely used in optogenetics. Photoactivation triggers a trans-to-cis isomerization of a covalently bound retinal. Ensuing conformational changes open a cation-selective channel. We explore the structural dynamics in the early photocycle leading to channel opening by classical (MM) and quantum mechanical (QM) molecular simulations. With QM/MM simulations, we generated a protein-adapted force field for the retinal chromophore, which we validated against absorption spectra. In a 4-μs MM simulation of a dark-adapted ChR2 dimer, water entered the vestibules of the closed channel. Retinal all-trans to 13-cis isomerization, simulated with metadynamics, triggered a major restructuring of the charge cluster forming the channel gate. On a microsecond time scale, water penetrated the gate to form a membrane-spanning preopen pore between helices H1, H2, H3, and H7. This influx of water into an ion-impermeable preopen pore is consistent with time-resolved infrared spectroscopy and electrophysiology experiments. In the retinal 13-cis state, D253 emerged as the proton acceptor of the Schiff base. Upon proton transfer from the Schiff base to D253, modeled by QM/MM simulations, we obtained an early-M/P₂390–like intermediate. Rapid rotation of the unprotonated Schiff base toward the cytosolic side effectively prevents its reprotonation from the extracellular side. From MM and QM simulations, we gained detailed insight into the mechanism of ChR2 photoactivation and early events in pore formation. By rearranging the network of charges and hydrogen bonds forming the gate, water emerges as a key player in light-driven ChR2 channel opening.

Misfolded endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome in a process known as ER-associated protein degradation (ERAD). ERAD of misfolded luminal ER proteins (ERAD-L) is mediated by the Hrd1 complex, composed of the ubiquitin ligase Hrd1 and four additional proteins (Hrd3, Der1, Usa1, and Yos9). Wu et al. report a cryo–electron microscopy structure of the active Hrd1 complex from yeast and, based on this structure, developed a model for how substrates are recognized and retrotranslocated. They propose that Hrd3 and Yos9 jointly create a luminal binding site for misfolded glycoproteins. Hrd1 and Der1 form “half-channels” juxtaposed in a thinned section of the ER membrane, which allows a polypeptide loop of an ERAD-L substrate to move through it. Science , this issue p. eaaz2449 The Hrd1 complex retrotranslocates misfolded luminal endoplasmic reticulum proteins through “half-channels” juxtaposed in a thinned membrane. Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo–electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two “half-channels” with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.

Gasdermin-D (GSDMD) is the ultimate effector of pyroptosis, a form of programmed cell death associated with pathogen invasion and inflammation. After proteolytic cleavage by caspases, the GSDMD N-terminal domain (GSDMD NT ) assembles on the inner leaflet of the plasma membrane and induces the formation of membrane pores. We use atomistic molecular dynamics simulations to study GSDMD NT monomers, oligomers, and rings in an asymmetric plasma membrane mimetic. We identify distinct interaction motifs of GSDMD NT with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) and phosphatidylserine (PS) headgroups and describe their conformational dependence. Oligomers are stabilized by shared lipid binding sites between neighboring monomers acting akin to double-sided tape. We show that already small GSDMD NT oligomers support stable, water-filled, and ion-conducting membrane pores bounded by curled beta-sheets. In large-scale simulations, we resolve the process of pore formation from GSDMD NT arcs and lipid efflux from partial rings. We find that high-order GSDMD NT oligomers can crack under the line tension of 86 pN created by an open membrane edge to form the slit pores or closed GSDMD NT rings seen in atomic force microscopy experiments. Our simulations provide a detailed view of key steps in GSDMD NT -induced plasma membrane pore formation, including sublytic pores that explain nonselective ion flux during early pyroptosis.

Nonequilibrium pulling experiments provide detailed information about the thermodynamic and kinetic properties of molecules. We show that unperturbed free energy profiles as a function of molecular extension can be obtained rigorously from such experiments without using work-weighted position histograms. An inverse Weierstrass transform is used to relate the system free energy obtained from the Jarzynski equality directly to the underlying molecular free energy surface. An accurate approximation for the free energy surface is obtained by using the method of steepest descent to evaluate the inverse transform. The formalism is applied to simulated data obtained from a kinetic model of RNA folding, in which the dynamics consists of jumping between linker-dominated folded and unfolded free energy surfaces.

In typical force spectroscopy experiments, a small biomolecule is attached to a soft polymer linker that is pulled with a relatively large bead or cantilever. At constant force, the total extension stochastically changes between two (or more) values, indicating that the biomolecule undergoes transitions between two (or several) conformational states. In this paper, we consider the influence of the dynamics of the linker and mesoscopic pulling device on the force-dependent rate of the conformational transition extracted from the time dependence of the total extension, and the distribution of rupture forces in force-clamp and force-ramp experiments, respectively. For these different experiments, we derive analytic expressions for the observables that account for the mechanical response and dynamics of the pulling device and linker. Possible artifacts arise when the characteristic times of the pulling device and linker become comparable to, or slower than, the lifetimes of the metastable conformational states, and when the highly anharmonic regime of stretched linkers is probed at high forces. We also revisit the problem of relating force-clamp and force-ramp experiments, and identify a linker and loading rate-dependent correction to the rates extracted from the latter. The theory provides a framework for both the design and the quantitative analysis of force spectroscopy experiments by highlighting, and correcting for, factors that complicate their interpretation.

Diffusion on a low-dimensional free-energy surface is a remarkably successful model for the folding dynamics of small single-domain proteins. Complicating the interpretation of both simulations and experiments is the expectation that the effective diffusion coefficient D will in general depend on the position along the folding coordinate, and this dependence may vary for different coordinates. Here we explore the position dependence of D, its connection to protein internal friction, and the consequences for the interpretation of single-molecule experiments. We find a large decrease in D from unfolded to folded, for reaction coordinates that directly measure fluctuations in Cartesian configuration space, including those probed in single-molecule experiments. In contrast, D is almost independent of Q, the fraction of native amino acid contacts: Near the folded state, small fluctuations in position cause large fluctuations in Q, and vice versa for the unfolded state. In general, position-dependent free energies and diffusion coefficients for any two good reaction coordinates that separate reactant, product, and transition states, are related by a simple transformation, as we demonstrate. With this transformation, we obtain reaction coordinates with position-invariant D. The corresponding free-energy surfaces allow us to justify the assumptions used in estimating the speed limit for protein folding from experimental measurements of the reconfiguration time in the unfolded state, and also reveal intermediates hidden in the original free-energy projection. Lastly, we comment on the design of future single-molecule experiments that probe the position dependence of D directly.