The protein elastin imparts extensibility, elastic recoil, and resilience to tissues including arterial walls, skin, lung alveoli, and the uterus. Elastin and elastin-like peptides are hydrophobic, ...disordered, and undergo liquid-liquid phase separation upon self-assembly. Despite extensive study, the structure of elastin remains controversial. We use molecular dynamics simulations on a massive scale to elucidate the structural ensemble of aggregated elastin-like peptides. Consistent with the entropic nature of elastic recoil, the aggregated state is stabilized by the hydrophobic effect. However, self-assembly does not entail formation of a hydrophobic core. The polypeptide backbone forms transient, sparse hydrogen-bonded turns and remains significantly hydrated even as self-assembly triples the extent of non-polar side chain contacts. Individual chains in the assembly approach a maximally-disordered, melt-like state which may be called the liquid state of proteins. These findings resolve long-standing controversies regarding elastin structure and function and afford insight into the phase separation of disordered proteins.
Free energy simulations are a powerful tool for evaluating the interactions of molecular solutes with lipid bilayers as mimetics of cellular membranes. However, these simulations are frequently ...hindered by systematic sampling errors. This review highlights recent progress in computing free energy profiles for inserting molecular solutes into lipid bilayers. Particular emphasis is placed on a systematic analysis of the free energy profiles, identifying the sources of sampling errors that reduce computational efficiency, and highlighting methodological advances that may alleviate sampling deficiencies. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.
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•Free energy simulations of solute-bilayer systems are prone to sampling errors.•Errors occur during solute adsorption, insertion, and crossing the bilayer center.•Hidden sampling barriers often involve lipid headgroups.•Initial conformations can dramatically influence sampling errors.•Barriers at the bilayer center usually depend on the bilayer's bending modulus.
All molecular dynamics simulations are susceptible to sampling errors, which degrade the accuracy and precision of observed values. The statistical convergence of simulations containing atomistic ...lipid bilayers is limited by the slow relaxation of the lipid phase, which can exceed hundreds of nanoseconds. These long conformational autocorrelation times are exacerbated in the presence of charged solutes, which can induce significant distortions of the bilayer structure. Such long relaxation times represent hidden barriers that induce systematic sampling errors in simulations of solute insertion. To identify optimal methods for enhancing sampling efficiency, we quantitatively evaluate convergence rates using generalized ensemble sampling algorithms in calculations of the potential of mean force for the insertion of the ionic side chain analog of arginine in a lipid bilayer. Umbrella sampling (US) is used to restrain solute insertion depth along the bilayer normal, the order parameter commonly used in simulations of molecular solutes in lipid bilayers. When US simulations are modified to conduct random walks along the bilayer normal using a Hamiltonian exchange algorithm, systematic sampling errors are eliminated more rapidly and the rate of statistical convergence of the standard free energy of binding of the solute to the lipid bilayer is increased 3-fold. We compute the ratio of the replica flux transmitted across a defined region of the order parameter to the replica flux that entered that region in Hamiltonian exchange simulations. We show that this quantity, the transmission factor, identifies sampling barriers in degrees of freedom orthogonal to the order parameter. The transmission factor is used to estimate the depth-dependent conformational autocorrelation times of the simulation system, some of which exceed the simulation time, and thereby identify solute insertion depths that are prone to systematic sampling errors and estimate the lower bound of the amount of sampling that is required to resolve these sampling errors. Finally, we extend our simulations and verify that the conformational autocorrelation times estimated by the transmission factor accurately predict correlation times that exceed the simulation time scalesomething that, to our knowledge, has never before been achieved.
In recent years, atomistic molecular simulations have become a method of choice for studying the interaction of small molecules, peptides, and proteins with biological membranes. Here, we critically ...examine the statistical convergence of equilibrium properties in molecular simulations of two amino acid side-chain analogs, leucine and arginine, in the presence of a hydrated phospholipid bilayer. To this end, the convergence of the standard binding free energy for the reversible insertion of the solutes in the bilayer is systematically assessed by evaluating dozens of separate sets of umbrella sampling calculations for a total simulation time exceeding 400 μs. We identify rare and abrupt transitions in bilayer structure as a function of solute insertion depth. These transitions correspond to the slow reorganization of ionic interactions involving zwitterionic phospholipid headgroups when the solutes penetrate the lipid–water interface and when arginine is forced through the bilayer center. These rare events are shown to constitute hidden sampling barriers that limit the rate of convergence of equilibrium properties and result in systematic sampling errors. Our analysis demonstrates that the difficulty of attaining convergence for lipid bilayer-embedded solutes has, in general, been drastically underestimated. This information will assist future studies in improving accuracy by selecting a more appropriate reaction coordinate or by focusing computational resources on those regions of the reaction coordinate that exhibit slow convergence of equilibrium properties.
Determination of a high-resolution 3D structure of voltage-gated sodium channel Na VAb opens the way to elucidating the mechanism of ion conductance and selectivity. To examine permeation of Na ⁺ ...through the selectivity filter of the channel, we performed large-scale molecular dynamics simulations of Na VAb in an explicit, hydrated lipid bilayer at 0 mV in 150 mM NaCl, for a total simulation time of 21.6 μs. Although the cytoplasmic end of the pore is closed, reversible influx and efflux of Na ⁺ through the selectivity filter occurred spontaneously during simulations, leading to equilibrium movement of Na ⁺ between the extracellular medium and the central cavity of the channel. Analysis of Na ⁺ dynamics reveals a knock-on mechanism of ion permeation characterized by alternating occupancy of the channel by 2 and 3 Na ⁺ ions, with a computed rate of translocation of (6 ± 1) × 10 ⁶ ions⋅s ⁻¹ that is consistent with expectations from electrophysiological studies. The binding of Na ⁺ is intimately coupled to conformational isomerization of the four E177 side chains lining the extracellular end of the selectivity filter. The reciprocal coordination of variable numbers of Na ⁺ ions and carboxylate groups leads to their condensation into ionic clusters of variable charge and spatial arrangement. Structural fluctuations of these ionic clusters result in a myriad of ion binding modes and foster a highly degenerate, liquid-like energy landscape propitious to Na ⁺ diffusion. By stabilizing multiple ionic occupancy states while helping Na ⁺ ions diffuse within the selectivity filter, the conformational flexibility of E177 side chains underpins the knock-on mechanism of Na ⁺ permeation.
Freeze-trapping x-ray crystallography, nuclear magnetic resonance, and computational techniques reveal the distribution of states and their interconversion rates along the reaction pathway of a ...bacterial homodimeric enzyme, fluoroacetate dehalogenase (FAcD). The crystal structure of apo-FAcD exhibits asymmetry around the dimer interface and cap domain, priming one protomer for substrate binding. This asymmetry is dynamically averaged through conformational exchange on a millisecond time scale. During catalysis, the protomer conformational exchange rate becomes enhanced, the empty protomer exhibits increased local disorder, and water egresses. Computational studies identify allosteric pathways between protomers. Water release and enhanced dynamics associated with catalysis compensate for entropic losses from substrate binding while facilitating sampling of the transition state. The studies provide insights into how substrate-coupled allosteric modulation of structure and dynamics facilitates catalysis in a homodimeric enzyme.
The recent elucidation of atomistic structures of Cl
−
channel CFTR provides opportunities for understanding the molecular basis of cystic fibrosis. Despite having been activated through ...phosphorylation and provided with ATP ligands, several near-atomistic cryo-EM structures of CFTR are in a closed state, as inferred from the lack of a continuous passage through a hydrophobic bottleneck region located in the extracellular portion of the pore. Here, we present repeated, microsecond-long molecular dynamics simulations of human CFTR solvated in a lipid bilayer and aqueous NaCl. At equilibrium, Cl
−
ions enter the channel through a lateral intracellular portal and bind to two distinct cationic sites inside the channel pore but do not traverse the narrow, de-wetted bottleneck. Simulations conducted in the presence of a strong hyperpolarizing electric field led to spontaneous Cl
−
translocation events through the bottleneck region of the channel, suggesting that the protein relaxed to a functionally open state. Conformational changes of small magnitude involving transmembrane helices 1 and 6 preceded ion permeation through diverging exit routes at the extracellular end of the pore. The pore bottleneck undergoes wetting prior to Cl
−
translocation, suggesting that it acts as a hydrophobic gate. Although permeating Cl
−
ions remain mostly hydrated, partial dehydration occurs at the binding sites and in the bottleneck. The observed Cl
−
pathway is largely consistent with the loci of mutations that alter channel conductance, anion binding, and ion selectivity, supporting the model of the open state of CFTR obtained in the present study.
Voltage-gated sodium (Na
) channels initiate action potentials in excitable cells, and their function is altered by potent gating-modifier toxins. The α-toxin LqhIII from the deathstalker scorpion ...inhibits fast inactivation of cardiac Na
1.5 channels with IC
= 11.4 nM. Here we reveal the structure of LqhIII bound to Na
1.5 at 3.3 Å resolution by cryo-EM. LqhIII anchors on top of voltage-sensing domain IV, wedged between the S1-S2 and S3-S4 linkers, which traps the gating charges of the S4 segment in a unique intermediate-activated state stabilized by four ion-pairs. This conformational change is propagated inward to weaken binding of the fast inactivation gate and favor opening the activation gate. However, these changes do not permit Na
permeation, revealing why LqhIII slows inactivation of Na
channels but does not open them. Our results provide important insights into the structural basis for gating-modifier toxin binding, voltage-sensor trapping, and fast inactivation of Na
channels.
The heartbeat is initiated by voltage-gated sodium channel NaV1.5, which opens rapidly and triggers the cardiac action potential; however, the structural basis for pore opening remains unknown. Here, ...we blocked fast inactivation with a mutation and captured the elusive open-state structure. The fast inactivation gate moves away from its receptor, allowing asymmetric opening of pore-lining S6 segments, which bend and rotate at their intracellular ends to dilate the activation gate to ∼10 Å diameter. Molecular dynamics analyses predict physiological rates of Na+ conductance. The open-state pore blocker propafenone binds in a high-affinity pose, and drug-access pathways are revealed through the open activation gate and fenestrations. Comparison with mutagenesis results provides a structural map of arrhythmia mutations that target the activation and fast inactivation gates. These results give atomic-level insights into molecular events that underlie generation of the action potential, open-state drug block, and fast inactivation of cardiac sodium channels, which initiate the heartbeat.
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•Mutation of the fast inactivation gate IFM motif allows stable opening of the pore•The intracellular activation gate opens to ∼10 Å, sufficient to conduct hydrated Na+•Molecular dynamics analysis reveals Na+ conductance at approximately physiological rates•The antiarrhythmic drug propafenone binds the open state tightly and blocks the pore
Using a mutation to block fast sodium channel inactivation, the open-state structure of the primary cardiac sodium channel NaV1.5 was captured, revealing the molecular mechanisms for rapid opening and fast inactivation of the pore as well as the receptor site for high-affinity binding of the open-state sodium channel blocker propafenone.
Bacterial voltage-gated sodium channels (BacNavs) serve as models of their vertebrate counterparts. BacNavs contain conserved voltage-sensing and pore-forming domains, but they are homotetramers of ...four identical subunits, rather than pseudotetramers of four homologous domains. Here, we present structures of two NaVAb mutants that capture tightly closed and open states at a resolution of 2.8–3.2 Å. Introduction of two humanizingmutations in the S6 segment (NaVAb/FY: T206F and V213Y) generates a persistently closed form of the activation gate in which the intracellular ends of the four S6 segments are drawn tightly together to block ion permeation completely. This construct also revealed the complete structure of the four-helix bundle that forms the C-terminal domain. In contrast, truncation of the C-terminal 40 residues in NavAb/1–226 captures the activation gate in an open conformation, revealing the open state of a BacNav with intact voltage sensors. Comparing these structures illustrates the full range of motion of the activation gate, from closed with its orifice fully occluded to open with an orifice of ∼10 Å. Molecular dynamics and free-energy simulations confirm designation of NaVAb/1–226 as an open state that allows permeation of hydrated Na⁺, and these results also support a hydrophobic gating mechanism for control of ion permeation. These two structures allow completion of a closed–open–inactivated conformational cycle in a single voltage-gated sodium channel and give insight into the structural basis for state-dependent binding of sodium channel-blocking drugs.
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