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The hydrophobic gating model, in which ion permeation is inhibited by the hydrophobicity, rather than a physical occlusion of the nanopore, functions in various ion channels including potassium channels. Available research focused on the energy barriers for ion/water conduction due to the hydrophobicity, whereas how hydrophobic gating affects the function and structure of channels remains unclear. Here, we use potassium channels as examples and conduct molecular dynamics simulations to investigate this problem. Our simulations find channel activities (ion currents) highly correlated with cavity hydration level, implying insufficient hydration as a barrier for ion permeation. Enforced cavity dehydration successfully induces conformational transitions between known channel states, further implying cavity dewetting as a key step in the gating procedure of potassium channels utilizing different activation mechanisms. Our work reveals how the cavity dewetting is coupled to structural changes of potassium channels and how it affects channel activity. The conclusion may also apply to other ion channels. The hydrophobic gating is believed to function in various ion channels. Here, the authors use MD simulations to assess how dewetting of the channel pore modulates the function and conformational transition of the potassium channels.

Cell membranes actively participate in the regulation of protein structure and function. In this work, we conduct molecular dynamics simulations to investigate how different membrane environments affect protein structure and function in the case of MthK, a potassium channel. We observe different ion permeation rates of MthK in membranes with different properties, and ascribe them to a shift of the conformational equilibrium between two states of the channel that differ according to whether a transmembrane helix has a kink. Further investigations indicate that two key residues in the kink region mediate a crosstalk between two gates at the selectivity filter and the central cavity, respectively. Opening of one gate eventually leads to closure of the other. Our simulations provide an atomistic model of how lipid-protein interactions affect the conformational equilibrium of a membrane protein. The gating mechanism revealed for MthK may also apply to other potassium channels. Potassium (K+) channels, such as MthK, are essentional for many biological processes, but how lipid-protein interactions regulate ion permeation of K+ channels remained unclear. Here authors conducted molecular dynamics simulations of MthK and observed different ion permeation rates of MthK in membranes with different properties.

Human transthyretin (TTR) is implicated in several fatal forms of amyloidosis. Many mutations of TTR have been identified; most of these are pathogenic, but some offer protective effects. The molecular basis underlying the vastly different fibrillation behaviours of these TTR mutants is poorly understood. Here, on the basis of neutron crystallography, native mass spectrometry and modelling studies, we propose a mechanism whereby TTR can form amyloid fibrils via a parallel equilibrium of partially unfolded species that proceeds in favour of the amyloidogenic forms of TTR. It is suggested that unfolding events within the TTR monomer originate at the C-D loop of the protein, and that destabilising mutations in this region enhance the rate of TTR fibrillation. Furthermore, it is proposed that the binding of small molecule drugs to TTR stabilises non-amyloidogenic states of TTR in a manner similar to that occurring for the protective mutants of the protein. A number of disease-causing human transthyretin (TTR) mutations are known to lead to amyloid formation. Here the authors combine neutron crystallography, native mass spectrometry and modelling studies to characterize the T119M and S52P-TTR mutants, providing mechanistic insights into TTR amyloidosis.

Aquaporins and aquaglyceroporins form a family of pore proteins that facilitate the efficient and selective flux of small solutes across biological membranes. We studied the selectivity of aquaporin-1 (AQP1) and the bacterial glycerol facilitator, GlpF, for O₂, CO₂, NH₃, glycerol, urea, and water. Using molecular dynamics simulations, we calculated potentials of mean force for solute permeation along the aquaporin channels and compared them with the alternative pathway across the lipid bilayer. For small solutes permeating through AQP1, a remarkable anticorrelation between permeability and solute hydrophobicity was observed, whereas the opposite trend was observed for permeation through the membrane. This finding renders AQP1 a selective filter for small polar solutes, whereas GlpF was found to be highly permeable for small solutes and permeable for larger solutes. Surprisingly, not solute-channel but water-channel interactions were found to be the key determinant underlying the selectivity mechanism of aquaporins. Hence, a hydrophobic effect, together with steric restraints, determines the selectivity of aquaporins.

Potassium (K⁺) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K⁺ channels gated at their selectivity filter (SF), including many two-pore domain K⁺ (K2P) channels, voltage-gated hERG (human ether-à-go-go–related gene) channels and calcium (Ca2+)–activated big-conductance potassium (BK)–type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K⁺ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K⁺ channel activators and highlight a filter gating machinery that is conserved across different families of K⁺ channels with implications for rational drug design.

Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.

The accurate calculation of the binding free energy for arbitrary ligand–protein pairs is a considerable challenge in computer-aided drug discovery. Recently, it has been demonstrated that current state-of-the-art molecular dynamics (MD) based methods are capable of making highly accurate predictions. Conventional MD-based approaches rely on the first principles of statistical mechanics and assume equilibrium sampling of the phase space. In the current work we demonstrate that accurate absolute binding free energies (ABFE) can also be obtained via theoretically rigorous non-equilibrium approaches. Our investigation of ligands binding to bromodomains and T4 lysozyme reveals that both equilibrium and non-equilibrium approaches converge to the same results. The non-equilibrium approach achieves the same level of accuracy and convergence as an equilibrium free energy perturbation (FEP) method enhanced by Hamiltonian replica exchange. We also compare uni- and bi-directional non-equilibrium approaches and demonstrate that considering the work distributions from both forward and reverse directions provides substantial accuracy gains. In summary, non-equilibrium ABFE calculations are shown to yield reliable and well-converged estimates of protein–ligand binding affinity. Molecular dynamics-based approaches to calculate absolute protein–ligand binding free energy often rely on equilibrium free energy perturbation (FEP) protocols. Here, the authors study ligands binding to bromodomains and T4 lysozyme and find that both equilibrium and non-equilibrium approaches converge to the same results with the non-equilibrium method converging faster than FEP.

A recent molecular dynamics investigation into the stability of hemoglobin concluded that the unliganded protein is only stable in the T state when a solvent box is used in the simulations that is ten times larger than what is usually employed (El Hage et al., 2018). Here, we express three main concerns about that study. In addition, we find that with an order of magnitude more statistics, the reported box size dependence is not reproducible. Overall, no significant effects on the kinetics or thermodynamics of conformational transitions were observed.

Biological function of proteins is frequently associated with the formation of complexes with small-molecule ligands. Experimental structure determination of such complexes at atomic resolution, however, can be time-consuming and costly. Computational methods for structure prediction of protein/ligand complexes, particularly docking, are as yet restricted by their limited consideration of receptor flexibility, rendering them not applicable for predicting protein/ligand complexes if large conformational changes of the receptor upon ligand binding are involved. Accurate receptor models in the ligand-bound state (holo structures), however, are a prerequisite for successful structure-based drug design. Hence, if only an unbound (apo) structure is available distinct from the ligand-bound conformation, structure-based drug design is severely limited. We present a method to predict the structure of protein/ligand complexes based solely on the apo structure, the ligand and the radius of gyration of the holo structure. The method is applied to ten cases in which proteins undergo structural rearrangements of up to 7.1 A backbone RMSD upon ligand binding. In all cases, receptor models within 1.6 A backbone RMSD to the target were predicted and close-to-native ligand binding poses were obtained for 8 of 10 cases in the top-ranked complex models. A protocol is presented that is expected to enable structure modeling of protein/ligand complexes and structure-based drug design for cases where crystal structures of ligand-bound conformations are not available.

An all-atom protein force field, CHARMM36m, offers improved accuracy for simulating intrinsically disordered peptides and proteins. The all-atom additive CHARMM36 protein force field is widely used in molecular modeling and simulations. We present its refinement, CHARMM36m ( http://mackerell.umaryland.edu/charmm_ff.shtml ), with improved accuracy in generating polypeptide backbone conformational ensembles for intrinsically disordered peptides and proteins.