A molecular mechanism for proton-dependent gating in KcsA

Activation gating in KcsA is elicited by changes in intracellular proton concentration. Thompson et al. [1] identified a charge cluster around the inner gate that plays a key role in defining proton activation in KcsA. Here, through functional and spectroscopic approaches, we confirmed the role of this charge cluster and now provide a mechanism of pH-dependent gating. Channel opening is driven by a set of electrostatic interactions that include R117, E120 and E118 at the bottom of TM2 and H25 at the end of TM1. We propose that electrostatic compensation in this charge cluster stabilizes the closed conformation at neutral pH and that its disruption at low pH facilitates the transition to the open conformation by means of helix-helix repulsion.     

Mechanically induced titin kinase activation studied by force-probe molecular dynamics simulations

The conversion of mechanical stress into a biochemical signal in a muscle cell requires a force sensor. Titin kinase, the catalytic domain of the elastic muscle protein titin, has been suggested as a candidate. Its activation requires major conformational changes resulting in the exposure of its active site. Here, force-probe molecular dynamics simulations were used to obtain insight into the tension-induced activation mechanism. We find evidence for a sequential mechanically induced opening of the catalytic site without complete domain unfolding. Our results suggest the rupture of two terminal beta-sheets as the primary unfolding steps. The low force resistance of the C-terminal relative to the N-terminal beta-sheet is attributed to their different geometry. A subsequent rearrangement of the autoinhibitory tail is seen to lead to the exposure of the active site, as is required for titin kinase activity. These results support the hypothesis of titin kinase as a force sensor.     

A possible molecular mechanism of hanatoxin binding-modified gating in voltage-gated K+-channels

While S4 is known as the voltage sensor in voltage-gated potassium channels, the carboxyl terminus of S3 (S3C) is of particular interest concerning the site for gating modifier toxins like hanatoxin. The thus derived helical secondary structural arrangement for S3C, as well as its surrounding environment, has since been intensively and vigorously debated. Our previous structural analysis based on molecular simulation has provided sufficient information to describe reasonable docking conformation and further experimental designs (Lou et al., 2002. J. Mol. Recognit. 15: 175-179). However, if one only relies on such information, more advanced structure-functional interpretations for the roles S3C may play in the modification of gating behavior upon toxin binding will remain unknown. In order to have better understanding of the molecular details regarding this issue, we have performed the docking simulation with the S3C sequence from the hanatoxin-insensitive K+-channel, shaker, and analyzed the conformational changes resulting from such docking. Compared with other functional data from previous studies with respect to the proximity of the S3-S4 linker region, we suggested a significant movement of drk1 S3C, but not shaker S3C, in the direction presumably towards S4, which was comprehended as a possible factor interfering with S4 translocation during drk1 gating in the presence of toxin. In combination with the discussions for structural roles of the length of the S3-S4 linker, a possible molecular mechanism to illustrate the hanatoxin binding-modified gating is proposed.     

Towards a molecular understanding of titin.

Titin is at present the largest known protein (M(r) 3000 kDa) and its expression is restricted to vertebrate striated muscle. Single molecules span from M- to Z-lines and therefore over 1 micron. We have isolated cDNAs encoding five distant titin A-band epitopes, extended their sequences and determined 30 kb (1000 kDa) of the primary structure of titin. Sequences near the M-line encode a kinase domain and are closely related to the C-terminus of twitchin from Caenorhabditis elegans. This suggests that the function of this region in the titin/twitchin family is conserved throughout the animal kingdom. All other A-band sequences consist of 100 amino acid (aa) repeats predicting immunoglobulin-C2 and fibronectin type III globular domains. These domains are arranged into highly ordered 11 domain super-repeat patterns likely to match the myosin helix repeat in the thick filament. Expressed titin fragments bind to the LMM part of myosin and C-protein. Binding strength increases with the number of domains involved, indicating a cumulative effect of multiple binding sites for myosin along the titin molecule. We conclude that A-band titin is likely to be involved in the ordered assembly of the vertebrate thick filament.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

A mechanical model of the gating spring mechanism of stereocilia

The stereocilium is the basic sensory unit of nature's mechanotransducers, which include the cochlear and vestibular organs. In noisy environments, stereocilia display high sensitivity to miniscule stimuli, effectively dealing with a situation that is a design challenge in micro systems. The gating spring hypothesis suggests that the mechanical stiffness of stereocilia bundle is softened by tip-link gating in combination with active bundle movement, contributing to the nonlinear amplification of miniscule stimuli. To demonstrate that the amplification is induced mechanically by the gating as hypothesized, we developed a biomimetic model of stereocilia and fabricated the model at the macro scale. The model consists of an inverted pendulum array with bistable buckled springs at its tips, which represent the mechanically gated ion channel. Model simulations showed that at the moment of gating, instantaneous stiffness softening generates an increase in response magnitude, which then sequentially occurs as the number of gating increases. This amplification mechanism appeared to be robust to the change of model parameters. Experimental data from the fabricated macro model also showed a significant increase in the open probability and pendulum deflection at the region having a smaller input magnitude. The results demonstrate that the nonlinear amplification of miniscule stimuli is mechanically produced by stiffness softening from channel gating.     

Toward elucidating the heat activation mechanism of the TRPV1 channel gating by molecular dynamics simulation

As a key cellular sensor, the TRPV1 cation channel undergoes a gating transition from a closed state to an open state in response to various physical and chemical stimuli including noxious heat. Despite years of study, the heat activation mechanism of TRPV1 gating remains enigmatic at the molecular level. Toward elucidating the structural and energetic basis of TRPV1 gating, we have performed molecular dynamics (MD) simulations (with cumulative simulation time of 3 μs), starting from the high‐resolution closed and open structures of TRPV1 solved by cryo‐electron microscopy. In the closed‐state simulations at 30°C, we observed a stably closed channel constricted at the lower gate (near residue I679), while the upper gate (near residues G643 and M644) is dynamic and undergoes flickery opening/closing. In the open‐state simulations at 60°C, we found higher conformational variation consistent with a large entropy increase required for the heat activation, and both the lower and upper gates are dynamic with transient opening/closing. Through ensemble‐based structural analyses of the closed state versus the open state, we revealed pronounced closed‐to‐open conformational changes involving the membrane proximal domain (MPD) linker, the outer pore, and the TRP helix, which are accompanied by breaking/forming of a network of closed/open‐state specific hydrogen bonds. By comparing the closed‐state simulations at 30°C and 60°C, we observed heat‐activated conformational changes in the MPD linker, the outer pore, and the TRP helix that resemble the closed‐to‐open conformational changes, along with partial formation of the open‐state specific hydrogen bonds. Some of the residues involved in the above key hydrogen bonds were validated by previous mutational studies. Taken together, our MD simulations have offered rich structural and dynamic details beyond the static structures of TRPV1, and promising targets for future mutagenesis and functional studies of the TRPV1 channel. Proteins 2016; 84:1938–1949. © 2016 Wiley Periodicals, Inc.     

An energy-efficient gating mechanism in the acetylcholine receptor channel suggested by molecular and Brownian dynamics

Acetylcholine receptors mediate electrical signaling between nerve and muscle by opening and closing a transmembrane ion conductive pore. Molecular and Brownian dynamics simulations are used to shed light on the location and mechanism of the channel gate. Four separate 5 ns molecular dynamics simulations are carried out on the imaged structure of the channel, a hypothetical open structure with a slightly wider pore and a mutant structure in which a central ring of hydrophobic residues is replaced by polar groups. Water is found to partially evacuate the pore during molecular simulations of the imaged structure, whereas ions face a large energy barrier and do not conduct through the channel in Brownian dynamics simulations. The pore appears to be in a closed configuration despite containing an unobstructed pathway across the membrane as a series of hydrophobic residues in the center of the channel provide an unfavorable home to water and ions. When the channel is widened slightly, water floods into the channel and ions conduct at a rate comparable to the currents measured experimentally in open channels. The pore remains permeable to ions provided the extracellular end of the pore-lining helix is restrained near the putative open configuration to mimic the presence of the ligand binding domain. Replacing some of the hydrophobic residues with polar ones decreases the barrier for ion permeation but does not result in significant currents. The channel is posited to utilize an energy efficient gating mechanism in which only minor conformational changes of the hydrophobic region of the pore are required to create macroscopic changes in conductance.     

An ion gating mechanism of gastric H,K-ATPase based on molecular dynamics simulations

Gastric H,K-ATPase is an electroneutral transmembrane pump that moves protons from the cytoplasm of the parietal cell into the gastric lumen in exchange for potassium ions. The mechanism of transport against the established electrochemical gradients includes intermediate conformations in which the transferred ions are trapped (occluded) within the membrane domain of the pump. The pump cycle involves switching between the E1 and E2P states. Molecular dynamics simulations on homology models of the E2P and E1 states were performed to investigate the mechanism of K⁺ movement in this enzyme. We performed separate E2P simulations with one K⁺ in the luminal channel, one K⁺ ion in the occlusion site, two K⁺ ions in the occlusion site, and targeted molecular dynamics from E2P to E1 with two K⁺ ions in the occlusion site. The models were inserted into a lipid bilayer system and were stable over the time course of the simulations, and K⁺ ions in the channel moved to a consistent location near the center of the membrane domain, thus defining the occlusion site. The backbone carbonyl oxygen from residues 337 through 342 on the nonhelical turn of M4, as well as side-chain oxygen from E343, E795, and E820, participated in the ion occlusion. A single water molecule was stably bound between the two K⁺ ions in the occlusion site, providing an additional ligand and partial shielding the positive charges from one another. Targeted molecular dynamics was used to transform the protein from the E2P to the E1 state (two K⁺ ions to the cytoplasm). This simulation identified the separation of the water column in the entry channel as the likely gating mechanism on the luminal side. A hydrated exit channel also formed on the cytoplasmic side of the occlusion site during this simulation. Hence, water molecules became available to hydrate the ions. The movement of the M1M2 transmembrane segments, and the displacement of residues Q159, E160, Q110, and T152 during the conformational change, as well as the motions of E343 and L346, acted as the cytoplasmic-side gate.     

A loosened gating mechanism of RIG-I leads to autoimmune disorders

DDX58 encodes RIG-I, a cytosolic RNA sensor that ensures immune surveillance of nonself RNAs. Individuals with RIG-I_E510V and RIG-I_Q517H mutations have increased susceptibility to Singleton-Merten syndrome (SMS) defects, resulting in tissue-specific (mild) and classic (severe) phenotypes. The coupling between RNA recognition and conformational changes is central to RIG-I RNA proofreading, but the molecular determinants leading to dissociated disease phenotypes remain unknown. Herein, we employed hydrogen/deuterium exchange mass spectrometry (HDX-MS) and single molecule magnetic tweezers (MT) to precisely examine how subtle conformational changes in the helicase insertion domain (HEL2i) promote impaired ATPase and erroneous RNA proofreading activities. We showed that the mutations cause a loosened latch-gate engagement in apo RIG-I, which in turn gradually dampens its self RNA (Cap2 moiety:m7G cap and N_1-2-2′-O-methylation RNA) proofreading ability, leading to increased immunopathy. These results reveal HEL2i as a unique checkpoint directing two specialized functions, i.e. stabilizing the CARD2-HEL2i interface and gating the helicase from incoming self RNAs; thus, these findings add new insights into the role of HEL2i in the control of antiviral innate immunity and autoimmunity diseases.     

A kinetic molecular model of the reversible unfolding and refolding of titin under force extension.

Molecular elasticity is a physicomechanical property that is associated with a select number of polypeptides and proteins, such as the giant muscle protein, titin, and the extracellular matrix protein, tenascin. Both proteins have been the subject of atomic force microscopy (AFM), laser tweezer, and other in vitro methods for examining the effects of force extension on the globular (FNIII/Ig-like) domains that comprise each protein. In this report we present a time-dependent method for simulating AFM force extension and its effect on FNIII/Ig domain unfolding and refolding. This method treats the unfolding and refolding process as a standard three-state protein folding model (U right arrow over left arrow T right arrow over left arrow F, where U is the unfolded state, T is the transition or intermediate state, and F is the fully folded state), and integrates this approach within the wormlike chain (WLC) concept. We simulated the effect of AFM tip extension on a hypothetical titin molecule comprised of 30 globular domains (Ig or FNIII) and 25% Pro-Glu-Val-Lys (PEVK) content, and analyzed the unfolding and refolding processes as a function of AFM tip extension, extension rate, and variation in PEVK content. In general, we find that the use of a three-state protein-folding kinetic-based model and the implicit inclusion of PEVK domains can accurately reproduce the experimental force-extension curves observed for both titin and tenascin proteins. Furthermore, our simulation data indicate that PEVK domains exhibit extensibility behavior, assist in the unfolding and refolding of FNIII/Ig domains in the titin molecule, and act as a force "buffer" for the FNIII/Ig domains, particularly at low and moderate extension forces.     

Structural models of the MscL gating mechanism.

Three-dimensional structural models of the mechanosensitive channel of large conductance, MscL, from the bacteria Mycobacterium tuberculosis and Escherichia coli were developed for closed, intermediate, and open conformations. The modeling began with the crystal structure of M. tuberculosis MscL, a homopentamer with two transmembrane alpha-helices, M1 and M2, per subunit. The first 12 N-terminal residues, not resolved in the crystal structure, were modeled as an amphipathic alpha-helix, called S1. A bundle of five parallel S1 helices are postulated to form a cytoplasmic gate. As membrane tension induces expansion, the tilts of M1 and M2 are postulated to increase as they move away from the axis of the pore. Substantial expansion is postulated to occur before the increased stress in the S1 to M1 linkers pulls the S1 bundle apart. During the opening transition, the S1 helices and C-terminus amphipathic alpha-helices, S3, are postulated to dock parallel to the membrane surface on the perimeter of the complex. The proposed gating mechanism reveals critical spatial relationships between the expandable transmembrane barrel formed by M1 and M2, the gate formed by S1 helices, and "strings" that link S1s to M1s. These models are consistent with numerous experimental results and modeling criteria.     

A molecular dynamics study of gating in dioxolane-linked gramicidin A channels.

The gating transition of the RR and SS dioxolane ring-linked gramicidin A channels were studied with molecular dynamics simulations using a detailed atomic model. It was found that the probable reaction path, describing the transition of the ring from the exterior to the interior of the channel where it blocked the permeation pathway, involved several steps including the isomerization of the transpeptide plane dihedral angle of Val1. Reaction coordinates along this pathway were defined, and the transition rates between the stable conformers were calculated. It was found, in good accord with experimental observations, that the calculated blocking rate for the RR-linked channel was 280/s with a mean blocking time of 0.04 ms, whereas such blocking did not occur in the case of the SS-linked channel. An important observation is that the resulting lifetime for the blocked state of the RR-linked channel was in good accord with the experimental observations only when the calculations were performed in the presence of a potassium ion inside the channel.     

Steered molecular dynamics studies of titin I1 domain unfolding

The cardiac muscle protein titin, responsible for developing passive elasticity and extensibility of muscle, possesses about 40 immunoglobulin-like (Ig) domains in its I-band region. Atomic force microscopy (AFM) and steered molecular dynamics (SMD) have been successfully combined to investigate the reversible unfolding of individual Ig domains. However, previous SMD studies of titin I-band modules have been restricted to I27, the only structurally known Ig domain from the distal region of the titin I-band. In this paper we report SMD simulations unfolding I1, the first structurally available Ig domain from the proximal region of the titin I-band. The simulations are carried out with a view toward upcoming atomic force microscopy experiments. Both constant velocity and constant force stretching have been employed to model mechanical unfolding of oxidized I1, which has a disulfide bond bridging beta-strands C and E, as well as reduced I1, in which the disulfide bridge is absent. The simulations reveal that I1 is protected against external stress mainly through six interstrand hydrogen bonds between its A and B beta-strands. The disulfide bond enhances the mechanical stability of oxidized I1 domains by restricting the rupture of backbone hydrogen bonds between the A'- and G-strands. The disulfide bond also limits the maximum extension of I1 to approximately 220 A. Comparison of the unfolding pathways of I1 and I27 are provided and implications to AFM experiments are discussed.     

The molecular mechanism behind resistance of the kinase FLT3 to the inhibitor quizartinib

Fms‐like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is a drug target for leukemias. Several potent inhibitors of FLT3 exist, and bind to the inactive form of the enzyme. Unfortunately, resistance due to mutations in the kinase domain of FLT3 limits the therapeutic effects of these inhibitors. As in many other cases, it is not straightforward to explain why certain mutations lead to drug resistance. Extensive fully atomistic molecular dynamics (MD) simulations of FLT3 were carried out with an inhibited form (FLT‐quizartinib complex), a free (apo) form, and an active conformation. In all cases, both the wild type (wt) proteins and two resistant mutants (D835F and Y842H) were studied. Analysis of the simulations revealed that impairment of protein‐drug interactions cannot explain the resistance mutations in question. Rather, it appears that the active state of the mutant forms is perturbed by the mutations. It is therefore likely that perturbation of deactivation of the protein (which is necessary for drug binding) is responsible for the reduced affinity of the drug to the mutants. Importantly, this study suggests that it is possible to explain the source of resistance by mutations in FLT3 by an analysis of unbiased MD simulations.     

The genetics and molecular biology of the titin/connectin-like proteins of invertebrates

Conclusions A substantial amount of information has been gathered about the structure and function of twitchin/titin-related proteins in the invertebrates. This has been obtained through sequence analysis and the analysis of loss-offunction phenotypes inC. elegans andDrosophila. Nevertheless, a number of fascinating questions remain, including: (i) Why are these invertebrate proteins all of approx. 700–800 kDa? In terms of sarcomeric organization, what is the significance of this size? (ii) Why do three of these proteins consist of a mixture of Ig and Fn domains, whereas UNC-89 contains only Ig domains? This is even more interesting because the structures of Ig and Fn domains are very similar (118). What is the significance of the repeating pattern of groups of Ig and Fn domains (e.g. Fn-Fn-Ig)? (iii) How are twitchin and the synchronous muscle isoform of projectin situated on the surface of thick filaments? That is, do they form polymers or are they located at discrete locations with intervening gaps? (iv) What is the mechanism by which the fundamentally similar projectin isoforms get localized to different sarcomeric locations? (v) If the data onAplysia twitchin can be extended to the muscles of other invertebrates, what is the mechanism by which twitchin inhibits the rate of relaxation? How does phosphorylation of twitchin relieve this inhibition? (vi) What are the substrates for the protein kinase domains of nematode twitchin and insect projectin? If rMLCs are indeed the substrates, how would and why does this phosphorylation take place for the IFM isoform of projectin, which resides primarily in the I band? If rMLCs are the substrates, given the stoichiometry of approx. 1∶50 for twitchin:myosin, and the likely fixed position of twitchin along the thick filament, how does phosphorylation of just a few rMLCs result in a physiological effect (e.g. inhibition of relaxation)? What is the true activator for the twitchin and projectin kinases? (vii) How does UNC-89 participate in M-line assembly? (viii) What are the biochemical and physiological functions of intestinal brush border twitchin? A number of investigators will enjoy pursuing these and other questions for some time in the future.     

A NUDEL-dependent mechanism of neurofilament assembly regulates the integrity of CNS neurons

The cytoskeleton controls the architecture and survival of central nervous system (CNS) neurons by maintaining the stability of axons and dendrites. Although neurofilaments (NFs) constitute the main cytoskeletal network in these structures, the mechanism that underlies subunit incorporation into filaments remains a mystery. Here we report that NUDEL, a mammalian homologue of the Aspergillus nidulans nuclear distribution molecule NudE, is important for NF assembly, transport and neuronal integrity. NUDEL facilitates the polymerization of NFs through a direct interaction with the NF light subunit (NF-L). Knockdown of NUDEL by RNA interference (RNAi) in a neuroblastoma cell line, primary cortical neurons or post-natal mouse brain destabilizes NF-L and alters the homeostasis of NFs. This results in NF abnormalities and morphological changes reminiscent of neurodegeneration. Furthermore, variations in levels of NUDEL correlate with disease progression and NF defects in a mouse model of neurodegeneration. Thus, NUDEL contributes to the integrity of CNS neurons by regulating NF assembly.     

Cell walls: monitoring integrity with the kinase

For a plant to grow properly it has to detect and respond appropriately to a diverse set of signals that emanate from the cell wall. A novel receptor-like kinase has recently been identified as essential in this process.     

A mechanism for molecular asymmetry

Summary The origin of the molecular asymmetry of biological systems has been speculated upon extensively, and has been the object of numerous inconclusive experimental studies. That circularly polarized light (CPL) might have been the cause of this asymmetry was suggested in 1874 (van't Hoff 1897; Le Bel 1874). During the daylight morning (AM) there is a significant component of left (L) CPL in skylight, which reverses to right (R) CPL in the afternoon (PM) (Wolstencroft 1985). The rates or photochemical reactions of LCPL and RCPL are different for the R (right) and S (left) forms of chiral molecules (Flores et al. 1977). At sunset the ambient temperature at the surface of the earth is approximately 10°C higher than at sunrise. Most chemical reactions proceed faster at higher temperatures and for each 10°C rise in temperature chemical reaction rates increase by a factor of 1.8–4.1 (Taylor 1925). It is proposed that the combination of these four factors, LCPL in the AM compared to the RCPL in the PM, the different rates of photochemical reaction of the R and the S forms of an R-S racemic mixture with RCPL (and LCPL), the higher PM temperature, and the faster reaction rates in the PM could lead to a substantial deviation from equality in the degradation and formation of R and S enantiomeric forms of chiral molecules.