Selectivity filter ion binding affinity determines inactivation in a potassium channel

Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca²⁺-activated K⁺ channel from Methanobacterium thermoautotrophicum, the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K⁺ concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF’s four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K⁺ affinity (∼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K⁺, similar to KcsA, but that even a single K⁺ binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K⁺ concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK’s S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes.

Ion permeation gating within the selectivity filter (SF) of potassium (K⁺) channels has been proposed to control channel activity in different ways for different family members. In voltage-dependent K⁺ (K_V) channels, the SF has been proposed to underlie C-type inactivation (1–3), resulting in the progressive loss of current following the activation of a channel gate located near the intracellular side of the pore (4). C-type inactivation in K_V channels has been shown to be strongly dependent on the affinity of a particular binding site for permeant ions in the pore, and the affinity of these pore sites has been proposed to depend not only on the SF chemical composition, but also on regions outside of the SF (5–8). A structure-function model for this mechanism has been provided most specifically by studies of the proton-gated KcsA channel (9–11) where opening of the activation gate is correlated with a conformational constriction and a decrease in ion occupancy within the four K⁺ binding sites of the SF (named S1 to S4) (9, 12–15). Experimental and structural studies of KcsA in low K⁺ showed that the SF constriction consists of an outward flip of the carbonyl groups of the Gly77, in the middle of the signature sequence (TVGYG) of K⁺ channels, associated with a loss of K⁺ binding at site S2 of the SF (9, 14, 16–20). These conformational changes were accompanied by the binding of several water molecules behind the SF, stabilizing this constricted (also called flipped) state by sterically preventing the SF from switching back into its conductive state (16, 18–20). While some reports challenge this view (21, 22), this activation gate-coupled collapse of the SF is now generally accepted as the mechanism underlying C-type inactivation in K⁺ channels.Several types of ligand-dependent K⁺ channels, including those opened by binding Ca²⁺, such as the BK and MthK channels (23–27), do not exhibit traditional C-type inactivation, despite possessing an identical SF with K_V and KcsA channels. Furthermore, these channels have been proposed to actually gate at the SF (28–32) although a recent cryogenic electron microscopy (cryo-EM) structure of MthK in the absence of calcium (33) revealed a steric closure at the bundle crossing inner gate region, suggesting that there may be two gates involved in calcium gating. Nevertheless, at this time, the structural correlates of SF gating and the difference from inactivation are unknown.In the present study, we set out to first investigate whether we can capture different gating states of MthK by obtaining X-ray structures of its pore (Fig. 1A) in wide-ranging concentrations of K⁺. MthK channels have been previously shown to display a decrease in activity with depolarization, which is further augmented when external K⁺ concentration is lowered, a signature of SF gating and a hallmark of C-type inactivation (34). We reasoned that K⁺ titration of MthK pore structures may provide insights into the molecular causes for K⁺-dependent SF gating and will indicate whether it shares features with the C-type inactivation observed in KcsA (such as a collapsed SF). Unlike KcsA, MthK SF did not collapse in similarly low K⁺ concentrations, suggesting that the clue to why MthK does not display traditional C-type inactivation may lie in understanding the molecular underpinnings that contribute to SF conformational change. Thus, we next investigated the dependence of SF conformation on ion occupancy and used molecular dynamics (MD) simulations to reveal a uniquely low affinity central S2 site in MthK, which may play a lead role in the SF-based channel closure. Overall, our results illustrate how the exact same sequence and structure of the SF in a K⁺ channel can lead to slight variations in K⁺ binding site chemistry, which in turn can lead to distinct functional phenotypes.Open in a separate windowFig. 1.Structure of the MthK pore in different K⁺ concentrations. (A) Overall architecture of wild-type MthK pore structure (three subunits of the tetrameric pore shown for clarity) crystallized with 150 mM K⁺. The SF is highlighted by dashed lines. Alignment of this structure with that crystallized in 6 mM K⁺ yields an all-atom root-mean-square deviation (RMSD) value of 0.25 Å. (B) K⁺-omit electron density maps (2F_o – F_c contoured at 2.0 σ) for SF atoms from two opposing subunits. Structures were solved in 150, 50, 11, and 6 mM [K⁺], as indicated. Crystallographic statistics are in SI Appendix, Table S1. (C) MD system with the MthK (ribbons) embedded in a lipid bilayer (gray sticks) bathed in 200 mM KCl (K⁺ as green spheres, Cl⁻ as blue spheres, and water as red and white sticks).     

Oxidation of NADPH on Kvbeta1 inhibits ball-and-chain type inactivation by restraining the chain

The Kv1 family voltage-dependent K(+) channels assemble with cytosolic β subunits (Kvβ), which are composed of a flexible N terminus followed by a structured core domain. The N terminus of certain Kvβs inactivates the channel by blocking the ion conduction pore, and the core domain is a functional enzyme that uses NADPH as a cofactor. Oxidation of the Kvβ-bound NADPH inhibits inactivation and potentiates channel current, but the mechanism behind this effect is unknown. Here we show that after oxidation, the core domain binds to part of the N terminus, thus restraining it from blocking the channel. The interaction is partially mediated by two negatively charged residues on the core domain and three positively charged ones on the N terminus. These results provide a molecular basis for the coupling between the cellular redox state and channel activity, and establish Kvβ as a target for pharmacological control of Kv1 channels.     

Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase

The ubiquitin ligase SCF(Cdc4) (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. Composite dynamic interactions of low affinity sites may be a general mechanism to establish phosphorylation thresholds in biological responses.     

Slow inactivation of calcium channels in the cardiac Purkinje fiber

Voltage-dependent calcium channels exist in a wide variety of cells and participate in diverse aspects of cellular function [6]. In the heart, ion movement through these channels underlies impulse conduction in the atrioventricular and sinoatrial nodes, contributes to regulation of some ionic conductances, maintains the plateau phase of the ventricular and Purkinje fiber action potentials, and is linked to activation of contractile protein [4, 7, 11, 13]. The activation of this channel has been assumed to resemble activation of sodium channels but recent investigations have raised questions about the nature of its inactivation. In cardiac Purkinje fibers, Marban and Tsien [10] find that inactivation of this conductance is regulated, at least in part, by intracellular calcium, as is calcium channel inactivation in Helix and Aplysia neurones [14, 15] and Paramecium [2].In this paper, we report experiments designed to investigate another characteristic of calcium channel inactivation in the cardiac Purkinje fiber. Using a new procedure to reduce overlapping outward currents, we find a very slow component of inactivation in this channel. Time constants for this slow inactivation are on the order of seconds, are not steeply voltage dependent and are not regulated by calcium ion entry.     

Molecular determinants of fast Ca2+-dependent inactivation and gating of the Orai channels

Ca²⁺ influx by store-operated Ca²⁺ influx channels (SOCs) mediates many cellular functions regulated by Ca²⁺, and excessive SOC-mediated Ca²⁺ influx is cytotoxic and associated with disease. One form of SOC is the CRAC current that is mediated by Orai channels activated by STIM1. A fundamental property of the native CRAC and of the Orais is fast Ca²⁺-dependent inactivation, which limits Ca²⁺ influx to guard against cellular damage. The molecular mechanism of this essential regulatory mechanism is unknown. We report here the fast Ca²⁺-dependent inactivation is mediated by three conserved glutamates in the C termini (CT) of Orai2 and Orai3, which show prominent fast Ca²⁺-dependent inactivation compared with Orai1. Transfer of the CT between the Orais transfers both the extent of channel opening and the mode of fast Ca²⁺-dependent inactivation. Fast Ca²⁺-dependent inactivation of the Orais also requires a domain of STIM1; fragments of STIM1 that efficiently open Orai channels do not evoke fast inactivation unless they include an anionic sequence that is C-terminal to the STIM1-Orai activating region (SOAR). Our studies suggest that Orai CT are necessary and sufficient to control pore opening and uncover the molecular mechanism of fast Ca²⁺-dependent inactivation that has implications for Ca²⁺ influx by SOC in physiological and pathological states.     

On the selective ion binding hypothesis for potassium channels

The mechanism by which K(+) channels select for K(+) over Na(+) ions has been debated for the better part of a century. The prevailing view is that K(+) channels contain highly conserved sites that selectively bind K(+) over Na(+) ions through optimal coordination. We demonstrate that a series of alternating sites within the KcsA channel selectivity filter exists, which are thermodynamically selective for either K(+) (cage made from two planes of oxygen atoms) or Na(+) ions (a single plane of four oxygen atoms). By combining Bennett free energy perturbation calculations with umbrella sampling, we show that when K(+) and Na(+) are both permitted to move into their preferred positions, the thermodynamic preference for K(+) over Na(+) is significantly reduced throughout the entire selectivity filter. We offer a rationale for experimental measures of thermodynamic preference for K(+) over Na(+) from Ba(2+) blocking data, by demonstrating that the presence of Ba(2+) ions exaggerates K(+) over Na(+) thermodynamic stability due to the different binding locations of these ions. These studies reveal that K(+) channel selectivity may not be associated with the thermodynamics of ions in crystallographic K(+) binding sites, but requires consideration of the kinetic barriers associated with the different multi-ion permeation mechanisms.     

Transmembrane allosteric coupling of the gates in a potassium channel

It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98–103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.Potassium channel activation and inactivation is fundamental to many physiological functions including muscle contraction and the generation of synaptic action potentials (1). KcsA is a 160-residue pH-activated homotetrameric K⁺ channel isolated from the soil bacterium Streptomyces lividans (2, 3) with high sequence homology and functional similarity to mammalian potassium channels (4). It has provided an excellent model for studies of ion-conduction by X-ray crystallography (3, 5, 6), electrophysiology (7, 8), and NMR (9–21). Like many potassium channels, it exhibits (4, 6, 22, 23) slow, spontaneous inactivation involving the residues near the extracellular selectivity filter subsequent to channel activation. Recent results from X-ray crystallography and molecular dynamics suggest that the gates are coupled and that inactivation is prompted by channel opening, mediated via a series of intrasubunit steric contacts involving F103 with T74, T75, and M96 and an intersubunit contact with the neighboring I100 side chain (4–6, 24, 25). In separate experiments, the extracellular gate has been observed to respond directly to ambient [K⁺]: at high [K⁺] it exists in a conductive form, and at low K⁺ it collapses into a nonconductive state (3). Our NMR studies suggest that the low [K⁺] state and the low pH inactivated state may be similar; this conclusion is supported by the effect of the mutation E71A and the pattern of chemical shift perturbations in the selectivity filter when the ion is depleted (9, 19). Meanwhile, X-ray crystallography studies suggest that mutants (E71A) unable to undergo inactivation are also unable to expel ions (26).An established similarity of the low pH and the low [K⁺] states would clarify the importance of allosteric coupling and have the practical consequence that the well-behaved low K⁺ state could serve as a useful structural proxy for the otherwise fleeting inactivated state. For these reasons we tested this correspondence using NMR experiments. If the low K⁺ state is similar to the inactivated state of KcsA achieved by lowering the pH, it is expected that structural changes indicative of channel opening observed at low [K⁺] would occur not only in the selectivity filter but also in the pH gate and the hinge region. However, some studies imply that these two gates might be uncoupled or weakly coupled. For example, X-ray crystallographic studies of KcsA, where K⁺ sensitivity was largely isolated to the selectivity filter (3). In this work, we asked whether, by contrast, full-length wild-type KcsA (160 aa) reconstituted into hydrated lipid bilayers exhibits global structural changes upon ion expulsion suggestive of channel opening. To accomplish this, nearly complete ¹³C and ¹⁵N chemical shift assignments were obtained for the transmembrane and loop regions from four-dimensional (4D) solid-state nuclear magnetic resonance (SSNMR) (27), providing numerous reporters for conformational change during ion binding. In low [K⁺] ion conditions at neutral pH, not only does KcsA expel the K⁺ ions from the inner selectivity filter sites, but the channel also exhibits chemical shift perturbations at the pH gate and the hinge of the inner transmembrane helix, suggesting features akin to the inhibited state that is present at low pH and high [K⁺]. That these two distinct conditions result in a nearly identical state of the channel offers strong evidence for transmembrane allostery in the inactivation process.     

Quantifying the topography of the intrinsic energy landscape of flexible biomolecular recognition

Biomolecular functions are determined by their interactions with other molecules. Biomolecular recognition is often flexible and associated with large conformational changes involving both binding and folding. However, the global and physical understanding for the process is still challenging. Here, we quantified the intrinsic energy landscapes of flexible biomolecular recognition in terms of binding–folding dynamics for 15 homodimers by exploring the underlying density of states, using a structure-based model both with and without considering energetic roughness. By quantifying three individual effective intrinsic energy landscapes (one for interfacial binding, two for monomeric folding), the association mechanisms for flexible recognition of 15 homodimers can be classified into two-state cooperative “coupled binding–folding” and three-state noncooperative “folding prior to binding” scenarios. We found that the association mechanism of flexible biomolecular recognition relies on the interplay between the underlying effective intrinsic binding and folding energy landscapes. By quantifying the whole global intrinsic binding–folding energy landscapes, we found strong correlations between the landscape topography measure Λ (dimensionless ratio of energy gap versus roughness modulated by the configurational entropy) and the ratio of the thermodynamic stable temperature versus trapping temperature, as well as between Λ and binding kinetics. Therefore, the global energy landscape topography determines the binding–folding thermodynamics and kinetics, crucial for the feasibility and efficiency of realizing biomolecular function. We also found “U-shape” temperature-dependent kinetic behavior and a dynamical cross-over temperature for dividing exponential and nonexponential kinetics for two-state homodimers. Our study provides a unique way to bridge the gap between theory and experiments.Biomolecules realize their functions through interacting with other molecules. Fully understanding the manner in which a protein participates in the process of biomolecular recognition is the basis for studying cellular activity. The first proposal of the binding mechanism was given by Fischer, called “lock-and-key,” to explain the rigid biomolecular docking (1). However, more and more experimental evidence has been accumulating in favor of the idea that protein binding often associates conformational changes. In this regard, considering the local configurational plasticity in protein–protein interactions, two scenarios have been proposed. One is named “induced fit” (2) and the other is called “conformational selection” (3). Furthermore, increasing recent evidence demonstrates that some isolated proteins are found to be disordered at physiological conditions. These proteins, known as “intrinsically disordered proteins” (IDPs), have refreshed our understanding of protein folding and function (4–6). The unstructured characteristic provides binding of IDPs with the advantage of multiple targeted partners, high association rates, high specificity, and moderate affinity (7, 8). For IDPs, the global conformational changes are always associated in their binding, known as “binding induced folding.” By investigating the synchronization of binding and folding, the association mechanism of IDPs can be classified into cooperative “coupled binding–folding” as well as noncooperative “binding prior to folding” and “folding prior to binding” (Fig. 1). As a result, it is now recognized that not only the well-defined 3D structure but also the conformational flexibility are critical pieces of information to determine the function of protein.Open in a separate windowFig. 1.The schematic diagram of three typical association mechanisms for IDPs. The diagonal line represents the cooperative process with binding and folding strongly coupled. The noncooperative processes are represented by the two lines along the rectangular edge, corresponding to binding prior to folding (up) and folding prior to binding (down), respectively.The energy landscape theory has been proposed to help understand the dynamics of protein folding (9–12). The shape of the folding energy landscape of naturally evolved proteins appears to be minimally frustrated and funneled so that the Levinthal’s paradox (13) can be solved with folding going through multiple routes toward the native structure rather than one single specific pathway (14–16). The folding landscape is widely studied in experiments and simulations (17–21) and has deepened our understanding of protein folding. As folding can be regarded as self-binding, the flexible recognition with large global conformational changes can be regarded as a process of binding coupled with folding—i.e., binding–folding. Therefore, binding and folding are analogous to each other except for the chain connectivity. It is expected that Levinthal’s paradox also exists in flexible protein binding. Therefore, it is feasible to extend the folding energy landscape concept to the binding dynamics to solve the conformational search problem in flexible binding and investigate the function of protein (22–27). The funneled binding landscape indicates that naturally evolved binding also follows the principle of minimal frustration, resulting in a reasonable physiological time for realizing the function of protein with vital activity.However, protein binding involving at least two chains is certainly different from protein folding in some respects. The global binding–folding energy landscape is expected to be a combination or interactions of an interfacial binding energy landscape and two monomeric folding energy landscapes (28). The flexible binding–folding energy landscape, as a result of the delicate combination or balance of folding and binding, controls the way a protein realizes its function during the protein–protein associations. For the noncooperative folding prior to binding scenario, the monomeric folding energy landscapes are expected to be more funneled than the interfacial binding energy landscapes, and vice versa for the binding prior to folding scenario. The cooperative coupled binding–folding scenario is an intermediary between the two noncooperative scenarios. Notice that the global binding–folding energy landscapes do not require that the three individual binding and folding energy landscapes are necessarily all funneled. For IDPs, they do not fold to a specific 3D structure, and therefore, their individual folding energy landscapes will be highly rugged. However, IDPs realize their functions by folding to ordered structures upon binding to their targets (29, 30). In other words, coupled with binding, the individual folding energy landscapes with functional rearrangements have been changed with strong bias toward the native binding structure during the associations. In conclusion, the binding–folding energy landscapes are the underlying key factors governing the protein–protein interactions and control the realization of protein’s function.In our work, we focused on the flexible biomolecular recognition in terms of binding–folding dynamics of 15 homodimers, which are formed by two identical monomers each (SI Appendix, Fig. S7). We quantified the whole global intrinsic landscape and the three individual intrinsic energy landscapes (one for interfacial binding, two for monomeric folding) from underlying density of states (DOS) extracted from the binding–folding dynamics using a structure-based model both with and without considering energetic roughness. We showed that the topography of each individual effective landscape and the whole global landscape of flexible recognition in terms of binding–folding can be quantified by a dimensionless ratio Λ of the energy gap between native state and average of nonnative states versus roughness modulated by the entropy. We found that the association mechanism of flexible recognition strongly relies on the interplay between the topographies of the underlying effective intrinsic binding and folding energy landscapes. This interplay changes with different strengths of nonnative interactions. We also showed that the whole global landscape topography measure is strongly correlated with the thermodynamics characterized by the binding transition temperature versus the glassy trapping temperature and the kinetics characterized by the binding time. By investigating the kinetics of Troponin C site, which is a two-state homodimer, we demonstrated that the temperature-dependent kinetic behavior is under the control of the topography of energy landscapes. The results are consistent with the previous analytical theories and experiments (31–33). Therefore, our work gives strong evidence that the topography of the intrinsic energy landscape is the key to understanding the binding–folding mechanism in terms of both thermodynamics and kinetics. Since the thermodynamics and kinetics of binding–folding dynamics can be explicitly measured by experiments (31, 34–36), and the underlying physical observable quantities are found to be strongly dependent on the theoretical energy landscape topography, our simulation findings can be regarded as the quantitative connections between the experiments and theory. This provides a unique way to bridge the theory and experimental measurements of flexible biomolecular recognition.     

Combinatorial augmentation of voltage-gated KCNQ potassium channels by chemical openers

Noninactivating potassium current formed by KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) subunits resembles neuronal M-currents which are activated by voltage and play a critical role in controlling membrane excitability. Activation of voltage-gated potassium channels by a chemical opener is uncommon. Therefore, the mechanisms of action are worthy further investigation. Retigabine and zinc pyrithione are two activators for KCNQ channels but their molecular interactions with KCNQ channel remain largely elusive. Here we report that retigabine and zinc pyrithione recognize two different sites of KCNQ2 channels. Their agonistic actions are noncompetitive and allow for simultaneous binding of two different activators on the same channel complex, hence giving rise to combinatorial potentiation with characteristic properties of both openers. Examining their effects on mutant channels, we showed zinc pyrithione is capable of opening nonconductive channels and coapplication of zinc pyrithione and retigabine could restore a disease mutant channel similar to wild type. Our results indicate two independent activator binding sites present in KCNQ channels. The resultant combinatorial potentiation by multiple synthetic chemical openers indicates that KCNQ channels are accessible to various types and combinations of pharmacological regulation.     

Molecular mechanism of pH sensing in KcsA potassium channels

The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Despite prior attempts to determine the mechanism responsible for pH gating, the proton sensor has remained elusive. We have constructed a KcsA channel mutant that remains open up to pH 9.0 by replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge. A series of individual and combined mutations were investigated by using single-channel recordings in lipid bilayers. We propose that these residues are the proton-binding sites and at neutral pH they form a complex network of inter- and intrasubunit salt bridges and hydrogen bonds near the bundle crossing that greatly stabilize the closed state. In our model, these residues change their ionization state at acidic pH, thereby disrupting this network, modifying the electrostatic landscape near the channel gate, and favoring channel opening.     

大鼠心房肌细胞三磷酸腺苷敏感钾通道的牵张敏感性

目的研究成年大鼠心房肌细胞三磷酸腺苷敏感钾通道(KATP)的牵张敏感性。方法应用单通道膜片钳技术在开放细胞贴附模式下记录大鼠心房肌细胞上KATP通道的电活动,并研究KATP通道对机械刺激的反应。结果根据通道的激活方式、药理学特性、I-V曲线、反转电位及整流特性,确定所记录的通道为KATP通道。进一步研究表明该通道的开放概率对负压刺激呈现强度依赖性。结论大鼠心房肌细胞上的KATP通道对牵张刺激敏感。     

Structure of CrgA,a cell division structural and regulatory protein from Mycobacterium tuberculosis,in lipid bilayers

The 93-residue transmembrane protein CrgA in Mycobacterium tuberculosis is a central component of the divisome, a large macromolecular machine responsible for cell division. Through interactions with multiple other components including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA, CrgA facilitates the recruitment of the proteins essential for peptidoglycan synthesis to the divisome and stabilizes the divisome. CrgA is predicted to have two transmembrane helices. Here, the structure of CrgA was determined in a liquid–crystalline lipid bilayer environment by solid-state NMR spectroscopy. Oriented-sample data yielded orientational restraints, whereas magic-angle spinning data yielded interhelical distance restraints. These data define a complete structure for the transmembrane domain and provide rich information on the conformational ensembles of the partially disordered N-terminal region and interhelical loop. The structure of the transmembrane domain was refined using restrained molecular dynamics simulations in an all-atom representation of the same lipid bilayer environment as in the NMR samples. The two transmembrane helices form a left-handed packing arrangement with a crossing angle of 24° at the conserved Gly39 residue. This helix pair exposes other conserved glycine and alanine residues to the fatty acyl environment, which are potential sites for binding CrgA’s partners such as CwsA and FtsQ. This approach combining oriented-sample and magic-angle spinning NMR spectroscopy in native-like lipid bilayers with restrained molecular dynamics simulations represents a powerful tool for structural characterization of not only isolated membrane proteins, but their complexes, such as those that form macromolecular machines.Better understanding of cell division in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), will generate new opportunities for pharmaceutical development. CrgA, a transmembrane (TM) protein, is a central component of the Mtb divisome (1). CrgA has homologs in other actinomycetes (2, 3), but not in the two bacteria, Escherichia coli and Bacillus subtilis, with better characterized cell division mechanisms. Conversely, many cell division proteins in the latter organisms, such as FtsA, FtsN, FtsL, and ZipA, appear to have no homologs in Mtb. CrgA is localized at the poles and septum, and interacts with multiple cell division proteins, including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA. One function of these interactions is to stabilize the divisome (1, 4). The interaction with CwsA, a protein that is unique to mycobacteria (5), might coordinate elongation at the poles and division at midcell (4). Moreover, CrgA appears to have an important role in peptidoglycan (PG) formation during cell division, by recruiting PG synthases to the divisome (4). Reduced production of CrgA results in elongated cells and reduced growth rate (1), and loss of CrgA impairs PG synthesis (5). In addition to CwsA, the Mtb divisome involves other atypical players such as FipA (FhaB), ChiZ, and MtrB (6–8), and thus there is much yet to be learned about the participants in mycobacterial cell division (9). Here, we determined the structure of CrgA in a lipid bilayer environment using solid-state NMR (ssNMR) spectroscopy.TB is a devastating human disease that kills ∼1.3 million people each year with 8.6 million new cases diagnosed annually worldwide (10). Rising extreme drug-resistant Mtb strains do not succumb to the frontline antibiotics, generating a dire need for new drugs (11). Pathways critical for bacterial survival such as DNA replication and cell division include numerous potential drug targets and represent a major focus for structural biology. Also, TB treatment is expensive and significantly toxic and requires an extensive period caused by Mtb’s ability to exist in a latent state. Hence, there are additional motivations for characterizing the proteins associated with its survival in active and nonreplicative persistent states.CrgA was first described from Streptomyces as being required for sporulation through coordinating several aspects of its reproductive growth (2, 3). The Mtb CrgA consists of 93 residues, with two predicted TM helices (12) (TM1: residues 29–51; and TM2: residues 66–88; Fig. 1A). The N-terminal 17 residues are predicted to be disordered by the software PONDR (13); the C terminus is predicted to be just five residues, whereas the loop between the TM helices is predicted to be just 14 residues. The predicted TM1 sequence contains a pair of conserved tryptophan residues (W32 and W47) that appear from the sequence to be positioned for anchoring the helix to the membrane interfacial regions. A second pair of conserved tryptophan residues is at positions 73 and 92. Because the TM2 prediction has W73 eight residues into the helix and W92 four residues beyond the end of the predicted helix, this prediction may not be as accurate. Both predicted helices contain a number of other conserved residues, whereas the loop between the helices is much more variable both in length and in composition (SI Appendix, Fig. S1).Fig. 1.Amino acid sequence and ssNMR spectra of full-length Mtb CrgA membrane protein. (A) The sequence of the expressed CrgA with a C-terminal His₆ tag. The predicted transmembrane (TM) helical residues (TMHMM, version 2.0) are indicated by red lettering. ( ...Surprisingly, this small membrane protein binds a large number of other proteins, all of which are transmembrane proteins except for FtsZ. In particular, FtsI, with a single TM helix, is a transpeptidase responsible for synthesis of the septal PG (1). A crgA-deletion mutant results in the loss of septal and polar localization of FtsI, suggesting the importance of CrgA for PG synthesis through its recruitment of FtsI. CwsA also contains a single TM helix. A crgA and cwsA double-deletion mutant showed the importance of the corresponding gene products for cell wall synthesis and cell shape maintenance (8).The CrgA TM helices contain a number of conserved glycine and alanine residues (SI Appendix, Fig. S1). Although glycine residues are known to be helix breakers in water-soluble proteins, in TM helices, they may allow local helix bending in the low dielectric membrane environment where intrahelical hydrogen bonds are strengthened for maintaining the overall integrity of the helical structure. In addition, glycine and alanine residues permit close approach of adjacent helical backbones, resulting in backbone–backbone electrostatic and side-chain–side-chain van der Waals interactions that stabilize the tertiary structure. Therefore, glycines may allow helical membrane proteins to sacrifice secondary structural stability for tertiary structural stability (14–16). This is needed because the amino acid composition in the interior of membrane proteins is more hydrophobic than the interior of water-soluble proteins where there are more frequent tertiary hydrogen bonds than in TM domains (17). In addition, conserved glycine residues are rarely found on the fatty-acyl exposed surface of multihelix membrane proteins (16). In such a location, they would expose their hydrophilic backbone atoms to the low dielectric environment of the protein. If present, it is a strong indication that they are exposed for a required function such as binding another protein. Interestingly, E. coli FtsQ is thought to localize to the divisome through interactions with other components via its single TM helix (18).Only a couple of full-length Mtb membrane protein structures have been determined. One is an X-ray structure of the mechanosensitive channel of large conductance, and the other is a single TM helix protein, Rv1761 (19, 20). In addition, water-soluble domains of other Mtb membrane proteins have been characterized such as those from PknB and FtsX (21–24). Although X-ray crystallographers have focused on large membrane proteins, the majority of the 1,162 ORFs of the Mtb genome code for small helical membrane proteins containing one to three TM helices with <40-kDa molecular weight (25). Structure–function studies of these small membrane proteins are essential for understanding Mtb cell division and other cellular processes. Small polytopic membrane protein structures are stabilized not just by interactions between their TM helices, but also by interactions with their membrane environment. Consequently, it is necessary to solve their structures in an appropriate membrane mimetic environment, one that possesses many of the restraining influences of the native membrane such as a relatively fixed hydrophobic thickness, a dramatic lateral pressure profile, and a hydrophobic core essentially devoid of water (26, 27).For the structure determination of CrgA, here we used both oriented-sample (OS) and magic-angle spinning (MAS) ssNMR to characterize the full-length protein in lipid bilayers. All ssNMR spectroscopy was performed on fully hydrated liquid–crystalline lipid bilayer preparations of CrgA. The use of such bilayer preparations for supporting the native-like conformation of the M2 protein from Influenza A has been validated with the comparison of spectra from synthetic bilayers and from cellular membranes where the protein has been inserted by the cellular machinery and never removed from this environment or exposed to a detergent environment (28). Multiple recent membrane protein structures have now been determined by OS ssNMR (29–34), and the first membrane protein structure has been obtained from MAS ssNMR (35). OS ssNMR generates information on the orientations of peptide planes with respect to the bilayer normal, and for a TM helix it yields the tilt angle of the helix relative to the lipid bilayer normal and rotational orientation about the helical axis along the entire length of helix. However, it does not directly provide information on the helix–helix packing interface. The latter information can be ascertained by relatively few distance restraints between the helices, as the degrees of freedom for packing the helices have been minimized by the orientational restraints, to just the relative rotation around the bilayer normal and relative translation in the bilayer plane. The combination of OS and MAS ssNMR thus allows the complete determination of the helical TM domain structure.Based on the OS and MAS data, we refined the structure using restrained molecular dynamics simulations in an all-atom representation of the same lipid bilayer environment as in the protein samples. The two TM helices both have a tilt angle of 13° but are tilted in nearly opposite directions such that they form a left-handed packing arrangement with a crossing angle of 24° at the conserved Gly39 residue. The two-helix TM domain exposes other conserved glycine and alanine residues that potentially form binding sites for TM helices of CrgA binders. Much of the N-terminal region is disordered, but a nine-residue motif therein appears to form an amphipathic helix. In the interhelical loop, a short segment appears to be disordered while a 12-residue motif appears to form a β-hairpin in the membrane interface. The C terminus comprises just two residues. Overall, the structure suggests how CrgA serves as a platform where other proteins of the divisome assemble.     

多发性硬化炎症细胞钾离子通道的研究进展

多发性硬化(MS)是中枢神经系统常见的慢性炎症性自身免疫性疾病,目前病因及发病机制尚不清楚。近年研究发现,MS多种炎性细胞的钾离子通道参与了其自身免疫性发病过程,控制炎性细胞上钾离子通道为MS的治疗提供了新的途径。本文就MS自身免疫性炎症细胞上钾离子通道的研究进展作一综述。     

Allosteric gating mechanism underlies the flexible gating of KCNQ1 potassium channels

KCNQ1 (Kv7.1) is a unique member of the superfamily of voltage-gated K(+) channels in that it displays a remarkable range of gating behaviors tuned by coassembly with different β subunits of the KCNE family of proteins. To better understand the basis for the biophysical diversity of KCNQ1 channels, we here investigate the basis of KCNQ1 gating in the absence of β subunits using voltage-clamp fluorometry (VCF). In our previous study, we found the kinetics and voltage dependence of voltage-sensor movements are very similar to those of the channel gate, as if multiple voltage-sensor movements are not required to precede gate opening. Here, we have tested two different hypotheses to explain KCNQ1 gating: (i) KCNQ1 voltage sensors undergo a single concerted movement that leads to channel opening, or (ii) individual voltage-sensor movements lead to channel opening before all voltage sensors have moved. Here, we find that KCNQ1 voltage sensors move relatively independently, but that the channel can conduct before all voltage sensors have activated. We explore a KCNQ1 point mutation that causes some channels to transition to the open state even in the absence of voltage-sensor movement. To interpret these results, we adopt an allosteric gating scheme wherein KCNQ1 is able to transition to the open state after zero to four voltage-sensor movements. This model allows for widely varying gating behavior, depending on the relative strength of the opening transition, and suggests how KCNQ1 could be controlled by coassembly with different KCNE family members.     

On the Prevalence and Potential Functionality of an Intrinsic Disorder in the MERS-CoV Proteome

Middle East respiratory syndrome is a severe respiratory illness caused by an infectious coronavirus. This virus is associated with a high mortality rate, but there is as of yet no effective vaccine or antibody available for human immunity/treatment. Drug design relies on understanding the 3D structures of viral proteins; however, arriving at such understanding is difficult for intrinsically disordered proteins, whose disorder-dependent functions are key to the virus’s biology. Disorder is suggested to provide viral proteins with highly flexible structures and diverse functions that are utilized when invading host organisms and adjusting to new habitats. To date, the functional roles of intrinsically disordered proteins in the mechanisms of MERS-CoV pathogenesis, transmission, and treatment remain unclear. In this study, we performed structural analysis to evaluate the abundance of intrinsic disorder in the MERS-CoV proteome and in individual proteins derived from the MERS-CoV genome. Moreover, we detected disordered protein binding regions, namely, molecular recognition features and short linear motifs. Studying disordered proteins/regions in MERS-CoV could contribute to unlocking the complex riddles of viral infection, exploitation strategies, and drug development approaches in the near future by making it possible to target these important (yet challenging) unstructured regions.     

Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors)

P2X receptors are trimeric cation channels with widespread roles in health and disease. The recent crystal structure of a P2X4 receptor provides a 3D view of their topology and architecture. A key unresolved issue is how ions gain access to the pore, because the structure reveals two different pathways within the extracellular domain. One of these is the central pathway spanning the entire length of the extracellular domain and covering a distance of ≈70 Å. The second consists of three lateral portals, adjacent to the membrane and connected to the transmembrane pore by short tunnels. Here, we demonstrate the preferential use of the lateral portals. Owing to their favorable diameters and equivalent spacing, the lateral portals split the task of ion supply threefold and minimize an ion''s diffusive path before it succumbs to transmembrane electrochemical gradients.     

Connecting sequence features within the disordered C-terminal linker of Bacillus subtilis FtsZ to functions and bacterial cell division

Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT in Bacillus subtilis FtsZ (Bs-FtsZ). Molecular simulations show that the CT17 of Bs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical coupling analysis of more than 1,000 sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain, whereas the CTL is under independent selection. Despite this, we discover the conservation of nonrandom sequence patterns within CTLs across orthologs. To test how the nonrandom patterns of CTLs mediate CTT–core interactions and modulate FtsZ functionalities, we designed Bs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core–CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysis in vitro and protein degradation, aberrant assembly, and disruption of cell division in vivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass nonrandom, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain.

Intrinsically disordered regions (IDRs) contribute to a multitude of protein functions (1–4). IDRs often have autoregulatory roles when tethered to folded domains either as tails or as linkers between folded domains (5–12). Of particular interest are IDRs tethered to folded domains that are enzymes (7, 13, 14). Several studies demonstrate that IDRs tethered to folded domains can function as autoregulators (12), specifically as autoinhibitors of enzymatic activities (13, 15, 16). One such example is the C-terminal tail (CTT) of the essential GTPase that controls and regulates bacterial cell division (17). The CTT encompasses a disordered C-terminal linker (CTL) and an alpha-helix-forming C-terminal peptide.Cell division in bacteria is initiated by assembly of the cytokinetic ring at the nascent division site (18–26). Polymers formed by the essential GTPase filamenting temperature-sensitive mutant Z (FtsZ) are the foundation of this ring, which is also known as the Z-ring (27–32). FtsZ is a prokaryotic homolog of tubulin. It forms single-stranded protofilaments upon binding GTP in vitro (33). Linear polymers of FtsZ, which also undergo bundling via lateral associations, serve as a platform for the cell division machinery composed of at least 30 different proteins (19, 32, 34–39). FtsZ polymers also undergo treadmilling in vivo, driven by the turnover of subunits that occurs on the order of seconds (40).Previous in vitro experiments showed that FtsZ polymerization belongs to a class of phase transitions known as reversible polymerization (41). A defining hallmark of reversible phase transitions, with subunit concentration as the conserved order parameter, is the presence of at least one threshold concentration for the occurrence of a specific phase transition. Cohan et al. recently identified two distinct threshold concentrations for Bacillus subtilis FtsZ (Bs-FtsZ) phase transitions occurring in the presence of GTP (17). In agreement with previous work on Escherichia coli FtsZ, Bs-FtsZ forms single-stranded protofilaments when the first threshold concentration, denoted as c_A, is crossed (42–47). The second threshold concentration, denoted as c_B where c_B > c_A, characterizes the threshold for bundling of protofilaments.Bs-FtsZ encompasses two domains: a folded N-terminal core and a CTT (Fig. 1A). The core domain forms a complete GTPase upon dimerization whereby the T7 loop of one protomer is inserted into the nucleotide binding site of the complementary protomer. The interface between the T7 loop and the nucleotide binding site is the active site for GTP hydrolysis (48). The CTT is further composed of an intrinsically disordered CTL and a 17-residue C-terminal peptide (CT17). The CT17 was previously termed CTP (17), and it includes a conserved “constant region” and a “variable region” (CTV) (30, 49, 50). The CT17 can form alpha-helical conformations (51–53) and is thus an alpha-molecular recognition element (54) that enables a precise network of homotypic and heterotypic protein–protein interactions. Whereas the CT17 includes a conserved region (33), the CTL is hypervariable across orthologs, varying in length, amino acid composition, and sequence (33, 49, 55, 56). Mutations in the CTL and the CTV of Bs-FtsZ disrupt lateral interactions between protofilaments (49, 56).Open in a separate windowFig. 1.Modular architecture of B. subtilis FtsZ includes a disordered CTT. (A) The electrostatic potential (103) is mapped onto the core domain in red and blue for regions of negative and positive potential, respectively. The T7 loop is highlighted in green. The CTT includes a CTL that connects the 17-residue C-terminal peptide (CT17) to the core domain. (B) The CTT is predicted to be disordered using IUPRED (60). The CTT sequence is shown with negatively and positively charged residues of the CTL in red and blue, respectively. The CT17 sequence is shown in gray. (C) Ensemble-averaged secondary structure contents of the CTT obtained from atomistic simulations. (D) UV-CD spectra of the four Bs-FtsZ constructs. See Materials and Methods for details on scaling of the [θ*].Consistent with previous work (57–59), Cohan et al. showed, through systematic deletions of each module, that the core domain of Bs-FtsZ is the main driver of GTP binding-induced polymerization (17). Deletion of the CT17 (ΔCT17), previously referred to as ΔCTP, increases c_A while also shifting c_B upward by at least threefold. Internal deletion of the CTL (ΔCTL) decreases c_A and this construct forms mini rings stabilized by cohesive interactions of the CT17. Overall, the CTL weakens the driving forces for linear polymerization and bundling, whereas the CT17 appears to be the primary driver of lateral associations. Deletion of the CTT (ΔCTT) lowers c_A by over an order of magnitude and forms long, single-stranded polymers. Cohan et al. also showed that ΔCTT is the most efficient GTPase, whereas the wild-type Bs-FtsZ is the least efficient enzyme of the four constructs studied (17).The picture that emerges is of the CTT as an autoregulator of Bs-FtsZ assembly and an autoinhibitor of enzymatic activity (17). Here, we uncover a molecular-level, mechanistic understanding of how the distinctive functions of CTTs are achieved.