Interdomain communication in a homodimeric ABC transporter.

ABC transporters are found in all organisms and almost every cellular compartment. They mediate the transport of various solutes across membranes, energized by ATP binding and hydrolysis. Dysfunctions can result in severe diseases, such as cystic fibrosis or antibiotic resistance. In type IV ABC transporters, each of the two nucleotide-binding domains is connected to a transmembrane domain by two coupling helices, which are part of cytosolic loops. Although there are many structural snapshots of different conformations, the interdomain communication is still enigmatic. Therefore, we analyzed the function of three conserved charged residues in the intracytosolic loop 1 of the human homodimeric, lysosomal peptide transporter TAPL (transporter associated with antigen processing-like). Substitution of D278 in coupling helix 1 by alanine interrupted peptide transport by impeding ATP hydrolysis. Alanine substitution of R288 and D292, both localized next to the coupling helix 1 extending to transmembrane helix 3, reduced peptide transport but increased basal ATPase activity. Surprisingly, the ATPase activity of the R288A variant dropped in a peptide-dependent manner, whereas ATPase activity of wildtype and D292A was unaffected. Interestingly, R288A and D292A mutants did not differentiate between ATP and GTP in respect of hydrolysis. However, in contrast to wildtye TAPL, only ATP energized peptide transport. In sum, D278 seems to be involved in bidirectional interdomain communication mediated by network of polar interactions, whereas the two residues in the cytosolic extension of transmembrane helix 3 are involved in regulation of ATP hydrolysis, most likely by stabilization of the outward-facing conformation.

Current View of Ligand and Lipid Recognition by the Menthol Receptor TRPM8.

Transient receptor potential (TRP) melastatin member 8 (TRPM8), which is a calcium-permeable ion channel, functions as the primary molecular sensor of cold and menthol in humans. Recent cryoelectron microscopy (cryo-EM) studies of TRPM8 have shown distinct structural features in its architecture and domain assembly compared with the capsaicin receptor TRP vanilloid member 1 (TRPV1). Moreover, ligand-bound TRPM8 structures have uncovered unforeseen binding sites for both cooling agonists and membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. These complex structures unveil the molecular basis of cooling agonist sensing by TRPM8 and the allosteric role of PI(4,5)P2 in agonist binding for TRPM8 activation. Here, we review the recent advances in TRPM8 structural biology and investigate the molecular principles governing the distinguishing role of TRPM8 as the evolutionarily conserved menthol receptor.

Viral packaging ATPases utilize a glutamate switch to couple ATPase activity and DNA translocation.

Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate-switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free-energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in ATP- and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an active to an inactive pose upon ATP hydrolysis and that a residue assigned as the glutamate switch is necessary for regulating this transition. Furthermore, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental measurements. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the structural coupling predicted from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway that couples chemical and mechanical events in viral DNA packaging motors.

Allosteric gate modulation confers K+ coupling in glutamate transporters.

Excitatory amino acid transporters (EAATs) mediate glial and neuronal glutamate uptake to terminate synaptic transmission and to ensure low resting glutamate concentrations. Effective glutamate uptake is achieved by cotransport with 3 Na+ and 1 H+ , in exchange with 1 K+ . The underlying principles of this complex transport stoichiometry remain poorly understood. We use molecular dynamics simulations and electrophysiological experiments to elucidate how mammalian EAATs harness K+ gradients, unlike their K+ -independent prokaryotic homologues. Glutamate transport is achieved via elevator-like translocation of the transport domain. In EAATs, glutamate-free re-translocation is prevented by an external gate remaining open until K+  binding closes and locks the gate. Prokaryotic GltPh contains the same K+ -binding site, but the gate can close without K+ . Our study provides a comprehensive description of K+ -dependent glutamate transport and reveals a hitherto unknown allosteric coupling mechanism that permits adaptions of the transport stoichiometry without affecting ion or substrate binding.

Coupling of Ca2+ and voltage activation in BK channels through the αB helix/voltage sensor interface.

Large-conductance Ca2+ and voltage-activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high-affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved.

Mapping co-regulatory interactions among ligand-binding sites in ryanodine receptor 1.

Ryanodine receptor 1 (RyR1) is an intracellular calcium ion (Ca2+ ) release channel required for skeletal muscle contraction. Although cryo-electron microscopy identified binding sites of three coactivators Ca2+ , ATP, and caffeine (CFF), the mechanism of co-regulation and synergy of these activators is unknown. Here, we report allosteric connections among the three ligand-binding sites and pore region in (i) Ca2+ bound-closed, (ii) ATP/CFF bound-closed, (iii) Ca2+ /ATP/CFF bound-closed, and (iv) Ca2+ /ATP/CFF bound-open RyR1 states. We identified two dominant networks of interactions that mediate communication between the Ca2+ -binding site and pore region in Ca2+ bound-closed state, which partially overlapped with the pore communications in ATP/CFF bound-closed RyR1 state. In Ca2+ /ATP/CFF bound-closed and -open RyR1 states, co-regulatory interactions were analogous to communications in the Ca2+ bound-closed and ATP/CFF bound-closed states. Both ATP- and CFF-binding sites mediate communication between the Ca2+ -binding site and the pore region in Ca2+ /ATP/CFF bound-open RyR1 structure. We conclude that Ca2+ , ATP, and CFF propagate their effects to the pore region through a network of overlapping interactions that mediate allosteric control and molecular synergy in channel regulation.

Bacterial Transcriptional Regulators: A Road Map for Functional, Structural, and Biophysical Characterization.

The different niches through which bacteria move during their life cycle require a fast response to the many environmental queues they encounter. The sensing of these stimuli and their correct response is driven primarily by transcriptional regulators. This kind of protein is involved in sensing a wide array of chemical species, a process that ultimately leads to the regulation of gene transcription. The allosteric-coupling mechanism of sensing and regulation is a central aspect of biological systems and has become an important field of research during the last decades. In this review, we summarize the state-of-the-art techniques applied to unravel these complex mechanisms. We introduce a roadmap that may serve for experimental design, depending on the answers we seek and the initial information we have about the system of study. We also provide information on databases containing available structural information on each family of transcriptional regulators. Finally, we discuss the recent results of research about the allosteric mechanisms of sensing and regulation involving many transcriptional regulators of interest, highlighting multipronged strategies and novel experimental techniques. The aim of the experiments discussed here was to provide a better understanding at a molecular level of how bacteria adapt to the different environmental threats they face.

Probing the correlation between ligand efficacy and conformational diversity at the α1A-adrenoreceptor reveals allosteric coupling of its microswitches.

G protein-coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.

Inverted allosteric coupling between activation and inactivation gates in K+ channels.

The selectivity filter and the activation gate in potassium channels are functionally and structurally coupled. An allosteric coupling underlies C-type inactivation coupled to activation gating in this ion-channel family (i.e., opening of the activation gate triggers the collapse of the channel's selectivity filter). We have identified the second Threonine residue within the TTVGYGD signature sequence of K+ channels as a crucial residue for this allosteric communication. A Threonine to Alanine substitution at this position was studied in three representative members of the K+-channel family. Interestingly, all of the mutant channels exhibited lack of C-type inactivation gating and an inversion of their allosteric coupling (i.e., closing of the activation gate collapses the channel's selectivity filter). A state-dependent crystallographic study of KcsA-T75A proves that, on activation, the selectivity filter transitions from a nonconductive and deep C-type inactivated conformation to a conductive one. Finally, we provide a crystallographic demonstration that closed-state inactivation can be achieved by the structural collapse of the channel's selectivity filter.

Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein.

The allosteric interplay between distant functional sites present in a single protein provides for one of the most important regulatory mechanisms in biological systems. While the design of ligand-binding sites into proteins remains challenging, this holds even truer for the coupling of a newly engineered binding site to an allosteric mechanism that regulates the ligand affinity. Here it is shown how computational design algorithms enabled the introduction of doxycycline- and doxorubicin-binding sites into the serine proteinase inhibitor (serpin) family member α1-antichymotrypsin. Further engineering allowed exploitation of the proteinase-triggered serpin-typical S-to-R transition to modulate the ligand affinities. These design variants follow strategies observed in naturally occurring plasma globulins that allow for the targeted delivery of hormones in the blood. By analogy, we propose that the variants described in the present study could be further developed to allow for the delivery of the antibiotic doxycycline and the anticancer compound doxorubicin to tissues/locations that express specific proteinases, such as bacterial infection sites or tumor cells secreting matrix metalloproteinases.

Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements.

The allosteric coupling between activation and inactivation processes is a common feature observed in K+ channels. Particularly, in the prokaryotic KcsA channel the K+ conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation pKa of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.

Investigating dual Ca2+ modulation of the ryanodine receptor 1 by molecular dynamics simulation.

The ryanodine receptors (RyR) are essential to calcium signaling in striated muscles. A deep understanding of the complex Ca2+ -activation/inhibition mechanism of RyRs requires detailed structural and dynamic information for RyRs in different functional states (eg, with Ca2+ bound to activating or inhibitory sites). Recently, high-resolution structures of the RyR isoform 1 (RyR1) were solved by cryo-electron microscopy, revealing the location of a Ca2+ binding site for activation. Toward elucidating the Ca2+ -modulation mechanism of RyR1, we performed extensive molecular dynamics simulation of the core RyR1 structure in the presence and absence of activating and solvent Ca2+ (total simulation time is >5 µs). In the presence of solvent Ca2+ , Ca2+ binding to the activating site enhanced dynamics of RyR1 with higher inter-subunit flexibility, asymmetric inter-subunit motions, outward domain motions and partial pore dilation, which may prime RyR1 for subsequent channel opening. In contrast, the solvent Ca2+ alone reduced dynamics of RyR1 and led to inward domain motions and pore contraction, which may cause inhibition. Combining our simulation with the map of disease mutation sites in RyR1, we constructed a wiring diagram of key domains coupled via specific hydrogen bonds involving the mutation sites, some of which were modulated by Ca2+ binding. The structural and dynamic information gained from this study will inform future mutational and functional studies of RyR1 activation and inhibition by Ca2+ .

Structure and allosteric coupling of type â ¡ antitoxin CopASO.

Toxin-antitoxin (TA) systems play critical roles in the environment adaptation of bacteria. Allosteric coupling between the N-terminal DNA-binding domain and the C-terminal toxin-binding domain of antitoxins contributes to conditional cooperativity in the functioning of type II TA. Herein, using circular dichroism (CD), nuclear magnetic resonance (NMR), X-ray crystallography, and size exclusion chromatography (SEC), the structure and DNA binding of CopASO, a newly identified type II antitoxin in Shewanella oneidensis, were investigated. Our data show that CopASO is a typical RHH antitoxin with an ordered N-terminal domain and a disordered C-terminal domain, and furthermore indicate that the C-terminal domain facilitates DNA binding of the N-terminal domain, which in turn induces the C-terminal domain to fold and associate.

New Structures and Gating of Voltage-Dependent Potassium (Kv) Channels and Their Relatives: A Multi-Domain and Dynamic Question.

Voltage-dependent potassium channels (Kv channels) are crucial regulators of cell excitability that participate in a range of physiological and pathophysiological processes. These channels are molecular machines that display a mechanism (known as gating) for opening and closing a gate located in a pore domain (PD). In Kv channels, this mechanism is triggered and controlled by changes in the magnitude of the transmembrane voltage sensed by a voltage-sensing domain (VSD). In this review, we consider several aspects of the VSD⁻PD coupling in Kv channels, and in some relatives, that share a common general structure characterized by a single square-shaped ion conduction pore in the center, surrounded by four VSDs located at the periphery. We compile some recent advances in the knowledge of their architecture, based in cryo-electron microscopy (cryo-EM) data for high-resolution determination of their structure, plus some new functional data obtained with channel variants in which the covalent continuity between the VSD and PD modules has been interrupted. These advances and new data bring about some reconsiderations about the use of exclusively a classical electromechanical lever model of VSD⁻PD coupling by some Kv channels, and open a view of the Kv-type channels as allosteric machines in which gating may be dynamically influenced by some long-range interactional/allosteric mechanisms.

Side chain flexibility and coupling between the S4-S5 linker and the TRP domain in thermo-sensitive TRP channels: Insights from protein modeling.

The transient receptor potential (TRP) superfamily is subdivided into several subfamilies on the basis of sequence similarity, which is highly heterogeneous but shows a molecular architecture that resembles the one present in members of the Kv channel superfamily. Because of this diversity, they produce a large variety of channels with different gating and permeability properties. Elucidation of these particular features necessarily requires comparative studies based on structural and functional data. The present study aims to compilate, analyze, and determine, in a coherent way, the relationship between intrinsic side-chain flexibility and the allosteric coupling in members of the TRPV, TRPM, and TRPC families. Based on the recently determined structures of TRPV1 and TRPV2, we have generated protein models for single subunits of TRPV5, TRPM8, and TRPC5 channels. With these models, we focused our attention on the apparently crucial role of the GP dipeptide at the center of the S4-S5 linker and discussed its role in the interaction with the TRP domain, specifically with the highly-conserved Trp during this coupling. Our analysis suggests an important role of the S4-S5L flexibility in the thermosensitivity, where heat-activated channels possess rigid S4-S5 linkers, whereas cold-activated channels have flexible ones. Finally, we also present evidence of the key interaction between the conserved Trp residue of the TRP box and of several residues in the S4-S5L, importantly the central Pro. Proteins 2017; 85:630-646. © 2016 Wiley Periodicals, Inc.

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery.

Caseinolytic peptidase P (ClpP), a double-ring peptidase with 14 subunits, collaborates with ATPases associated with diverse activities (AAA+) partners to execute ATP-dependent protein degradation. Although many ClpP enzymes self-assemble into catalytically active homo-tetradecamers able to cleave small peptides, the Mycobacterium tuberculosis enzyme consists of discrete ClpP1 and ClpP2 heptamers that require a AAA+ partner and protein-substrate delivery or a peptide agonist to stabilize assembly of the active tetradecamer. Here, we show that cyclic acyldepsipeptides (ADEPs) and agonist peptides synergistically activate ClpP1P2 by mimicking AAA+ partners and substrates, respectively, and determine the structure of the activated complex. Our studies establish the basis of heteromeric ClpP1P2 assembly and function, reveal tight coupling between the conformations of each ring, show that ADEPs bind only to one ring but appear to open the axial pores of both rings, provide a foundation for rational drug development, and suggest strategies for studying the roles of individual ClpP1 and ClpP2 rings in Clp-family proteolysis.

Selective allosteric coupling in core chemotaxis signaling complexes.

Bacterial chemotaxis is mediated by signaling complexes that sense chemical gradients and direct bacteria to favorable environments by controlling a histidine kinase as a function of chemoreceptor ligand occupancy. Core signaling complexes contain two trimers of transmembrane chemoreceptor dimers, each trimer binding a coupling protein CheW and a protomer of the kinase dimer. Core complexes assemble into hexagons, and these form hexagonal arrays. The notable cooperativity and amplification in bacterial chemotaxis is thought to reflect allosteric interactions in cores, hexagons, and arrays, but little is known about this presumed allostery. We investigated allostery in core complexes assembled with two chemoreceptor species, each recognizing a different ligand. Chemoreceptors were inserted in Nanodiscs, which rendered them water soluble and allowed isolation of individual complexes. Neighboring dimers in receptor trimers influenced one another's operational ligand affinity, indicating allosteric coupling. However, this coupling did not include the key function of kinase inhibition. Our data indicated that only one receptor dimer could inhibit kinase as a function of ligand occupancy. This selective allosteric coupling corresponded with previously identified structural asymmetry: only one dimer in a trimer contacts kinase and only one CheW. We suggest one of these dimers couples ligand occupancy to kinase inhibition. Additionally, we found that kinase protomers are allosterically coupled, conveying inhibition across the dimer interface. Because kinase dimers connect core complex hexagons, allosteric communication across dimer interfaces provides a pathway for receptor-generated kinase inhibition in one hexagon to spread to another, providing a crucial step for the extensive amplification characteristic of chemotactic signaling.

Investigation of allosteric coupling in human ß2-adrenergic receptor in the presence of intracellular loop 3.

BACKGROUND: This study investigates the allosteric coupling that exists between the intra- and extracellular parts of human ß2-adrenergic receptor (ß2-AR), in the presence of the intracellular loop 3 (ICL3), which is missing in all crystallographic experiments and most of the simulation studies reported so far. Our recent 1 µs long MD run has revealed a transition to the so-called very inactive state of the receptor, in which ICL3 packed under the G protein's binding cavity and completely blocked its accessibility to G protein. Simultaneously, an outward tilt of transmembrane helix 5 (TM5) caused an expansion of the extracellular ligand-binding site. In the current study, we performed independent runs with a total duration of 4 µs to further investigate the very inactive state with packed ICL3 and the allosteric coupling event (three unrestrained runs and five runs with bond restraints at the ligand-binding site). RESULTS: In all three independent unrestrained runs (each 500 ns long), ICL3 preserved its initially packed/closed conformation within the studied time frame, suggesting an inhibition of the receptor's activity. Specific bond restraints were later imposed between some key residues at the ligand-binding site, which have been experimentally determined to interact with the ligand. Restraining the binding site region to an open state facilitated ICL3 closure, whereas a relatively constrained/closed binding site hindered ICL3 packing. However, the reverse operation, i.e. opening of the packed ICL3, could not be realized by restraining the binding site region to a closed state. Thus, any attempt failed to free the ICL3 from its locked state due to the presence of persistent hydrogen bonds. CONCLUSIONS: Overall, our simulations indicated that starting with very inactive states, the receptor stayed almost irreversibly inhibited, which in turn decreased the overall mobility of the receptor. Bond restraints which represented the geometric restrictions caused by ligands of various sizes when bound at the ligand-binding site, induced the expected conformational changes in TM5, TM6 and consequently, ICL3. Still, once ICL3 was packed, the allosteric coupling became ineffective due to strong hydrogen bonds connecting ICL3 to the core of the receptor.

Structure and function of the thermoTRP channel pore.

Temperature-sensitive transient receptor potential (TRP) channels are structurally similar to other tetrameric cation channels, but can be potently activated by heat. Recent studies suggest that the pore-forming region directly participates in activation gating. In this chapter, we summarize major findings from both structural and functional studies concerning the gating role of the pore region, focusing in particular on TRPV1. The emerging picture is that the peripheral S1-S4 region of TRPV1 is rigid and plays a supporting role for the pore to undergo conformational rearrangements. This places the pore region in the center of activation gating.

Phosphopeptide binding to the N-SH2 domain of tyrosine phosphatase SHP2 correlates with the unzipping of its central ß-sheet.

SHP2 is a tyrosine phosphatase that plays a regulatory role in multiple intracellular signaling cascades and is known to be oncogenic in certain contexts. In the absence of effectors, SHP2 adopts an autoinhibited conformation with its N-SH2 domain blocking the active site. Given the key role of N-SH2 in regulating SHP2, this domain has been extensively studied, often by X-ray crystallography. Using a combination of structural analyses and molecular dynamics (MD) simulations we show that the crystallographic environment can significantly influence the structure of the isolated N-SH2 domain, resulting in misleading interpretations. As an orthogonal method to X-ray crystallography, we use a combination of NMR spectroscopy and MD simulations to accurately determine the conformation of apo N-SH2 in solution. In contrast to earlier reports based on crystallographic data, our results indicate that apo N-SH2 in solution primarily adopts a conformation with a fully zipped central ß-sheet, and that partial unzipping of this ß-sheet is promoted by binding of either phosphopeptides or even phosphate/sulfate ions.