Energy landscape views for interplays among folding,binding, and allostery of calmodulin domains

Ligand binding modulates the energy landscape of proteins, thus altering their folding and allosteric conformational dynamics. To investigate such interplay, calmodulin has been a model protein. Despite much attention, fully resolved mechanisms of calmodulin folding/binding have not been elucidated. Here, by constructing a computational model that can integrate folding, binding, and allosteric motions, we studied in-depth folding of isolated calmodulin domains coupled with binding of two calcium ions and associated allosteric conformational changes. First, mechanically pulled simulations revealed coexistence of three distinct conformational states: the unfolded, the closed, and the open states, which is in accord with and augments structural understanding of recent single-molecule experiments. Second, near the denaturation temperature, we found the same three conformational states as well as three distinct binding states: zero, one, and two calcium ion bound states, leading to as many as nine states. Third, in terms of the nine-state representation, we found multiroute folding/binding pathways and shifts in their probabilities with the calcium concentration. At a lower calcium concentration, “combined spontaneous folding and induced fit” occurs, whereas at a higher concentration, “binding-induced folding” dominates. Even without calcium binding, we observed that the folding pathway of calmodulin domains can be modulated by the presence of metastable states. Finally, full-length calmodulin also exhibited an intriguing coupling between two domains when applying tension.Protein folding and conformational dynamics have often been characterized by the energy landscape of proteins (1–5). The energy landscape is dependent on the molecular physiochemistry and thus is modulated by many factors, such as chemical modification and ligand binding. Ligand binding, in turn, is dependent on the conformation of proteins. Thus, folding, binding, and allosteric conformational dynamics are mutually correlated. Despite their obvious correlation in concept, it has been very challenging to characterize how they are indeed coupled for any single proteins. Here, we address, in depth, how these three types of dynamics, folding, binding, and allosteric conformational dynamics, are coupled from the energy landscape perspective for a specific protein, calmodulin (CaM).CaM is a ubiquitous calcium-binding messenger protein involved in signal transduction (6) and, more importantly here, has been a model protein to investigate folding, binding, and allostery. Full-length CaM has two nearly symmetric globular domains connected by a flexible central helix (7, 8). Each domain is composed of paired EF hands containing two Ca²⁺-binding sites (Fig. 1A). Upon binding to Ca²⁺, each CaM domain undergoes substantial conformational change from a closed state to an open state, exposing a hydrophobic patch that can bind with target proteins and regulate downstream processes (9). CaM has been frequently used as a model in studying the folding of multidomain proteins (10, 11), allosteric transitions (12–14), slow conformational dynamics around physiological temperatures (15–18), metal ion binding (19, 20), and correlation between inherent flexibility and protein functions (21, 22). For example, using structure-based coarse-grained (CG) simulations, Chen and Hummer elucidated the coexistence of an unfolded state, a closed state, and an open state around physiological temperatures for the C-terminal domain of CaM (CaM-C) without Ca²⁺ binding (15), which reconciles some seemingly contradictory experimental observations on the slow conformational dynamics of CaM.Open in a separate windowFig. 1.(A) Three-dimensional structure of calmodulin domain at closed [Protein Data Bank (PDB) code: 1cfd] and open states (PDB code:1cll). Calcium ions are represented by yellow spheres. (B) Schematic of coupling among folding, calcium binding, and allosteric motions for the CaM domain. Due to the conformational transitions between open and closed states, in addition to the direct folding pathway (red solid arrow), folding to the most stable state may involve an alternative pathway via a metastable state (green arrow plus blue arrow). The calcium binding can modulate the relative stability of the conformational states and therefore the population of folding pathways. O, C, and U represent open, closed, and unfolded states, respectively.More recently, Rief and coworkers studied the Ca²⁺-dependent folding of CaM based on a new generation technique of single-molecule force spectroscopy, which can probe the reversible folding/unfolding transitions with near equilibrium conditions (10, 23, 24). Their results revealed that at high Ca²⁺ concentrations, the folding pathway of the CaM domain proceeds via a transition state capable of binding Ca²⁺ ions, demonstrating the coupling between Ca²⁺ binding and CaM folding. All these computational and experimental works provided unprecedented understanding of many aspects of the folding and allosteric transitions of CaM. However, a full picture of the coupling among folding, Ca²⁺ binding, and allosteric motions, as schematically shown in Fig. 1B, is still lacking. Particularly, two fundamental issues arising from the allostery and Ca²⁺-binding characteristics of CaM remain elusive: (i) How does the allosteric feature of the energy landscape contribute to the folding complexity? And (ii) how can the folding mechanism of CaM be modulated by Ca²⁺ binding?Motivated by previous computational and experimental studies (15, 23), in this work we investigated the folding coupled with Ca²⁺ binding and allosteric motions of the isolated CaM domains as well as the full-length CaM. To do so, we first integrated computational tools developed for folding, ligand binding, and allosteric motions together. The proposed CG protein model was used for the subsequent series of molecular dynamics (MD) simulations. First, corresponding to Rief’s experiments, we performed MD simulations of isolated CaM domains with pretensions, which gave consistent results with the experiments and, in addition, provided the direct structural assignment on the experimentally observed states. Second, at a higher temperature, without pretension we performed reversible folding/unfolding simulations for a wide range of Ca²⁺ concentrations. The conformational and ligand-binding energy landscape revealed as many as nine distinctive states. Then, we analyzed the binding-coupled folding reactions in terms of the nine states, finding multiple routes and their modulation by Ca²⁺ concentrations. Interestingly, as the Ca²⁺ concentration increases, the CaM domain folding mechanism switches from “combined spontaneous folding and induced fit” to “binding-induced folding,” which accords with the scenario deduced from single-molecule force spectroscopy experiments. Finally, the effects of tension on the conformational fluctuations of the full-length CaM are discussed.     

Conserved properties of individual Ca2+-binding sites in calmodulin

Calmodulin (CaM) is a Ca²⁺-sensing protein that is highly conserved and ubiquitous in eukaryotes. In humans it is a locus of life-threatening cardiomyopathies. The primary function of CaM is to transduce Ca²⁺ concentration into cellular signals by binding to a wide range of target proteins in a Ca²⁺-dependent manner. We do not fully understand how CaM performs its role as a high-fidelity signal transducer for more than 300 target proteins, but diversity among its four Ca²⁺-binding sites, called EF-hands, may contribute to CaM’s functional versatility. We therefore looked at the conservation of CaM sequences over deep evolutionary time, focusing primarily on the four EF-hand motifs. Expanding on previous work, we found that CaM evolves slowly but that its evolutionary rate is substantially faster in fungi. We also found that the four EF-hands have distinguishing biophysical and structural properties that span eukaryotes. These results suggest that all eukaryotes require CaM to decode Ca²⁺ signals using four specialized EF-hands, each with specific, conserved traits. In addition, we provide an extensive map of sites associated with target proteins and with human disease and correlate these with evolutionary sequence diversity. Our comprehensive evolutionary analysis provides a basis for understanding the sequence space associated with CaM function and should help guide future work on the relationship between structure, function, and disease.Eukaryotes use Ca²⁺ in numerous intracellular signaling pathways. Calmodulin (CaM) is a highly versatile Ca²⁺ signaling protein that is essential for at least dozens of cellular processes in eukaryotic cells. In humans it binds to more than 300 targets (1–3). Humans have three genes that encode identical CaM proteins, but mutations in just one of the three copies can cause disease (4–8), as can altered gene expression (9). Although CaM has been extensively studied, many details about its function are still poorly understood. The high evolutionary conservation along with the wide range of targets brings up the question of how a single Ca²⁺-binding protein displays both selectivity and flexibility in the context of its various signaling pathways.CaM binds Ca²⁺ at four, nonidentical sites that contain the structural motif called an EF-hand (10, 11), each of which contains an acidic Ca²⁺-coordinating loop, or “EF-loop” (Fig. 1A). The EF-loop spans 12 amino acids and provides at least six oxygen atoms for coordinating Ca²⁺ (12). The coordinating oxygen atoms are provided by the side chains at the first, third, fifth, and 12th positions of the EF-loop, and an oxygen from a main chain carbonyl group is provided at the seventh position (10). Water molecules participate in the Ca² coordination geometry (13). CaM functions as a sensor over a broad range of Ca²⁺ signals that vary in amplitude, duration, and location. Although biophysical and evolutionary sequence studies have resulted in a general understanding of the bulk properties of EF-hand–binding sites, the implications of differences in Ca²⁺ affinity among the four EF-hands deserves a thorough investigation.Open in a separate windowFig. 1.(A) Example of a Ca²⁺-bound EF-hand structure from PDBID 1CLL. A cartoon of an EF-hand peptide chain threads through a semitransparent representation of its molecular surface. The surface is the interface between molecular atoms and solvent rendered in PyMOL. Only atoms nearest the Ca²⁺ are shown and are depicted as spheres—green for Ca²⁺ and red for oxygens. A Ca²⁺-coordinating water is depicted as a semitransparent red sphere. Helices are gray, and the EF-loop is tan. (B) Maximum likelihood branch lengths of CaM and tubulin constrained to match the species tree in Torruella et al. (40). This tree covers much of eukaryotic diversity. Holozoa and Holomycota include animals and fungi, respectively, and their closely related protist lineages. SARPAE is described in the text. Both proteins are highly constrained, but whereas tubulin’s rate has been fairly consistent across eukaryotes, CaM underwent a dramatic speed-up in Ascomycete fungi, which include the model system S. cerevisiae.Previous reports showed that the large family of EF-hand proteins likely arose from a founder protein with a single EF-hand in the most recent common ancestor of all extant eukaryotes (11, 14–18). Different EF-hand–containing proteins bind Ca²⁺ with different affinities, suggesting that a protein with multiple EF-hands, such as CaM, may bind Ca²⁺ with a different affinity at each site (19–28). It has therefore been suggested that CaM’s four sites display different affinities and perhaps cooperativity (29, 30). We therefore hypothesized that CaM’s four, nonidentical loops may generate some of their functional flexibility by binding Ca²⁺ using different physical properties and explored whether such differences could be discerned in the evolutionary record.Evolutionary analyses can provide mechanistic insight into how CaM is used as a Ca²⁺ sensor across eukaryotes. Prior work showed that the protein sequence of CaM is evolving at a faster pace in fungal species (11, 31–33), reflecting the fact that although CaM is essential in Saccharomyces cerevisiae, the cells can survive with all four EF-hands ablated (34). However, previous evolutionary studies focused on a small subset of eukaryotes, either because few sequences were available at the time of publication or because the study was focused on a particular lineage. The vast expansion of taxonomic coverage in sequence databases, and the recent availability of new NMR and X-ray crystal structures of CaM, therefore demands a more comprehensive analysis. Unfortunately, CaM is a small, ancient, and highly conserved protein and therefore does not contain enough information to infer phylogenetic tree topologies. Kretsinger and Nakayama and coworkers (11, 16, 17, 35), for instance, found little correspondence between phylogenies inferred from protein, DNA, or intron–exon structure.To overcome this hurdle, we used a variety of techniques to explore sequence and structural conservation in CaM across eukaryotes. Our approach allows us to address several key questions: (i) How fast is CaM diverging in different phyla? (ii) How does the function of a site, or its association with disease, correlate with sequence conservation? (iii) What properties of the EF-hands are conserved over deep evolutionary time, and how might this correspond to functional plasticity?     

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.Although proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein–protein interactions (2–4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5–11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein’s structural dynamics link to its functional mechanisms (12–21).A case in point is the nitric oxide synthase (NOS) enzymes (22–24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached “oxygenase” domain, which then enables NO synthesis to begin (22, 25–27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation (Fig. 1 A and B) (28, 30–36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25, 28, 32, 34, 35, 37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38–42).Open in a separate windowFig. 1.(A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome c reductase activity of nNOSr proteins in their CaM-bound and CaM-free states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark cyan, E827C/Q1268C CL nNOSr labeled.NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25, 27). CaM binding to NOS enzymes increases electron transfer from NADPH through the reductase domain and also triggers electron transfer from the FMN domain to the NOS heme as is required for NO synthesis (31, 32). The ability of CaM, or similar signaling proteins, to regulate electron transfer reactions in enzymes is unusual, and the mechanism is a topic of interest and intensive study. It has long been known that CaM binding alters NOSr structure such that, on average, it populates a more open conformation (43, 44). Recent equilibrium studies have detected a buildup of between two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26, 36, 37, 39, 40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34, 36, 45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations. Indeed, computer modeling has indicated that a shift toward more open conformations as is induced by CaM binding to nNOS should, on its own, actually diminish electron flux through nNOS and through certain related flavoproteins (38). Despite its importance, measuring enzyme conformational fluctuation dynamics is highly challenging, and as far as we know, there have been no direct measures on the NOS enzymes or on related flavoproteins, nor studies on how CaM binding might influence the conformational fluctuation dynamics in NOS.To address this gap, we used single-molecule fluorescence energy resonance transfer (FRET) spectroscopy to characterize individual molecules of nNOSr that had been labeled at two specific positions with Cyanine 3 (Cy3) donor and Cyanine 5 (Cy5) acceptor dye molecules, regarding their conformational states distribution and the associated conformational fluctuation dynamics, which in turn enabled us to determine how CaM binding impacts both parameters. This work provides a unique perspective and a novel study of the NOS enzymes and within the broader flavoprotein family, which includes the mammalian enzymes methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR), and reveals how CaM’s control of the conformational behaviors may regulate the electron transfer reactions of NOS catalysis.     

Time-resolved DEER EPR and solid-state NMR afford kinetic and structural elucidation of substrate binding to Ca2+-ligated calmodulin

Recent advances in rapid mixing and freeze quenching have opened the path for time-resolved electron paramagnetic resonance (EPR)-based double electron-electron resonance (DEER) and solid-state NMR of protein–substrate interactions. DEER, in conjunction with phase memory time filtering to quantitatively extract species populations, permits monitoring time-dependent probability distance distributions between pairs of spin labels, while solid-state NMR provides quantitative residue-specific information on the appearance of structural order and the development of intermolecular contacts between substrate and protein. Here, we demonstrate the power of these combined approaches to unravel the kinetic and structural pathways in the binding of the intrinsically disordered peptide substrate (M13) derived from myosin light-chain kinase to the universal eukaryotic calcium regulator, calmodulin. Global kinetic analysis of the data reveals coupled folding and binding of the peptide associated with large spatial rearrangements of the two domains of calmodulin. The initial binding events involve a bifurcating pathway in which the M13 peptide associates via either its N- or C-terminal regions with the C- or N-terminal domains, respectively, of calmodulin/4Ca²⁺ to yield two extended “encounter” complexes, states A and A*, without conformational ordering of M13. State A is immediately converted to the final compact complex, state C, on a timescale τ ≤ 600 μs. State A*, however, only reaches the final complex via a collapsed intermediate B (τ ∼ 1.5 to 2.5 ms), in which the peptide is only partially ordered and not all intermolecular contacts are formed. State B then undergoes a relatively slow (τ ∼ 7 to 18 ms) conformational rearrangement to state C.

Calmodulin (CaM) is a universal eukaryotic calcium sensor that plays a central role in calcium signaling (1). Binding of two Ca²⁺ ions per CaM domain exposes methionine-rich hydrophobic patches that prime the system for high-affinity binding to a wide range of protein partners (2). Free calcium–loaded calmodulin (CaM/4Ca²⁺) is predominantly extended (3–5), although sparsely populated, highly transient compact states are sampled (6, 7), but clamps down upon target substrates like two hands capturing a rope in the final complex (8–11) (Fig. 1). Concomitant conformational changes involve the transition of a largely helical interdomain linker to a long flexible loop and the adoption of a helical conformation by the intrinsically disordered substrate. Although CaM has been the subject of extensive biophysical studies (12–16), current structural knowledge is limited to calcium-free, calcium-loaded, and calcium-loaded/peptide-bound states as separate entities, with little experimental information about the molecular mechanisms that connect the latter two states. For example, it is not known whether substrates bind first to the N-terminal domain (NTD) or C-terminal domain (CTD) of CaM/4Ca²⁺, whether identifiable intermediate states exist, whether there is a single predominant pathway for complex formation, or at what stage in the process the CaM binding regions of target protein substrates become conformationally ordered. To address this knowledge gap, we have performed time-resolved electron paramagnetic resonance (EPR)-based double electron-electron resonance (DEER) and solid-state (ss) NMR studies that jointly elucidate the process of CaM/4Ca²⁺–peptide complex formation in quantitative kinetic and structural terms. This work relies on three technological advances: 1) rapid mixing and freeze quenching to sequentially trap the state of the reaction mixture on the millisecond timescale (17–24), thereby permitting time-resolved DEER EPR and ssNMR measurements of protein–substrate interactions; 2) the application of phase-memory time (T_m) filtering (25) to quantitatively extract species populations from DEER data (26) so that time-dependent probability distance distributions between pairs of spin labels can be monitored; and 3) quantitative analysis of ssNMR ¹³C-¹³C correlation spectra to provide residue-specific information on the appearance of structural order and the development of intermolecular contacts between substrate and protein (21, 23).Open in a separate windowFig. 1.Schematic overview of the time-resolved DEER EPR and ssNMR experiments. The predominant extended conformation of free CaM/4Ca²⁺ seen in the crystal structure is shown in Upper Left (3); the locations of the R1 nitroxide spin labels (transparent red spheres) at A17C–R1 and A128C–R1 in the NTD (blue) and CTD (purple), respectively, and of the relevant methionine residues (gray balls) are indicated. The intrinsically disordered M13 peptide, comprising the CaM binding site of skMLCK, is shown in Upper Right, with ¹³C-labeled residues in the N- and C-terminal halves of the peptides shown as blue and mauve balls, respectively. The structure of the final CaM/4Ca²⁺–M13 complex (8, 9) is shown in Lower, with the helical M13 peptide in red. The reaction time is controlled by the flow rate through the mixer and the flight distance from the mixer nozzle to the spinning copper disk cooled to 77K.     

Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl? channels

Ca²⁺-activated chloride currents carried via transmembrane proteins TMEM16A and TMEM16B regulate diverse processes including mucus secretion, neuronal excitability, smooth muscle contraction, olfactory signal transduction, and cell proliferation. Understanding how TMEM16A/16B are regulated by Ca²⁺ is critical for defining their (patho)/physiological roles and for rationally targeting them therapeutically. Here, using a bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP)—we discovered that Ca²⁺-free calmodulin (apoCaM) is preassociated with TMEM16A/16B channel complexes. The resident apoCaM mediates two distinct Ca²⁺-dependent effects on TMEM16A, as revealed by expression of dominant-negative CaM₁₂₃₄. These effects are Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI). CDI and CDSA are independently mediated by the N and C lobes of CaM, respectively. TMEM16A alternative splicing provides a mechanism for tuning apoCaM effects. Channels lacking splice segment b selectively lost CDI, and segment a is necessary for apoCaM preassociation with TMEM16A. The results reveal multidimensional regulation of TMEM16A/16B by preassociated apoCaM and introduce ChIMP as a versatile tool to probe the macromolecular complex and function of Ca²⁺-activated chloride channels.Calcium (Ca²⁺)-activated chloride (Cl^–) channels (CaCCs) broadly expressed in mammalian cells regulate diverse physiological functions including: epithelial mucus secretion (1, 2), neuronal excitability (3–5), smooth muscle contraction (6), olfactory transduction (7, 8), and cell proliferation (9, 10). Drugs targeting CaCCs are being pursued as therapies for hypertension, cystic fibrosis, asthma, and cancer (1, 9, 11).Three laboratories independently identified the transmembrane protein TMEM16A as the molecular component of a CaCC (12–14). TMEM16A belongs to a protein family with 10 members encoded by distinct genes (15–18). There is universal agreement that TMEM16A, and the closely related TMEM16B, are bona fide CaCCs (2, 12–14, 19). Consistent with this, TMEM16A knockout mice displayed defective CaCC activity in a variety of epithelia (20–22), and the olfactory CaCC current was completely abolished in TMEM16B knockout mice (23). Hydropathy analyses suggest TMEM16 proteins have a similar topology with cytosolic N and C termini and eight predicted transmembrane helices (2, 19). Human TMEM16A has four alternatively spliced segments (a–d), differential inclusion of which modify voltage and Ca²⁺ sensitivity of resultant channel splice variants (24).CaCCs are highly sensitive to intracellular [Ca²⁺], displaying graded increases in Cl⁻ current (I_Cl) amplitude as [Ca²⁺]_i is raised from resting levels (∼100 nM) to the 1- to 2-μM range. In some cases, high [Ca²⁺]_i (>10 μM) leads to decreased I_Cl amplitude (inactivation) (25–27). The Ca²⁺ sensor(s) for Ca²⁺-dependent activation and inactivation (CDA and CDI) of TMEM16A/16B is unknown. There are two possible nonexclusive mechanisms: (i) direct Ca²⁺ binding to the channel or (ii) Ca²⁺ binding through a separate Ca²⁺-sensing protein. The TMEM16A sequence does not reveal any canonical Ca²⁺-binding EF hand motifs (14, 16, 17). A sequence in the first intracellular loop of TMEM16A resembling the “Ca²⁺ bowl” in large conductance Ca²⁺-activated K⁺ (BK) channels was disqualified by mutagenesis as the Ca²⁺ sensor responsible for CDA of TMEM16A (28). A revised TMEM16A topological model suggests the originally predicted extracellular loop 4 is located intracellularly (29), and mutating E702 and E705 within this loop markedly alter Ca²⁺ sensitivity of TMEM16A (29, 30).Some reports have suggested involvement of calmodulin (CaM) in distinct aspects of Ca²⁺-dependent regulation of CaCCs. Tian et al. reported that inhibiting CaM with trifluoperazine or J-8 markedly suppressed CDA of TMEM16A(abc) in HEK293 cells, and mapped the CaM binding site to splice segment b (31). They concluded that CaM is essential for TMEM16A activation. However, this suggestion is contradicted by the robust CDA of TMEM16A(ac), a splice variant lacking the putative CaM binding site on splice segment b (2, 24). Recently, Ca²⁺–CaM was found to bind TMEM16A(ac) in a Ca²⁺-dependent manner and result in an increased permeability of the channel to HCO₃⁻ (32). Deleting the Ca²⁺–CaM binding site did not affect CDA of TMEM16A(ac). Ca²⁺–CaM regulation of TMEM16A HCO₃⁻ permeability conforms to a traditional signaling mode where Ca²⁺ binds to freely diffusing CaM to form a Ca²⁺–CaM complex that then interacts with a target protein.There are several examples of an alternative mode of CaM signaling in which Ca²⁺-free CaM (apoCaM) is preassociated with target proteins under resting [Ca²⁺]_i conditions and acts as a resident Ca²⁺ sensor to regulate function of the host protein in response to increased [Ca²⁺]_i (33). This mode of CaM signaling is used as the activating mechanism for small conductance K⁺ channels (34) and Ca²⁺-dependent regulation of high voltage-gated Ca²⁺ (Ca_V1 and Ca_V2) channels (35, 36). A distinguishing feature of this mode of CaM signaling is that it is impervious to pharmacological inhibitors of Ca²⁺–CaM, but can be eliminated in dominant negative fashion by a CaM mutant, CaM₁₂₃₄, in which all four EF hands have been mutated so they no longer bind Ca²⁺ (34, 36). Whether apocalmodulin preassociates with TMEM16A/TMEM16B channel complexes and participates in Ca²⁺-dependent regulation of these channels is controversial (30, 37, 38).Using a recently developed bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP) (39)—we discovered that apoCaM is functionally preassociated with TMEM16A/16B channel complexes. Whereas the resident apoCaM is not necessary for CDA, it does mediate two distinct Ca²⁺-dependent processes in TMEM16A. First, it causes a leftward shift in the Ca²⁺-dependent activation curve at [Ca²⁺]_i ≤ 1 μM, an effect we term Ca²⁺-dependent “sensitization” of activation (CDSA). Second, it is the Ca²⁺ sensor for CDI observed at [Ca²⁺]_i > 10 μM. The two opposite effects are independently mediated by the two lobes of preassociated apoCaM— Ca²⁺ occupancy of the N lobe leads to CDI, whereas the C lobe mediates CDSA. Alternative splicing of TMEM16A provides a mechanism for regulating apoCaM binding and signaling. TMEM16A splice variants lacking segment a lost apoCaM binding altogether, eliminating both CDSA and CDI, whereas variants specifically lacking segment b were selectively deficient in CDI. Finally, TMEM16A variants defective in apoCaM binding displayed dramatically decreased trafficking to the cell surface.     

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study’s in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions.Transport of protein across the cell membrane is a complex process that usually involves multipart translocation machineries. Many protein toxins from poisonous plants or from pathogenic bacteria are able to penetrate into the cytosol of their target cells where they exert their toxic effects. Some of these toxins exploit the endogenous cellular machinery of endocytosis and intracellular sorting to gain access to the cell cytosol, but others carry their own translocation apparatus (1–4). These latter toxins provide a unique opportunity to analyze the molecular mechanisms and the physicochemical principles underlying polypeptide transport across biological membranes. Studies combining structural, biochemical, and electrophysiological approaches have begun to unravel the various strategies developed by these toxins to deliver their catalytic moieties across the cell membranes (5–10).The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the few known toxins able to invade eukaryotic cells through a mechanism of direct translocation across the plasma membrane of the target cells (11–13). CyaA is an essential virulence factor of B. pertussis that is secreted by virulent bacteria and able to enter into eukaryotic cells, where, on activation by endogenous calmodulin (CaM), it catalyzes high-level synthesis of cAMP, which in turn alters cellular physiology (14–16). CyaA is a 1,706-residue-long bifunctional protein organized in a modular fashion (Fig. 1A); the ATP-cyclizing, CaM-activated catalytic domain (AC) is located in the 400 amino-proximal residues, whereas the carboxyl-terminal 1,306 residues are responsible for the hemolytic phenotype of B. pertussis (17–20).Open in a separate windowFig. 1.Principle of CyaA translocation assay on tBLM/CaM assembly. (A) Scheme of CyaA toxin structure showing the three major domains: the catalytic domain, AC; the hydrophobic region, H, responsible for insertion of CyaA into the membrane; and the Ca²⁺-binding, RTX-containing domain, RD. (B) Schematic illustration of the approach used to monitor CyaA translocation across the tBLM. (C) Schematic representation of the SPR sample cell cross-section and tBLM/CaM construction.The C-terminal “hemolysin” moiety contains, between residues 500 and 750, several hydrophobic segments that are predicted to adopt alpha-helical structures and to insert into membranes to create the cation-selective pores responsible for the hemolytic activity (20, 21). The C-terminal part of the molecule (RD; residues 1,000–1,706) is involved in toxin binding to a specific cellular receptor (CD11b/CD18) (22, 23). This domain consists of approximately 40 copies of a calcium-binding, glycine- and aspartate-rich nonapeptide repeat (residues 1,014–1,613) characteristic of a large family of bacterial cytolysins known as repeat-in-toxin (RTX) toxins (11, 13, 24, 25).The CyaA toxin is synthesized as an inactive precursor, proCyaA, which is converted into the active toxin form (CyaA) on specific acylation of two lysine residues (26, 27). Then CyaA is secreted across the bacterial envelope by a dedicated type I secretion machinery and binds to the CD11b/CD18 integrin expressed by a subset of leukocytes including neutrophils, macrophages, and dendritic cells (22, 28–30). However, CyaA can also invade a wide variety of cells that do not express this receptor, albeit with a lower efficiency (19, 31–35).The most unique property of CyaA is its capability to deliver its N-terminal catalytic domain directly across the plasma membrane of the eukaryotic target cells, a process that occurs independently of the CD11b/CD18 receptor (11–13). It is believed that CyaA first inserts its hydrophobic segments into the plasma membrane and then delivers its catalytic domain across the plasma membrane into the cell cytosol (19, 31, 32) (Fig. 1B). Previous studies have shown that the translocation process is dependent on the temperature (occurring only above 15 °C), the membrane potential of the target cells, and the presence of calcium ions in the mM range (32, 36). Inside the cell, on binding to CaM with a subnanomolar affinity, CyaA is stimulated by more than 1,000-fold and exhibits a high catalytic rate (k_cat > 2,000 s⁻¹) to produce supraphysiologic levels of cAMP (12, 19, 37).How the hydrophilic CyaA catalytic domain of approximately 400 residues is able to pass across the hydrophobic barrier of the plasma membrane remains largely unknown, and whether specific eukaryotic proteins and/or cell membrane components are involved in this process is also unclear (19, 32, 35, 38, 39). To characterize the molecular mechanisms of CyaA translocation across the membrane, we performed a functional in vitro assay that exploits a recently designed biomimetic membrane assembly composed of a bilayer membrane (tBLM) tethered over an amino-grafted gold surface derivatized with CaM (40). This multilayer biomimetic assembly exhibits the fundamental feature of an authentic biological membrane in creating a continuous, yet fluid phospholipidic barrier between two distinct compartments: a cis side, corresponding to the extracellular milieu, and a trans side, marked by the cytosolic protein CaM (Fig. 1C). We monitored the binding of CyaA to the tBLM by surface plasmon resonance (SPR) spectroscopy, and detected the translocation of the catalytic domain across the bilayer by CyaA activation by the immobilized CaM. With this highly sensitive assay, translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and application of a negative transmembrane potential, in agreement with previous studies on eukaryotic cells (36).Our results demonstrate that CyaA does not require any specific eukaryotic components apart from CaM to translocate across a membrane. They also suggest that the catalytic domain may be electrophoretically transported across the bilayer in a calcium-dependent manner. This study provides a direct in vitro demonstration of a toxin translocation across a tBLM (41) and suggests that the biomimetic tBLM/CaM structure may be a useful tool for characterizing the molecular mechanisms of protein translocation across biological membranes under precisely defined conditions.     

Membrane translocation of TRPC6 channels and endothelial migration are regulated by calmodulin and PI3 kinase activation

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels leading to inhibition of endothelial cell (EC) migration in vitro and delayed EC healing of arterial injuries in vivo. The precise mechanism through which lysoPC activates TRPC6 channels is not known, but calmodulin (CaM) contributes to the regulation of TRPC channels. Using site-directed mutagenesis, cDNAs were generated in which Tyr⁹⁹ or Tyr¹³⁸ of CaM was replaced with Phe, generating mutant CaM, Phe⁹⁹-CaM, or Phe¹³⁸-CaM, respectively. In ECs transiently transfected with pcDNA3.1-myc-His-Phe⁹⁹-CaM, but not in ECs transfected with pcDNA3.1-myc-His-Phe¹³⁸-CaM, the lysoPC-induced TRPC6-CaM dissociation and TRPC6 externalization was disrupted. Also, the lysoPC-induced increase in intracellular calcium concentration was inhibited in ECs transiently transfected with pcDNA3.1-myc-His-Phe⁹⁹-CaM. Blocking phosphorylation of CaM at Tyr⁹⁹ also reduced CaM association with the p85 subunit and subsequent activation of phosphatidylinositol 3-kinase (PI3K). This prevented the increase in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the translocation of TRPC6 to the cell membrane and reduced the inhibition of EC migration by lysoPC. These findings suggest that lysoPC induces CaM phosphorylation at Tyr⁹⁹ by a Src family kinase and that phosphorylated CaM activates PI3K to produce PIP3, which promotes TRPC6 translocation to the cell membrane.Endothelial cell (EC) migration is required for healing after arterial injuries, such as those that occur with angioplasties. Oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC), the major lysophospholipid of oxidized low-density lipoprotein, are abundant in plasma and atherosclerotic lesions and inhibit EC migration (1). A brief influx of calcium is required to initiate EC migration (2), but lysoPC causes a prolonged influx of Ca²⁺ that disrupts the cytoskeletal dynamics required for normal EC migration (3, 4). Specifically, lysoPC activates canonical transient receptor potential 6 (TRPC6) channels, as shown by patch clamp recording, with Ca²⁺ influx (3). The increased [Ca²⁺]_i initiates events that result in TRPC5 channel activation (3). The later activation of TRPC5 compared with TRPC6 and the failure of TRPC6 and TRPC5 to coimmunoprecipitate indicates that they do not form a heteromeric complex. The importance of this pathway is found in TRPC6-deficient EC, where lysoPC has little effect on EC migration (3). Furthermore, a high cholesterol diet markedly inhibits endothelial healing in wild-type (WT) mice, but has no effect in TRPC6-deficient (TRPC6^−/−) mice (5). The mechanism of TRPC6 activation by lysoPC is not fully elucidated, limiting the ability to block this important pathway.Calmodulin (CaM), a small, highly conserved, intracellular calcium-binding protein (6), binds to TRPC channels and regulates their activation. TRPC proteins, including TRPC6, possess a C-terminal CaM-binding domain that overlaps with a binding site for the inositol trisphosphate receptor, and CaM and the inositol triphosphate receptor compete for binding at this site (7). Removal of CaM from the common binding site results in activation of TRP3 channels (8). TRPC proteins contain additional binding sites for CaM and other Ca²⁺-binding proteins, indicating a complex regulatory mechanism in response to changes in [Ca²⁺]_i that includes positive and negative regulation of channels (9). In addition to CaM regulating TRPC proteins by direct binding, CaM-dependent kinases activate TRPC channels (10). CaM activity and peptide binding affinity is altered by its phosphorylation state and bound Ca²⁺, and Ca²⁺ can regulate the phosphorylation of CaM (11). LysoPC activates tyrosine kinases, including Src family tyrosine kinases (12), and Src family kinases can phosphorylate CaM (13). The role of CaM and CaM phosphorylation in TRPC6 channel activation or in EC migration is incompletely understood.TRPC6 channel activation generally requires externalization; however, the mechanism of TRPC6 channel translocation to the plasma membrane is not clear. In HEK cells overexpressing TRPC6, stimulation of Gq protein-coupled receptors causes TRPC6 externalization and localization to caveolae or lipid rafts by an exocytotic mechanism (14). In smooth muscle cells, phosphatidylinositol 3-kinase (PI3K) is involved in carbachol-induced TRPC6 externalization (15). The mechanism by which lysoPC induces TRPC6 externalization in EC is unknown.PI3K produces phosphatidylinositol (3,4,5)-trisphosphate (PIP3) from phosphatidylinositol (4,5)-bisphosphate (PIP2) in the inner leaflet of the plasma membrane and participates in numerous intracellular signaling processes, including TRPC6 activation (16). PI3K is composed of a p85 regulatory subunit (p85α, p85β, or p55γ) and a p110 catalytic subunit (p110α, p110β, p110γ, or p110δ), and activity can be influenced by CaM association (17).The purpose of the present study is to explore the underlying mechanism of lysoPC-induced TRPC6 activation. We identify a mechanism in which phosphorylation of CaM at Tyr⁹⁹ plays a key role in lysoPC-induced TRPC6 externalization and inhibition of EC migration.     

Class-specific interactions between Sis1 J-domain protein and Hsp70 chaperone potentiate disaggregation of misfolded proteins

Protein homeostasis is constantly being challenged with protein misfolding that leads to aggregation. Hsp70 is one of the versatile chaperones that interact with misfolded proteins and actively support their folding. Multifunctional Hsp70s are harnessed to specific roles by J-domain proteins (JDPs, also known as Hsp40s). Interaction with the J-domain of these cochaperones stimulates ATP hydrolysis in Hsp70, which stabilizes substrate binding. In eukaryotes, two classes of JDPs, Class A and Class B, engage Hsp70 in the reactivation of aggregated proteins. In most species, excluding metazoans, protein recovery also relies on an Hsp100 disaggregase. Although intensely studied, many mechanistic details of how the two JDP classes regulate protein disaggregation are still unknown. Here, we explore functional differences between the yeast Class A (Ydj1) and Class B (Sis1) JDPs at the individual stages of protein disaggregation. With real-time biochemical tools, we show that Ydj1 alone is superior to Sis1 in aggregate binding, yet it is Sis1 that recruits more Ssa1 molecules to the substrate. This advantage of Sis1 depends on its ability to bind to the EEVD motif of Hsp70, a quality specific to most of Class B JDPs. This second interaction also conditions the Hsp70-induced aggregate modification that boosts its subsequent dissolution by the Hsp104 disaggregase. Our results suggest that the Sis1-mediated chaperone assembly at the aggregate surface potentiates the entropic pulling, driven polypeptide disentanglement, while Ydj1 binding favors the refolding of the solubilized proteins. Such subspecialization of the JDPs across protein reactivation improves the robustness and efficiency of the disaggregation machinery.

Molecular chaperones are involved in the maintenance of protein homeostasis by aiding correct protein folding (1). Yet severe stress conditions induce excessive protein misfolding and aggregation (2). Upon stress relief, the return to the proteostasis is mediated by the Hsp70 chaperone with cochaperones, including J-domain proteins (JDPs/Hsp40s), which together restore the native state of misfolded polypeptides trapped in aggregates (3–5). The JDP–Hsp70 system acts alone in metazoans or in cooperation with an Hsp100 disaggregase in most other eukaryotes and bacteria (5, 6).Protein disaggregation and refolding starts with a recognition of misfolded polypeptides within an aggregate by a JDP, and then, its J-domain interacts with the nucleotide-binding domain of Hsp70, inducing ATP hydrolysis which triggers the closure of the Hsp70’s substrate-binding domain over the aggregated substrate (7, 8). The aggregate-bound Hsp70 interacts with an Hsp100 disaggregase, and this interaction allosterically activates Hsp100 and tethers it to the aggregate (9–16). Subsequently, in an ATP-driven process, Hsp100 disentangles and translocates polypeptides from aggregates (17–21), which enables their correct refolding, spontaneous or with an assistance of Hsp70 and its cochaperones (22, 23).JDPs are the major regulators of the Hsp70 activity and substrate specificity (3, 24, 25). In yeast Saccharomyces cerevisiae, a general Hsp70 chaperone, Ssa1, is recruited to protein disaggregation by two main cytosolic JDPs, Ydj1 and Sis1, assigned to the Class A and Class B, respectively (3, 4, 26). Both Ydj1 and Sis1 comprise a helical, highly conserved J-domain, a flexible, mostly unstructured G/F region, two beta-barrel peptide-binding domains, CTDI and CTDII, and a C-terminal dimerization domain (27–33). Ydj1 additionally features a Zn-binding domain located in the first part of the CTDI region of the protein, which is distinctive for the Class A JDPs (32, 34).Despite the structural similarities, the two JDPs are functionally nonredundant. Sis1 is essential, and Ydj1 is required for growth above 34 °C (26, 27, 35, 36). Overexpression of Sis1 suppresses the phenotype caused by the deletion of YDJ1, while Ydj1 overexpression is not sufficient to suppress the deletion of SIS1 (26, 27, 35–37). The two JDPs show different specificities toward amorphous and amyloid aggregates (35, 38) and different populations of amorphous aggregates formed in vitro (4, 24).Recent reports shed more light on the JDPs’ divergence. Both JDPs form homodimers, which differ in the structural orientation of the J-domain: In Sis1, the J-domain is restrained from Hsp70 binding by the interaction with the Helix 5 in the G/F region (26, 33, 39–41). Such autoinhibition, which also occurs in most human Class B JDPs, is released through the interaction with the C-terminal EEVD motif of Hsp70 (33, 42). This regulation is important for the disassembly of amyloid fibrils by the human JDP–Hsp70 system (43), but its role in the handling of stress-related, amorphous aggregates is not clear. Despite the breadth of data on Hsp70 mechanisms, we still lack understanding of how the disparate features of the JDPs impact Hsp70 functioning in protein disaggregation.Here, we investigate individual steps of protein disaggregation in the context of functional differences between Sis1 and Ydj1. Using various biochemical approaches, we show that the two JDPs drive different modes of Ssa1 binding to aggregated substrates, which dictate diverse kinetics of their disaggregation by Hsp104. The distinctive performance of Sis1 is associated with its interaction with the C terminus of Hsp70. Our results suggest that the bivalent interaction with the Class B JDP conditions aggregate remodeling by the Hsp70 system, resulting in enhanced Hsp104-dependent protein recovery. Our data indicate a mechanism by which the Class A and B JDPs contribute to the disaggregation efficacy in a complex and divergent manner.     

IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through tyrosine hydroxylase phosphorylation

Inositol 1,4,5-trisphosphate receptor (IP₃R) binding protein released with IP₃ (IRBIT) contributes to various physiological events (electrolyte transport and fluid secretion, mRNA polyadenylation, and the maintenance of genomic integrity) through its interaction with multiple targets. However, little is known about the physiological role of IRBIT in the brain. Here we identified calcium calmodulin-dependent kinase II alpha (CaMKIIα) as an IRBIT-interacting molecule in the central nervous system. IRBIT binds to and suppresses CaMKIIα kinase activity by inhibiting the binding of calmodulin to CaMKIIα. In addition, we show that mice lacking IRBIT present with elevated catecholamine levels, increased locomotor activity, and social abnormalities. The level of tyrosine hydroxylase (TH) phosphorylation by CaMKIIα, which affects TH activity, was significantly increased in the ventral tegmental area of IRBIT-deficient mice. We concluded that IRBIT suppresses CaMKIIα activity and contributes to catecholamine homeostasis through TH phosphorylation.Inositol 1,4,5-trisphosphate receptor (IP₃R) binding protein released with IP₃ (IRBIT) was originally identified as a molecule that interacts with the intracellular calcium channel, IP₃R. IRBIT binds to and suppresses IP₃R activity in the resting state by blocking IP₃ access to IP₃R (1, 2). Our group and others have reported that IRBIT contributes to electrolyte transport, mRNA processing, and the maintenance of genomic integrity (3–9) through its interaction with multiple targets. However, little is known about the physiological role of IRBIT in the brain, where it is most highly expressed (1).Calcium calmodulin (CaM) dependent kinase II alpha (CaMKIIα) is a Ser/Thr kinase that is abundant in the central nervous system and is activated by the binding of Ca²⁺–CaM. CaMKIIα phosphorylates various target proteins and is involved in the regulation of synaptic transmission and plasticity (10, 11). CaMKIIα is expressed in the hippocampus, neocortex, thalamus, hypothalamus, olfactory bulb, cerebellum, and basal ganglia (12, 13). Many studies involving mutant mice and also pharmacological studies have indicated that CaMKIIα activity is essential for the acquisition of memory and learning (14, 15). In addition, the appropriate regulation of CaMKIIα is required for cognitive function and mood control (16–18). Thus, aberrant CaMKIIα activity is associated with several neuronal disorders such as schizophrenia, autism spectrum disorder, attention-deficit hyperactivity disorder (ADHD), and drug addiction, in which hyperactivity and social abnormalities are frequently observed (19–23). However, the precise mechanism linking CaMKIIα dysregulation and mental disorders is poorly understood.Recent behavioral studies using knockout (KO) mouse models or pharmacological approaches have revealed that the dysregulation of dopamine (DA) systems is correlated with a hyperactive phenotype and social abnormalities (24–26). The catecholamines, DA and norepinephrine (NE) are biosynthesized from the amino acids phenylalanine and tyrosine. The sequence of steps starts with the enzyme, tyrosine hydroxylase (TH). Thus, TH is the rate-limiting enzyme for both DA and NE synthesis. The appropriate regulation of TH activity is important for the maintenance of normal brain function and mental state (27).In this study, we identified CaMKIIα as an IRBIT-interacting molecule in the central nervous system. IRBIT binds to and suppresses the kinase activity of CaMKIIα by inhibiting the binding of CaM to CaMKIIα. In addition, we found that mice deficient in IRBIT present with hyperactivity and social abnormalities. In IRBIT KO mice, we observed increased catecholamine levels and hyperphosphorylation of Ser19 on TH, which is known to enhance TH activity and increase the biosynthesis of DA and NE (27, 28). Thus, we have concluded that IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through TH phosphorylation.     

H-Ras forms dimers on membrane surfaces via a protein–protein interface

The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein–protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization K_d is measured to be ∼1 × 10³ molecules/µm², and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.In mammalian signal transduction, Ras functions as a binary switch in fundamental processes including proliferation, differentiation, and survival (1). Ras is a network hub; various upstream signaling pathways can activate Ras-GDP to Ras-GTP, which subsequently selects between multiple downstream effectors to elicit a varied but specific biochemical response (2, 3). Signaling specificity is achieved by a combination of conformational plasticity in Ras itself (4, 5) and dynamic control of Ras spatial organization (6, 7). Isoform-specific posttranslational lipidation targets the main H-, N-, and K-Ras isoforms to different subdomains of the plasma membrane (8–10). For example, H-Ras localizes to cholesterol-sensitive membrane domains, whereas K-Ras does not (11). A common C-terminal S-farnesyl moiety operates in concert with one (N-Ras) or two (H-Ras) palmitoyl groups, or with a basic sequence of six lysines in K-Ras4B (12), to provide the primary membrane anchorage. Importantly, the G-domain (residues 1–166) and the hypervariable region (HVR) (residues 167–189) dynamically modulate the lipid anchor localization preference to switch between distinct membrane populations (13). For example, repartitioning of H-Ras away from cholesterol-sensitive membrane domains is necessary for efficient activation of the effector Raf and GTP loading of the G-domain promotes this redistribution by a mechanism that requires the HVR (14). However, the molecular details of the coupling between lipid anchor partitioning and nucleotide-dependent protein–membrane interactions remain unclear.In addition to biochemical evidence for communication between the C-terminal membrane binding region and the nucleotide binding pocket, NMR and IR spectroscopic observations suggest that the HVR and lipid anchor membrane insertion affects Ras structure and orientation (15–17). Molecular dynamics (MD) modeling of bilayer-induced H-Ras conformations has identified two nucleotide-dependent states, which differ in HVR conformation, membrane contacts, and G-domain orientation (18). In vivo FRET measurements are consistent with a reorientation of Ras with respect to the membrane upon GTP binding (19, 20). Further modeling showed that the membrane binding region and the canonical switch I and II regions communicate across the protein via long-range side-chain interactions (21) in a conformational selection mechanism (22). Whereas these allosteric modes likely contribute to Ras partitioning and reorientation in vivo, direct functional consequences on Ras protein–protein interactions are poorly understood.Members of the Ras superfamily of small GTPases are widely considered to be monomeric (23). However, several members across the Ras GTPase subfamilies are now known to dimerize (24–28), and a class of small GTPases that use dimerization instead of GTPase activating proteins (GAPs) for GTPase activity has been identified (29). Recently, semisynthetic natively lipidated N-Ras was shown to cluster on supported membranes in vitro, in a manner broadly consistent with molecular mechanics (MM) modeling of dimers (30). For Ras, dimerization could be important because Raf, which is recruited to the membrane by binding to Ras, requires dimerization for activation. Soluble Ras does not activate Raf in vitro (31), but because artificial dimerization of GST-fused H-Ras leads to Raf activation in solution, it has been hypothesized that Ras dimers exist on membranes (32). However, presumed dimers were only detected after chemical cross-linking (32), and the intrinsic oligomeric properties of Ras remain unknown.Here, we use a combination of time-resolved fluorescence spectroscopy and microscopy to characterize H-Ras(C118S, 1–181) and H-Ras(C118S, 1–184) [referred to as Ras(C181) and Ras(C181,C184) from here on] anchored to supported lipid bilayers. By tethering H-Ras to membranes at cys181 (or both at cys181 and cys184) via a membrane-miscible lipid tail, we eliminate effects of lipid anchor clustering while preserving the HVR region between the G-domain and the N-terminal palmitoylation site at cys181 (or cys184), which is predicted to undergo large conformational changes upon membrane binding and nucleotide exchange (18). Labeling is achieved through a fluorescent Atto488-linked nucleotide. Fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA) show that H-Ras forms surface density-dependent clusters. Photon counting histogram (PCH) analysis and single-molecule tracking (SMT) reveal that H-Ras clusters are dimers and that no higher-order oligomers are formed. A Y64A point mutation in the loop between beta strand 3 (β3) and alpha helix 2 (α2) abolishes dimer formation, suggesting that the corresponding switch II (SII) region is either part of, or allosterically coupled to, the dimer interface. The 2D dimerization K_d is measured to be on the order of 1 × 10³ molecules/µm², within the broad range of Ras surface densities measured in vivo (10, 33–35). Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution, suggesting that a membrane-induced structural change in H-Ras leads to dimerization. Comparing singly lipidated Ras(C181) and doubly lipidated Ras(C181,C184) reveals that dimer formation is insensitive to the details of HVR lipidation, suggesting that dimerization is a general property of H-Ras on membrane surfaces.     

Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating

The human ERG (hERG) K⁺ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS–CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.

Members of the KCNH superfamily of voltage-gated K⁺ channels contribute to neuronal excitability, cardiac repolarization, and cellular proliferation and are linked to human disease (1–12). In particular, the human ERG (hERG) channel has a crucial role in repolarization of the cardiac action potential; channel malfunction, either from genetic alterations or from unwanted pharmacological channel block, results in long QT syndrome (LQTS), a condition associated with cardiac arrhythmias and sudden death (2, 13, 14).KCNH channels are characterized by a “non-domain swapped” architecture, where the voltage-sensor domain interfaces with the pore domain in the same subunit (15). While the domain architecture is present in other channels (16), what distinguishes KCNH channels is the conserved cytosolic assembly formed by the N-terminal PAS (Per-Arnt-Sim) domain and the C-terminal cyclic nucleotide binding homology (CNBh) domain (17–24). PAS domains are widespread in nature, sensing light, redox potential, or small molecules and mediating protein–protein interactions (25–27). CNBh domains closely resemble CNB domains but lack the ability to bind nucleotides (28–32). Instead, the C-terminal tail of the CNBh domain acts as an intrinsic ligand, occupying the same position as a cyclic nucleotide in a bona fide CNB domain (30, 31).It is well established that PAS and CNBh domains interact with each other in the channel (15, 22, 23, 33, 34). Mutations that interfere with this interaction, disrupt the CNBh intrinsic ligand, or destabilize the fold of the domains give rise to changes in hERG gating. Many are associated with type 2 LQTS (LQT2) (29–31, 35–41). It is also clear that hERG function is modulated by variation in the number of PAS/CNBh assemblies present in individual channels, resulting from heteromers of two isoforms, one isoform with the PAS domain (hERG1a) and another without (hERG1b) (42–47).Further clues about the role of PAS and CNBh domains have been provided by ligands that target the cytosolic channel domains. We have demonstrated that allosteric modulation of hERG channel function through the PAS domain is possible by using scFv (single-chain variable fragment) antibodies (scFv2.10 and scFv2.12) that bind to PAS at distinct epitopes (48). scFv2.10 binds to residues R4 and R5 of hERG, in the PAS-cap region that spans the first 25 residues of the channel N terminus, just before the globular region of the PAS domain. In contrast, scFv2.12 binds to a region in the globular domain. In addition, small-molecule screening campaigns have identified ligands of PAS and CNBh domains that affect channel function (49–52). These data suggest that the assembly formed by the PAS and CNBh domains has an important role in the mechanism of gating of KCNH channels.Comparison of the cryoelectron microscopy (cryo-EM) structures of hERG, with an open cytosolic gate, and the calmodulin-inhibited rat EAG (another KCNH channel), with a closed gate, provides clear insights about the role of the PAS-cap in gating (15, 34). The PAS-cap engages the channel gating machinery when the gate opens, with its N terminus trapped between the C-linker, the S4-S5 linker, and the S2–S3 cytosolic loop. When the channel closes, the C-linker moves away, widening the PAS-cap binding site and releasing the N terminus. In contrast, the cryo-EM structure comparison shows that the PAS/CNBh assembly is not altered, even relative to the crystal structure of the isolated complex (33), undergoing only a simple rigid-body rotation (15, 34, 53). The overall view is that that role of the PAS/CNBh assembly is limited to correctly position the N-terminal PAS-cap for engagement with the gating machinery.Here, we propose a model in which the PAS/CNBh domain assembly is an active participant in the mechanism of hERG channel gating, undergoing a stabilization/destabilization cycle during hERG gating. This proposal results from the characterization of the molecular basis for the functional effect of scFv2.12 on the hERG channel—defining the antibody’s epitope, determining the impact of antibody binding on the PAS interaction with the CNBh domain in vitro and in the cell, and finally from monitoring the association of a fluorescent scFv2.12 antibody to the PAS domain during hERG gating.     

DNA self-organization controls valence in programmable colloid design

Just like atoms combine into molecules, colloids can self-organize into predetermined structures according to a set of design principles. Controlling valence—the number of interparticle bonds—is a prerequisite for the assembly of complex architectures. The assembly can be directed via solid “patchy” particles with prescribed geometries to make, for example, a colloidal diamond. We demonstrate here that the nanoscale ordering of individual molecular linkers can combine to program the structure of microscale assemblies. Specifically, we experimentally show that covering initially isotropic microdroplets with N mobile DNA linkers results in spontaneous and reversible self-organization of the DNA into Z(N) binding patches, selecting a predictable valence. We understand this valence thermodynamically, deriving a free energy functional for droplet–droplet adhesion that accurately predicts the equilibrium size of and molecular organization within patches, as well as the observed valence transitions with N. Thus, microscopic self-organization can be programmed by choosing the molecular properties and concentration of binders. These results are widely applicable to the assembly of any particle with mobile linkers, such as functionalized liposomes or protein interactions in cell–cell adhesion.

Building blocks encoded with assembly rules harness thermal energy to put themselves together in a process called self-assembly (1, 2). These elements can be proteins (3, 4), DNA (5–8), or colloids (8–12). Akin to atoms and molecules, colloidal particles with well-defined shapes and interactions self-organize into bulk crystalline phases that minimize the free energy (13–19). More-complex objects with nonrepeating structures, such as protein folds or aperiodic crystals, require a prescribed limit to particle valence (20, 21). A fundamental goal is to fabricate structures with important technological applications (22). For example, colloidal self-assembly into a diamond lattice (10) or a quasicrystal (23, 24) is expected to exhibit photonic band gaps due to the materials’ interaction with light (25, 26). At its most complex, self-assembly of biological cells is a crucial part of the development of a living organism (27).Experimentally, valence control can be achieved by designing anisotropic sticky particles with patches to create colloidal clusters (28–30) or DNA origami that specifies the bond orientation (31, 32). Mixing particles with a given size and number ratio can result in steric valence control (33). Other proposed methods include the self-organization of nematic shells on spheres (34, 35) or the arrested phase separation of lipids on droplet surfaces (36). These processes are complex to experimentally realize, feature slow assembly kinetics due to the necessity of patch-to-patch binding, and require extensive purification (28).Unlike solid particles, droplets (37–40), lipid vesicles (41–46), and biological cells (47–50) allow any sticky binders to freely diffuse at the interface and segregate into adhesions with their neighbors. If the particles are Brownian or mobile, they can rearrange even after binding to reach the most favorable valence and geometry, avoiding kinetic bottlenecks. Angioletti-Uberti et al. (51) theoretically proposed that mobile ligands coupled with an additional repulsive potential—such as a steric brush—could yield colloidal valence selection in the bulk. More generally, the mobility and reversibility of linker binding between particles allows the system to optimize its equilibrium structure according to the laws of statistical mechanics. Not only is this strategy more robust than directed irreversible assembly, but it enables colloidal design based on the properties of molecular binders.Here, we derive and experimentally validate the free energy functional for droplet–droplet adhesion and predict the consequent thermodynamically stable valence for given control parameters. Moreover, we show that droplets recover their equilibrium valence in a matter of minutes after their bonds are broken. Our results are applicable to any functionalized particles with mobile binders, showing that molecular properties and concentration are sufficient to predetermine valence. Emulsions serve as a template for programmable solid materials because the droplets can be readily polymerized at any stage of the self-assembly process (52, 53).     

Engineered CaM2 modulates nuclear calcium oscillation and enhances legume root nodule symbiosis

The key physiological event essential to the establishment of nitrogen-fixing bacteria and phosphate-delivering arbuscular mycorrhizal symbioses is the induction of nuclear calcium oscillations that are required for endosymbioses. These regular fluctuations in nucleoplasmic calcium concentrations are generated by ion channels and a pump located at the nuclear envelope, including the CYCLIC NUCLEOTIDE GATED CHANNEL 15 (CNGC15). However, how the CNGC15s are regulated in planta to sustain a calcium oscillatory mechanism remains unknown. Here, we demonstrate that the CNGC15s are regulated by the calcium-bound form of the calmodulin 2 (holo-CaM2), which, upon release of calcium, provides negative feedback to close the CNGC15s. Combining structural and evolutionary analyses of CaM residues with bioinformatic analysis, we engineered a holo-CaM2 with an increased affinity for CNGC15s. In planta, the expression of the engineered holo-CaM2 accelerates the calcium oscillation frequency, early endosymbioses signaling and is sufficient to sustain over time an enhanced root nodule symbiosis but not an increased arbuscular mycorrhization. Together, these results reveal that holo-CaM2 is a component of endosymbiosis signaling required to modulate CNGC15s activity and the downstream root nodule symbiosis pathway.

Nutrient acquisition is fundamental to life. Plants have evolved strategies to overcome soil phosphate limitation and gain access to atmospheric dinitrogen by developing beneficial associations with arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria, respectively. Unlike other crops, the vast majority of legumes have mastered associations with both endosymbionts, positioning them as key crops to develop sustainable agricultural practices in both developed and developing countries (1).The entry of nitrogen-fixing bacteria, known as rhizobia, and AM fungi into legume roots is initiated by the recognition of the endosymbiont. Host plants have plasma-membrane receptor-like kinases (2–6) that recognize rhizobial elicitors, lipochitooligosaccharides (LCOs), also known as Nod factors (7), and mycorrhizal factors composed of derivatives of LCOs and shorter chain chitooligosaccharides (8, 9). Although rhizobial and AM elicitors are recognized by different complexes of receptor-like kinases (10, 11), both symbionts require the activation of calcium oscillations in root epidermal nuclei (9, 12, 13) to set off the endosymbiosis program. In the model legume Medicago truncatula, two types of nuclear envelope localized ion channels are required to generate the calcium oscillation; the DOESN’T MAKE INFECTIONS1 (DMI1) channel and paralogs of CYCLIC NUCLEOTIDE GATED CHANNEL 15 (CNGC15) (14), and the calcium pump, MCA8 (15). Similar to the animal CNGCs, plant CNGCs are tetrameric ion channels that can include different CNGC units (16, 17). In M. truncatula, CNGC15a, CNGC15b, and CNGC15c are all involved in nuclear calcium oscillation in the root epidermis, nodulation, and arbuscular mycorrhization, suggesting that the three units could assemble into a heterocomplex at the nuclear envelope (14). However, how CNGC15s are regulated in planta to sustain a calcium oscillatory mechanism remains unknown.In this study, we demonstrate that CNGC15s are regulated by the calcium-bound form of the calmodulin 2 (holo-CaM2) in planta, which shapes the oscillatory pattern of nucleoplasmic calcium concentration by providing negative feedback on CNGC15s to cause its closure. By engineering CaM2 to generate CaM2^R91A, which specifically increased holo-CaM2 binding affinity to each CNGC15 unit, we accelerated closure of CNGC15s and increased the calcium oscillation frequency. We further show that accelerating the calcium oscillation frequency was sufficient to accelerate the early endosymbiosis signaling and that the expression of CaM2^R91A resulted in an enhanced root nodule symbiosis but not enhanced AM colonization. Our data reveal differential regulation of rhizobia and AM endosymbioses by CaM2^R91A and suggest that modulating calcium signaling can be used as a strategy to positively impact symbiosis with nitrogen-fixing bacteria.     

Eicosanoid regulation of debris-stimulated metastasis

Cancer therapy reduces tumor burden via tumor cell death (“debris”), which can accelerate tumor progression via the failure of inflammation resolution. Thus, there is an urgent need to develop treatment modalities that stimulate the clearance or resolution of inflammation-associated debris. Here, we demonstrate that chemotherapy-generated debris stimulates metastasis by up-regulating soluble epoxide hydrolase (sEH) and the prostaglandin E₂ receptor 4 (EP4). Therapy-induced tumor cell debris triggers a storm of proinflammatory and proangiogenic eicosanoid-driven cytokines. Thus, targeting a single eicosanoid or cytokine is unlikely to prevent chemotherapy-induced metastasis. Pharmacological abrogation of both sEH and EP4 eicosanoid pathways prevents hepato-pancreatic tumor growth and liver metastasis by promoting macrophage phagocytosis of debris and counterregulating a protumorigenic eicosanoid and cytokine storm. Therefore, stimulating the clearance of tumor cell debris via combined sEH and EP4 inhibition is an approach to prevent debris-stimulated metastasis and tumor growth.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death and the most rapidly increasing cancer in the United States (1). Pancreatic cancer is the fourth leading cause of cancer-related deaths (2). Both of these cancer types are associated with a poor prognosis (1, 2). Despite the effectiveness of chemotherapy as a frontline cancer treatment, accumulating evidence from animal models suggests that chemotherapy may stimulate tumor growth and metastasis (3–22). The Révész effect, described in 1956, demonstrates that tumor cell death (“debris”) generated by cancer therapy, such as radiation, accelerates tumor engraftment (23). Follow-up studies have confirmed the Révész effect, whereby radiation-generated debris stimulates tumor growth via a proinflammatory response (24–29). Dead cell–derived mediators also stimulate tumor cell growth (30, 31). Notably, large numbers of cells are known to die in established tumors (32), which can lead to endogenous tumor-promoting debris in the tumor microenvironment (8, 33–35).Chemotherapy-generated tumor cell debris (e.g., apoptotic and necrotic cells) promotes tumor growth and metastasis via several mechanisms, including: 1) triggering a storm of proinflammatory and proangiogenic eicosanoids and cytokines (8, 9, 33, 35–38); 2) hijacking tumor-associated macrophages (TAMs) (37, 39); 3) inactivating M1-like TAMs (37); and 4) inducing immunosuppression and limiting antitumor immunity (40–42). Importantly, a metastatic phenotype and poor survival in cancer patients can be predicted by high levels of tumor cell debris (43–48). Thus, every attempt to induce tumor cell death is a double-edged sword as the resulting debris stimulates the growth of surviving tumor cells (8, 25, 33, 34, 35, 37, 38, 49–53). Tumor cells that survive treatment with chemotherapy or radiation undergo tumor cell repopulation (29). Yet, no strategy currently exists to stimulate the clearance or resolution of therapy-induced tumor cell debris and inflammation in cancer patients (35, 54).The failure to resolve inflammation-associated debris critically drives the pathogenesis of many human diseases, including cancer (8, 35, 55). Inflammation is regulated by a balance between inflammation-initiating eicosanoids (e.g., prostaglandins, leukotrienes, and thromboxanes) and specialized proresolving lipid autacoid mediators (SPMs; e.g., resolvins and lipoxins), which are endogenously produced in multiple tissues throughout the human body (56). Notably, arachidonic acid metabolites, collectively called eicosanoids, are potent mediators of inflammation and cancer metastasis (57, 58). Epoxyeicosatrienoic acids (EETs, also named EpETrEs), key eicosanoid regulators of angiogenesis, also stimulate inflammation resolution via macrophage-mediated phagocytosis of cell debris (59–64). Because EETs are rapidly metabolized by soluble epoxide hydrolase (sEH) to the less active dihydroxyeicosatrienoic acids (DiHETEs) (62), inhibition of sEH stabilizes EETs (62, 65). Indeed, sEH is a key therapeutic target for pain, as well as neurodegenerative and inflammatory diseases, including cancer (33, 35, 65–74). Thus, sEH regulates inflammatory responses (62). Importantly, sEH inhibition reduces the circulating levels and the expression of pancreatic mRNA of inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in experimental acute pancreatitis in mice (75). Chronic pancreatitis is essential for the induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice, suggesting that inflammation is a critical driver of pancreatic cancer (76, 77). Potent, selective inhibitors of sEH have been demonstrated to suppress human cancers (e.g., glioblastoma) and inflammation-induced carcinogenesis (67, 71). Similarly, inhibition of sEH can suppress inflammatory bowel disease-induced carcinogenesis and inflammation-associated pancreatic cancer (74, 78). In addition, a dual inhibitor of c-RAF and sEH suppresses chronic pancreatitis and murine pancreatic intraepithelial neoplasia in mutant K-Ras–initiated carcinogenesis (72, 73). Likewise, dual cyclooxygenase-2 (COX-2)/sEH inhibitors (e.g., PTUPB) potentiate the antitumor activity of chemotherapy and suppress primary tumor growth and metastasis via inflammation resolution (33, 35, 66, 70).Cancer therapy-induced debris can stimulate tumor growth and metastasis via prostaglandin E₂ (PGE₂) in the tumor microenvironment (25, 35, 79). PGE₂ exerts its biological activity via four G protein-coupled receptors: EP1, EP2, EP3, and EP4 (80). Among these, EP4 is upregulated in both tumor cells and immune cells (e.g., macrophages) and exhibits protumorigenic activity in many human malignancies (e.g., breast, prostate, colon, ovarian, and lung) by regulating angiogenesis, lymphangiogenesis, liver metastasis, and lymphatic metastasis (81–85). Interestingly, PGE₂ impairs macrophage phagocytosis of pathogens via EP4 receptor activation (86–88). Moreover, EP4 stimulates cancer proliferation, migration, invasion, and metastasis (89). EP4 gene silencing inhibits metastatic potential in vivo in preclinical models of breast, prostate, colon, and lung cancer (85, 90). Additionally, EP4 antagonists can suppress proinflammatory cytokines (e.g., C-C motif chemokine ligand 2 [CCL2], IL-6, and C-X-C chemokine motif 8 [CXCL8]), reduce inflammation-dependent bone metastasis, and diminish immunosuppression, while restoring antitumor immunity (91–93). In a clinical study, the EP4 antagonist E7046 increased the levels of T cells and tumor-infiltrating M2 macrophages in patients with advanced malignancies (94). Intriguingly, EP4 antagonists enhance the tumor response to chemotherapy by inducing extracellular vesicle-mediated clearance of cancer cells (95). Notably, EP4 antagonists reverse chemotherapy resistance or enhance immune-based therapies in various tumor types, including lymphoma, colorectal cancer, and lung cancer (80, 93, 96). Thus, targeting the EP4 receptor may be a strategy to suppress debris-stimulated tumor growth and metastasis.Here, we demonstrate that tumor cell debris generated by chemotherapy (e.g., gemcitabine) stimulates primary hepato-pancreatic cancer growth and metastasis when coinjected with a subthreshold (nontumorigenic) inoculum of tumor cells. Chemotherapy-generated debris upregulated sEH and EP4, which triggered a macrophage-derived storm of proinflammatory and proangiogenic mediators. Inhibitors of sEH and EP4 antagonists promoted inflammation resolution through macrophage phagocytosis of tumor cell debris and reduced proinflammatory eicosanoid and cytokine production in the tumor microenvironment. Altogether, our data show that the combined pharmacological abrogation of sEH and EP4 can prevent hepato-pancreatic cancer and metastatic progression.     

Kinesin processivity is gated by phosphate release

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or “gate,” the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P_i) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P_i state (by adding free P_i) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.Since its discovery nearly 30 years ago (1), kinesin-1—the founding member of the kinesin protein superfamily—has emerged as an important model system for studying biological motors (2, 3). During “hand-over-hand” stepping, kinesin dimers alternate between a two–heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one–head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the MT (6–8). A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2, 3, 10–17). This transition, called “NL docking,” is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2, 3, 10–17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2, 3, 10–17). Prevailing models of the kinesin mechanochemical cycle (2, 3, 10, 14, 15, 17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding. Nevertheless, these abstractions do not speak directly to the branching transitions that determine whether kinesin dissociates from the MT (off-pathway) or continues its processive reaction cycle (on-pathway). The distance moved by an individual motor before dissociating—the run length—is limited by unbinding from the MT. The propensity for a dimer to unbind involves a competition among multiple, force-dependent transitions in the two heads, which are not readily characterized by traditional structural or bulk biochemical approaches. Here, we implemented high-resolution single-molecule optical trapping techniques to determine transitions in the kinesin cycle that govern processivity.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Synergistic regulation of nonbinary molecular switches by protonation and light

We report a molecular switching ensemble whose states may be regulated in synergistic fashion by both protonation and photoirradiation. This allows hierarchical control in both a kinetic and thermodynamic sense. These pseudorotaxane-based molecular devices exploit the so-called Texas-sized molecular box (cyclo[2]-(2,6-di(1H-imidazol-1-yl)pyridine)[2](1,4-dimethylenebenzene); 1⁴⁺, studied as its tetrakis-PF₆ ⁻ salt) as the wheel component. Anions of azobenzene-4,4′-dicarboxylic acid (2H⁺•2) or 4,4′-stilbenedicarboxylic acid (2H⁺•3) serve as the threading rod elements. The various forms of 2 and 3 (neutral, monoprotonated, and diprotonated) interact differently with 1⁴⁺, as do the photoinduced cis or trans forms of these classic photoactive guests. The net result is a multimodal molecular switch that can be regulated in synergistic fashion through protonation/deprotonation and photoirradiation. The degree of guest protonation is the dominating control factor, with light acting as a secondary regulatory stimulus. The present dual input strategy provides a complement to more traditional orthogonal stimulus-based approaches to molecular switching and allows for the creation of nonbinary stimulus-responsive functional materials.

Multifactor regulation of biomolecular machines is essential to their ability to carry out various biological functions (1 –11). Construction of artificial molecular devices with multifactor regulation features may allow us to understand and simulate biological systems more effectively (12 –31). However, creating and controlling such synthetic constructs remains challenging (16, 32 –37). Most known systems involving multifactor regulation, including most so-called molecular switches and logic devices (38 –43), have been predicated on an orthogonal strategy wherein the different control factors that determine the distribution of accessible states do not affect one another (20, 44 –56). However, in principle, a greater level of control can be achieved by using two separate regulatory inputs that operate in synergistic fashion. Ideally, this could lead to hierarchical control where different states are specifically accessed by means of appropriately selected nonorthogonal inputs. However, to our knowledge, only a limited number of reports detailing controlled hierarchical systems have appeared (57). Furthermore, the balance between specific effects (e.g., kinetics vs. thermodynamics) under conditions of stimulus regulation is still far from fully understood (54). There is thus a need for new systems that can provide further insights into the underlying design determinants. Here we report a set of pseudorotaxane molecular shuttles that act as multimodal chemical switches subject to hierarchical control.     

Cellular heterogeneity profiling by hyaluronan probes reveals an invasive but slow-growing breast tumor subset

Tumor heterogeneity confounds cancer diagnosis and the outcome of therapy, necessitating analysis of tumor cell subsets within the tumor mass. Elevated expression of hyaluronan (HA) and HA receptors, receptor for HA-mediated motility (RHAMM)/HA-mediated motility receptor and cluster designation 44 (CD44), in breast tumors correlates with poor outcome. We hypothesized that a probe for detecting HA–HA receptor interactions may reveal breast cancer (BCa) cell heterogeneity relevant to tumor progression. A fluorescent HA (F-HA) probe containing a mixture of polymer sizes typical of tumor microenvironments (10–480 kDa), multiplexed profiling, and flow cytometry were used to monitor HA binding to BCa cell lines of different molecular subtypes. Formulae were developed to quantify binding heterogeneity and to measure invasion in vivo. Two subsets exhibiting differential binding (HA^−/low vs. HA^high) were isolated and characterized for morphology, growth, and invasion in culture and as xenografts in vivo. F-HA–binding amounts and degree of heterogeneity varied with BCa subtype, were highest in the malignant basal-like cell lines, and decreased upon reversion to a nonmalignant phenotype. Binding amounts correlated with CD44 and RHAMM displayed but binding heterogeneity appeared to arise from a differential ability of HA receptor-positive subpopulations to interact with F-HA. HA^high subpopulations exhibited significantly higher local invasion and lung micrometastases but, unexpectedly, lower proliferation than either unsorted parental cells or the HA^−/low subpopulation. Querying F-HA binding to aggressive tumor cells reveals a previously undetected form of heterogeneity that predicts invasive/metastatic behavior and that may aid both early identification of cancer patients susceptible to metastasis, and detection/therapy of invasive BCa subpopulations.Breast tumors display substantial heterogeneity driven by genetic and epigenetic mechanisms (1–3). These processes select and support tumor cell subpopulations with distinct phenotypes in proliferation, metastatic/invasive proclivity, and treatment susceptibility that contribute to clinical outcomes. Currently, there is a paucity of biomarkers to identify these subpopulations (3–12). Although detection of genetic heterogeneity may itself be a breast cancer (BCa) prognostic marker (3, 13–15), the phenotypes manifested from this diversity are context-dependent. Therefore, phenotypic markers provide additional powerful tools for biological information required to design diagnostics and therapeutics. Glycomic approaches have enormous potential for revealing tumor cell phenotypic heterogeneity because glycans are themselves highly heterogeneous and their complexity reflects the nutritional, microenvironmental, and genetic dynamics of the tumors (16–18).We used hyaluronan (HA) as a model carbohydrate ligand for probing heterogeneity in glycosaminoglycan–BCa cell receptor interactions. We reasoned this approach would reveal previously undetected cellular and functional heterogeneity linked to malignant progression because the diversity of cell glycosylation patterns, which can occur as covalent and noncovalent modifications of proteins and lipids as well as different sizes of such polysaccharides as HA, is unrivaled (16, 17, 19). In particular, tumor and wound microenvironments contain different sizes of HA polymers that bind differentially to cell receptors to activate signaling pathways regulating cell migration, invasion, survival, and proliferation (19–22).More than other related glycosaminoglycans, HA accumulation within BCa tumor cells and peritumor stroma is a predictor of poor outcome (23) and of the conversion of the preinvasive form of BCa, ductal carcinoma in situ, to an early invasive form of BCa (24). HA is a nonantigenic and large, relatively simple, unbranched polymer, but the manner in which it is metabolized is highly complex (19, 25). There are literally thousands of different HA sizes in remodeling microenvironments, including tumors. HA polymers bind to cells via at least six known receptors (16, 19, 20, 26–32). Two of these, cluster designation 44 (CD44) and receptor for HA-mediated motility/HA-mediated motility receptor (RHAMM/HMMR), form multivalent complexes with different ranges of HA sizes (19, 29, 33), and both receptors are implicated in BCa progression (19–21, 23, 29, 30, 33–36). Elevated CD44 expression in the peritumor stroma is associated with increased relapse (37), and in primary BCa cell subsets may contribute to tumor initiation and progression (38–40). Elevated RHAMM expression in BCa tumor subsets is a prognostic indicator of poor outcome and increased metastasis (22, 33, 41). RHAMM polymorphisms may also be a factor in BCa susceptibility (42, 43).We postulated that multivalent interactions resulting from mixture of a polydisperse population of fluorescent HA (F-HA) sizes, typical of those found in remodeling microenvironments of wounds and tumors (19, 20, 29), with cellular HA receptors would uncover a heterogeneous binding pattern useful for sorting tumor cells into distinct subsets. We interrogated the binding of F-HA to BCa lines of different molecular subtypes, and related binding/uptake patterns to CD44 and RHAMM display, and to tumor cell growth, invasion, and metastasis.