Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase

We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τ_G1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τ_G2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 μs with [NO] = 200 μM) forming a 6c heme with a diffusion-limited rate constant of 2 × 10⁸ M⁻¹⋅s⁻¹. In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ_5C1 = 0.66 μs and τ_5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1–50 μs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed.The soluble guanylate cyclase (sGC), localized in many different cell types, is the receptor of the endogenous messenger nitric oxide (NO) and catalyzes the formation of cGMP from GTP upon activation triggered by NO binding (1, 2). The diatomic messenger NO and sGC play a critical role in several physiological processes: regulation of vascular blood pressure and cardiovascular diseases (3), lung airway relaxation and pulmonary pathologies (4), immune response and inflammatory disorders (5), and tumor progression and apoptosis (6). Thus, sGC is a pharmacological target of very high interest, and several activators have been developed (7, 8), leading to the approval of riociguat for the treatment of pulmonary hypertension (9, 10). Because of its pharmacological interest, the mechanisms of activation, deactivation, and regulation of sGC must be deciphered at the molecular level. Despite numerous efforts, the 3D crystal structure of heterodimeric sGC remains unknown, but the heme domain of the sGC β1-subunit [called heme NO/oxygen-binding (H-NOX)] was modeled from the heme domain of bacterial NO sensors (11, 12) and the sGC catalytic α1-subunit was modeled from the catalytic α1-subunit of adenylate cyclase (13). Recently, the entire quaternary structure of sGC was reconstructed by inserting individual protein domains into the density envelope of entire single-sGC molecules observed by EM (14), revealing a high flexibility of the sGC dimer. Subsequently, the structural perturbations induced by NO binding were mapped at the domain interfaces (15). In the past decade, a diversity of molecular models and regulatory models have been proposed (16–23), with some including structural hypotheses and involving or not involving the hypothetical NO binding to the proximal heme side (vs. distal NO binding).The heterodimeric sGC (∼150 kDa) comprises a regulatory domain in the β-subunit that contains the prosthetic heme group for NO binding, which activates the synthesis of cGMP from GTP, whereas the interface between the C termini of both α- and β-subunits harbors the GTP-binding catalytic site, remote from the heme domain (Fig. 1). The first internal molecular event correlated with sGC activation is the cleavage of the heme-proximal His covalent bond induced by NO binding (24). This event triggers the structural allosteric changes within the protein that induce the increase of catalytic activity. Because the sensing heme domain and the catalytic site are separated by a helical domain (15), there are necessary structural changes for “cross-talk” between both, mediating the allosteric regulation. The NO concentration dependence of NO binding (16) and the modulation of the sGC activity by nucleotides (21), together with the possibility of its activation by artificial compounds (7), illustrate the complexity of sGC regulation when interacting with its messenger NO.Open in a separate windowFig. 1.Sequence domain organization of sGC and focus on the structure of the heme-sensing domain. GC, guanylate cyclase; PAS, period circadian protein-aryl hydrocarbon receptor-single minded protein.One critical aspect for understanding this allosteric mechanism is the identification of structural transitions and intermediate species, which must be addressed by time-resolved techniques. We have previously observed that after NO dissociation from the heme (25, 26), NO recombines geminately to the reactive 4-coordinate (4c) heme with a very high probability (97% of dissociated NO with time constant τ = 7.5 ps). However, the dynamics of the 3% NO population that does not rebind geminately have not been identified so far. Here, we focused on the transitions occurring after NO has left the heme pocket of sGC. We investigated the dynamics of the heme coordination by time-resolved absorption spectroscopy in a time range encompassing 11 orders of magnitude, from 1 ps to 0.2 s. Methodologically, the photodissociation of the ligand NO from the heme with a fast laser pulse allows one to displace the equilibrium and to simulate the thermal release of NO. Then, because NO can either rebind immediately from within the heme pocket or migrate within the protein core and eventually to the solution, from where it may rebind at a longer time, we could monitor each of the intermediate states of the heme during the dynamics of NO from the solution to the heme by exploring 11 time decades. We report the heme iron coordination dynamics triggered by NO release and binding, and we have identified the subsequent transitions, including the time range of the allosteric transition between the activated and resting states of sGC.     

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.     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

N-terminal domain of complexin independently activates calcium-triggered fusion

Complexin activates Ca²⁺-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca²⁺-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca²⁺-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca²⁺-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca²⁺-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca²⁺-triggered release.Neurotransmitter release occurs upon fusion of synaptic vesicles with the plasma membrane (1, 2). Synaptic vesicle fusion is orchestrated by the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) fusion proteins (3, 4), in conjunction with synaptotagmin, complexin, and other synaptic proteins. The Ca²⁺-sensor synaptotagmin is essential for Ca²⁺-triggered release (5–8). Neuronal SNARE proteins form a ternary complex consisting of synaptobrevin/vesicle-associated membrane protein (VAMP2), syntaxin, and synaptosomal-associated protein 25 (SNAP-25). The main isoform synaptotagmin-1 is involved in synchronous release, and forms a conserved Ca²⁺-independent interface with the ternary SNARE complex (9), along with Ca²⁺-dependent interactions with the plasma membrane, and potentially other interfaces with the SNARE complex (10). Complexin is a small cytosolic α-helical protein abundant in the presynaptic terminal (11) that interacts with the SNARE complex (12) and the membrane (13).Complexin has at least two functions: It “activates” (i.e., greatly enhances) Ca²⁺-triggered synchronous neurotransmitter release by cooperating with synaptotagmin, and it regulates spontaneous release in the presynaptic terminal (recently reviewed in refs. 14–16). The activating function of complexin is conserved across all species (mammals, Drosophila, and Caenorhabditis elegans) and different types of Ca²⁺-triggered synaptic vesicle fusion studied to date (11, 17–26). Complexin also regulates spontaneous neurotransmitter release, although this effect is less conserved among species and varies depending on experimental conditions: for example, in Drosophila, spontaneous release increases with knockout of complexin (27, 28). Likewise, knockdown of complexin in cultured cortical neurons increases spontaneous release, although knockout of complexin in mice only affects spontaneous release depending on the particular neuronal cell type (20, 23, 24, 29). Exactly how complexin can exhibit these dual effects on Ca²⁺-triggered and spontaneous synaptic vesicle fusion remains enigmatic; however, it is known that different domains of complexin play different roles in Ca²⁺-triggered and spontaneous vesicle fusion, as summarized in the following paragraphs.Here, we focus on the complexin-1 isoform (referred to as Cpx in the following). Cpx can be divided into four domains (Fig. 1A, Bottom) that are involved in different functions. The N-terminal domain (residues 1–27) of Cpx is important for activation of synchronous Ca²⁺-triggered release in murine neurons (20, 30, 31) and in isolated chromaffin cells (32). However, N-terminal truncation of Cpx in C. elegans neuromuscular junctions does not decrease Ca²⁺-triggered release, but rather increases spontaneous release (21, 22), perhaps suggesting that reduction of activation may have been masked by a simultaneous increase of spontaneous fusion in these previous experiments.Open in a separate windowFig. 1.Cpx (26–83) fragment reduces spontaneous fusion similar to wild-type Cpx. (A) Schematic diagram of the single-vesicle content mixing assay (35) (Methods) and domain diagrams of Cpx and Cpx fragments used in this figure. PM, vesicles with reconstituted syntaxin-1A and SNAP-25A that mimic the plasma membrane; SV, vesicles with reconstituted synaptobrevin-2 and synaptotagmin-1 that mimic synaptic vesicles. The bar graphs show the effects of 2 μM Cpx or Cpx fragments on the SV/PM vesicle association count during the first acquisition periods (Methods) (B), the average probability of spontaneous fusion events per second (C), the amplitude of the first 1-s time bin (probability of a fusion event in that bin) upon Ca²⁺ injection (D), and the decay rate (1/τ) of the histogram upon Ca²⁺ injection (E). The fusion probabilities and amplitudes were normalized with respect to the corresponding number of analyzed SV/PM vesicle pairs (Methods). Individual histograms are in Figs. S1 and ​andS2.S2. The error bars in B–D are SDs for multiple independent repeat experiments (20, 29, 30, 33–38). Although the accessory domain is required for regulating spontaneous release, mutations of this domain do not affect the activating function of Cpx for Ca²⁺-triggered release compared with wild-type neurons in rescue experiments of Cpx knockdown (23, 39).The central domain of Cpx (residues 49–70) is essential for all functions of complexins in all species studied to date, including priming (23, 24, 39, 40), inhibiting spontaneous release (18, 20–22, 35, 37, 38), and activation of Ca²⁺-triggered release (17, 18, 20, 22, 30, 31, 35, 41).The C-terminal domain binds to phospholipids (24, 42), and it is important for vesicle priming in neurons (24, 32, 43). Moreover, Cpx without the C-terminal domain does not reduce spontaneous release in neuronal cultures, but it still activates Ca²⁺-triggered release in neuronal cultures (24) and in a reconstituted system (35). The C-terminal domain is sensitive to membrane curvature, and it may thus localize Cpx to the synaptic membrane (13, 44).Structurally, in isolation, both the N- and C-terminal domains of Cpx are largely flexible, although the accessory and central domains have α-helical propensity (45). The α-helical central domain of Cpx binds to the groove between the synaptobrevin-2 and syntaxin-1A α-helices in the center of the neuronal SNARE complex (12, 46). Cpx has two conformations when bound to the ternary SNARE complex, one of which induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of Cpx (47).Cpx has been studied extensively with reconstituted systems (35, 38, 48–52). The single-vesicle fusion assay described by Lai et al. (35) qualitatively reproduced the effects of synaptotagmin-1 and Cpx in both spontaneous and Ca²⁺-triggered release that have been observed in cortical neuronal cultures (9, 35).Here, we conducted single-vesicle fusion and single-molecule membrane-binding experiments to obtain new insights into the function of the Cpx N-terminal domain. We found that the N-terminal domain can be physically separated from the accessory and central domains of Cpx and still preserve its role in activating Ca²⁺-triggered release. The N-terminal domain interacts with membranes, an interaction that is enhanced by the presence of SNARE complex. Moreover, the N-terminal domain of full-length Cpx can be functionally substituted by the fusion peptide of influenza virus hemagglutinin (HA), suggesting that similar fusion elements and principles are used in different contexts of biological membrane fusion.     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Multiaddressable molecular rectangles with reversible host–guest interactions: Modulation of pH-controlled guest release and capture

A series of multiaddressable platinum(II) molecular rectangles with different rigidities and cavity sizes has been synthesized by endcapping the U-shaped diplatinum(II) terpyridine moiety with various bis-alkynyl ligands. The studies of the host–guest association with various square planar platinum(II), palladium(II), and gold(III) complexes and the related low-dimensional gold(I) complexes, most of which are potential anticancer therapeutics, have been performed. Excellent guest confinement and selectivity of the rectangular architecture have been shown. Introduction of pH-responsive functionalities to the ligand backbone generates multifunctional molecular rectangles that exhibit reversible guest release and capture on the addition of acids and bases, indicating their potential in controlled therapeutics delivery on pH modulation. The reversible host–guest interactions are found to be strongly perturbed by metal–metal and π–π interactions and to a certain extent, electrostatic interactions, giving rise to various spectroscopic changes depending on the nature of the guest molecules. Their binding mode and thermodynamic parameters have been determined by 2D NMR and van’t Hoff analysis and supported by computational study.The study of metal–metal interactions has drawn enormous attention since the past two decades because of the intriguing spectroscopic and photophysical properties arising from the close proximity of the metal centers (1, 2). Square planar d⁸ platinum(II) complexes with coordination unsaturation are one of the important classes of metal complexes that have been extensively explored because of their capability to exhibit metal–metal interactions and display rich photophysical properties (3–26). Platinum(II) terpyridine complexes have been found to exhibit rich polymorphism in the solid state (16–20) owing to their square planar coordination geometry, which permits facile access to Pt(II)···Pt(II) interactions as well as π–π interactions between the chromophores. It was not until 2001 that the first successful synthesis of platinum(II) terpyridine alkynyl complexes, which possess enhanced solubility and luminescence compared with the chloro counterpart, was reported (16). Additional efforts have been devoted to the use of the system to respond to external stimuli, such as variation in solvent composition (17, 18), pH (19, 20), temperature (21, 22), addition of ionic (24–26), and polymeric species (27, 28), in which spectral changes induced by strong Pt(II)···Pt(II) and π−π interactions have been displayed.In the past few decades, enormous efforts have been devoted to the construction of molecular architectures by fusing the organic framework to the transition metal centers through self-assembly processes (29–57). There has been continuous interest in the construction of stimuli-responsive metallosupramolecular architectures with diverse sizes, shapes, and symmetries to rationalize the criteria for molecular recognition and impart them on unique areas of applications, such as stereoselective guest encapsulation and molecular transporting devices (45–65). Although such a variety of metal–organic macrocyclic architectures has been reported, those involving the use of noncovalent interactions other than those of hydrogen bonding, donor–acceptor, electrostatic, and hydrophobic–hydrophobic interactions as well as luminescence changes that depend on the nature of the guests, which would be attractive for chemo- and biosensing, have been rare and are rather underexplored. Examples of such systems that can exhibit reversible host–guest association are also limited.Since the discovery of anticancer properties of cisplatin in 1969 (58), the coordination chemistry and the development of related species with enhanced properties and reduced cytotoxicity have received enormous attention. Although the potency and cytotoxicity studies are important, the availability of the drugs and their transport and release to the site of action are equally important. Thus, the design of smart drug delivery systems has been an area of growing interest. The first phosphorescent molecular tweezers making use of the alkynylplatinum(II) terpyridine moiety have been reported by our group to show their host–guest interactions with transition metal complexes (57). However, the opened structures of the tweezers have limited their selectivity and functionality. To accomplish the controlled drug delivery functionalities, the first main strategy is to rigidify the molecular architecture of the host from tweezers to a rectangle, so that the guest molecules would be better accommodated within the cavity, which may lead to a more selective encapsulation of guests within a definite size and steric environment. The possibility of introducing responsive functionalities into the molecular rectangles, which may serve as models for the study of on-demand controlled guest capture and release systems, has also been explored. pH-sensitive pyridine moieties have, therefore, been incorporated into the backbone of the rectangle to modulate the reversible host–guest interaction within the constrained rectangle environment on protonation/deprotonation of the pyridine nitrogen atom to achieve multiaddressable functions that would not have been readily achievable with the molecular tweezers structure. Additionally, the use of various platinum and gold complexes as guest molecules, which have been shown to display anticancer therapeutic behavior (58–65), may lead to the design of a smart multiaddressable molecular rectangle system that could capture and release specific guest molecules under different pH conditions to achieve proof-of-principle on-demand controlled drug delivery. Herein, the design and synthesis of a series of alkynylplatinum(II) terpyridine molecular rectangles (Fig. 1) with different geometries, topologies and electronic properties are reported. Moreover, the encapsulation of various guest molecules is also investigated in detail to provide a proof-of-principle model for the design of artificial drug delivery systems with the modulation of drug release by pH.Open in a separate windowFig. 1.Molecular structures of rectangles 1−4.     

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.     

A family of starch-active polysaccharide monooxygenases

The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries.Polysaccharide monooxygenases (PMOs) are enzymes secreted by a variety of fungal and bacterial species (1–5). They have recently been found to oxidatively degrade chitin (6–8) and cellulose (8–14). PMOs have been shown to oxidize either the C1 or C4 atom of the β(1→4) glycosidic bond on the surface of chitin (6, 7) or cellulose (10–12, 14), resulting in the cleavage of this bond and the creation of new chain ends that can be subsequently processed by hydrolytic chitinases and cellulases. Several fungal PMOs were shown to significantly enhance the degradation of cellulose by hydrolytic cellulases (9), indicating that these enzymes can be used in the conversion of plant biomass into biofuels and other renewable chemicals.There are three families of PMOs characterized thus far: fungal PMOs that oxidize cellulose (9–12) (also known as GH61 and AA9); bacterial PMOs that are active either on chitin (6, 8) or cellulose (8, 13) (also known as CBM33 and AA10); and fungal PMOs that oxidize chitin (AA11) (7). Sequence homology between these three families is very low. Nevertheless, the available structures of PMOs from all three families reveal a conserved fold, including an antiparallel β-sandwich core and a highly conserved monocopper active site on a flat protein surface (Fig. 1A) (2, 6, 7, 9, 10, 15–17). Two histidine residues in a motif termed the histidine brace coordinate the copper center. The N-terminal histidine ligand binds in a bidentate mode, and its imidazole ring is methylated at the N_ε position in fungal PMOs (Fig. 1A).Open in a separate windowFig. 1.(A) Representative overall and active site structures of fungal PMOs (PDB ID code 2YET) (10). (B) Structure of cellulose (18, 19). Chitin also contains β(1→4) linkages and has similar crystalline higher order structure to cellulose. (C) Model structure of amylopectin (23–25). Hydrogen bonds are shown with green dashed lines.Considering the conserved structural features, it is not surprising that the currently known PMOs act on substrates with similar structures. Cellulose and chitin contain long linear chains of β(1→4) linked glucose units and N-acetylglucosamine units, respectively (Fig. 1B). The polymer chains form extensive hydrogen bonding networks, which result in insoluble and very stable crystalline structures (18–21). PMOs are thought to bind to the substrate with their flat active site surface, which orients the copper center for selective oxidation at the C1 or C4 position (6, 16, 22). Some bacterial chitin-binding proteins are cellulose-active PMOs (8, 13, 14), further suggesting that the set of PMO substrates is restricted to β(1→4) linked polymers of glucose and glucose derivatives.Here, we report on the identification of new families of PMOs that contain several key features of previously characterized PMOs, but act on substrates different from cellulose or chitin. A member of one of these novel families of PMOs, NCU08746, was shown to oxidatively cleave amylose, amylopectin, and starch. We designate the NCU08746 family as starch-active PMOs. Both amylose and amylopectin contain linear chains of α(1→4) linked glucose, whereas the latter also contains α(1→6) glycosidic linkages at branch points in the otherwise α(1→4) linked polymer. Unlike cellulose and chitin, amylose and amylopectin do not form microcrystals; instead, they exist in disordered, single helical, and double helical forms (23–27) (see Fig. 1C for example). Starch exists partially in nanocrystalline form, but lacks the flat molecular surfaces as those found in chitin and cellulose. The discovery of starch-active PMOs shows that this oxidative mechanism of glycosidic bond cleavage is more widespread than initially expected.     

Structure of BipA in GTP form bound to the ratcheted ribosome

BPI-inducible protein A (BipA) is a member of the family of ribosome-dependent translational GTPase (trGTPase) factors along with elongation factors G and 4 (EF-G and EF4). Despite being highly conserved in bacteria and playing a critical role in coordinating cellular responses to environmental changes, its structures (isolated and ribosome bound) remain elusive. Here, we present the crystal structures of apo form and GTP analog, GDP, and guanosine-3′,5′-bisdiphosphate (ppGpp)-bound BipA. In addition to having a distinctive domain arrangement, the C-terminal domain of BipA has a unique fold. Furthermore, we report the cryo-electron microscopy structure of BipA bound to the ribosome in its active GTP form and elucidate the unique structural attributes of BipA interactions with the ribosome and A-site tRNA in the light of its possible function in regulating translation.Bacterial protein synthesis involves four main translational GTPase (trGTPase) factors: initiation factor 2 (IF2), elongation factors Tu and G (EF-Tu and EF-G), and release factor 3 (RF3). These factors catalyze major steps in translation initiation, elongation (both decoding and mRNA–tRNA complex translocation), and termination, in a GTP-dependent manner. Several additional GTPase factors, including EF4 (formerly known as LepA), BipA, and RelA, have been revealed to be associated with ribosomes under stress conditions (1).Both EF4 and BipA are paralogs of EF-G (1–5). Although EF4 is highly conserved in bacteria (4), deletion of ef4 gene causes no evident phenotype in Escherichia coli under optimal growth conditions (6). However, EF4 was shown to notably improve protein synthesis under stress conditions (7). Qin et al. (2) reported a unique function of EF4 promoting the back translocation of the elongation complex by one codon, hence presumably providing a second chance for EF-G to carry out a correct translocation.BipA (BPI-inducible protein A) gene is highly conserved among bacterial and chloroplast genomes (4) and has been implicated in regulating a variety of cellular processes including bacterial virulence, symbiosis, various stress responses, resistance to host defenses, swarming motility, biofilm, and capsule formation (8–10). As is the case with EF4, BipA is not required under optimal growth conditions but becomes an essential factor for bacterial survival at low temperature, nutrient depletion, and various other stress conditions (1, 9). The diverse nature of these processes underscores the global regulatory properties of BipA. Similarity to classical trGTPases and EF4 led to the speculation that BipA affects translation through directly interacting with the ribosome. For example, wild-type (fully modified) ribosomes seem to depend on BipA for translation of specific mRNAs (11). Furthermore, as with EF4, overexpression of BipA inhibits transfer-messenger mRNA (tmRNA)-dependent peptide tagging activity of nonstop messages on ribosome (6). Thus, BipA likely functions as an elongation factor as well. Consistent with this notion, BipA is able to bind to 70S ribosome in a GTP-dependent manner and its GTPase activity is enhanced in the presence of ribosomes, a characteristic feature of classical trGTPase factors (5, 12). Salmonella enterica BipA has been shown to interact with either 70S ribosomes or 30S subunits depending on the relative abundance of GTP and of the stress alarmone guanosine-3′,5′-bisdiphosphate (ppGpp), respectively (12). In addition, a recent study links BipA to ribosome biogenesis because bipA gene deletion results in perturbed 50S subunit processing and assembly, particularly at low temperatures (13). Although the evidence for BipA involvement in ribosome biosynthesis and/or functioning in translation is mounting, its exact role remains elusive.As a member of the ribosome-dependent trGTPase family, BipA is proposed to share structural similarity with EF4 and EF-G (4, 5). Indeed, all three consist of five domains, of which the N-terminal G domain (nucleotide-binding domain), the β-barrel domain (domain II), and the two α/β-domains (domains III and IV) are topologically equivalent (5) (Fig. 1). EF-G has G′ domain inserted into its G domain and a unique domain IV, whereas unique C-terminal domains (CTDs) are present in BipA and EF4 (Fig. 1). Despite the similarity, the three proteins have distinct functions probably attributed to their varied domain arrangements and ribosome-binding modes.Open in a separate windowFig. 1.Comparison of domain arrangement and overall structure of EF-G, EF4, and BipA. (A) Structures of isolated EF-G and EF4 are obtained from Protein Data Bank (PDB ID codes: 2BM0 and 3CB4, respectively). Structure of BipA apo form is presented. Domain I (green), also known as the G domain, is the nucleotide-binding region. G′ domain insertion (dark blue) is a characteristic feature of the EF-G protein. Domain II (violet) contains the translation factor signature β-barrel motif. Domains III (yellow) and V (sky blue) contain α/β-motifs. EF-G has a unique domain IV (brown), whereas EF4 and BipA have unique C-terminal domains (warm pink and red, respectively). The same color scheme is used throughout this work. (B) Schematic diagram depicting the domain arrangement of EF-G, EF4, and BipA.Extensive structural studies (14–22) of EF-G bound to ribosome have generated a wealth of atomic or near-atomic resolution information on how EF-G, in particular the positioning of its domain IV in ribosome decoding center, facilitates translocation. Mutagenesis study of EF-G revealed that the highly conserved loops I and II of domain IV disrupt the interactions between the decoding center and the codon–anticodon duplex that act as the barrier for mRNA–tRNA complex translocation (23). Structural studies have also shed light on the molecular basis of how EF4 reverses EF-G catalyzed translocation through its CTD reaching into the PTC and interacting with the acceptor stem of the peptidyl-tRNA in the P site (24, 25). In contrast, structures of neither the isolated BipA nor BipA bound to ribosome, which could illuminate the molecular basis of BipA functioning in protein translation, have been characterized yet. Hence, we aimed to structurally characterize the various biologically relevant states of BipA on and off the ribosome, toward a better understanding of the detailed function of BipA. Note that during the revision process, a paper was published reporting the structure of isolated BipA (26).     

Structural insight into HIV-1 capsid recognition by rhesus TRIM5α

Tripartite motif protein isoform 5 alpha (TRIM5α) is a potent antiviral protein that restricts infection by HIV-1 and other retroviruses. TRIM5α recognizes the lattice of the retrovirus capsid through its B30.2 (PRY/SPRY) domain in a species-specific manner. Upon binding, TRIM5α induces premature disassembly of the viral capsid and activates the downstream innate immune response. We have determined the crystal structure of the rhesus TRIM5α PRY/SPRY domain that reveals essential features for capsid binding. Combined cryo-electron microscopy and biochemical data show that the monomeric rhesus TRIM5α PRY/SPRY, but not the human TRIM5α PRY/SPRY, can bind to HIV-1 capsid protein assemblies without causing disruption of the capsid. This suggests that the PRY/SPRY domain alone constitutes an important pattern-sensing component of TRIM5α that is capable of interacting with viral capsids of different curvatures. Our results provide molecular insights into the mechanisms of TRIM5α-mediated retroviral restriction.TRIM5α potently inhibits infection by HIV-1 and other retroviruses at an early postentry stage in a species-specific manner (1). Rhesus TRIM5α (rhTRIM5α) potently blocks HIV-1 infection (2). In contrast, human TRIM5α (huTRIM5α) only weakly inhibits HIV-1, but potently restricts N-tropic murine leukemia viruses (N-MLV) (3, 4). TRIM5α is able to induce premature capsid disassembly (5) and activate downstream innate immune responses upon recognizing the retroviral capsid lattice (6).TRIM5α is composed of RING, B-box 2, coiled-coil (CC), and B30.2 (PRY/SPRY) domains (Fig. 1A), similar to many tripartite/RBCC motif (TRIM) family members (7). The RING domain functions as an E3 ubiquitin ligase (8); the B-box 2 domain mediates formation of higher-order structure and self-association (9, 10); and the coiled-coil domain mediates dimerization (11) and facilitates the formation of the hexagonal lattice (12). The PRY/SPRY domain is essential for recognition of retroviral capsids and determines the specificity of restriction (13, 14). Two linker regions, L1 and L2, separate RING/B-box 2 and coiled-coil/(PRY/SPRY) domains, respectively (Fig. 1A). The antiviral potency of TRIM5α has been shown to correlate with its affinity for the viral capsid lattice (5). Interestingly, a single amino acid change from arginine to proline at residue 332 (R332P) in the PRY/SPRY domain of huTRIM5α conferred the ability to restrict HIV-1 (14–16).Open in a separate windowFig. 1.(A) Schematic depiction of dimeric TRIM5α. The four domains are colored differently and their respective molecular masses are indicated. The two linker regions are labeled L1 and L2. (B) Size-exclusion chromatograms of rhesus (solid) and human (dashed) MBP-TRIM5α PRY/SPRY. The molecular masses of protein standards are indicated at the top.HIV-1 capsid (CA) proteins can assemble into closed fullerene cones or helical tubes; other structurally homologous retrovirus CA proteins form cylindrical or spherical capsids (17–19). Despite the diverse array of retroviral capsids, different shapes are recognized by the same TRIM5α protein or highly homologous orthologs (20). The binding interaction requires an assembled capsid lattice as individual CA molecules do not have an appreciable affinity to TRIM5α (21). This broad, yet specific lattice pattern-sensing ability resides in the capsid-recognition PRY/SPRY domain of TRIM5α (5, 13, 14, 16, 22). A TRIM5α truncation construct containing the coiled-coil and the PRY/SPRY domains (CC-SPRY) is sufficient to bind and disrupt HIV-1 CA assembly (23). However, the lack of detailed structural data on TRIM5α PRY/SPRY poses an obstacle to understanding the pattern-sensing mechanism by which TRIM5α interacts with the retrovirus capsid. Here we report the crystal structure of the rhTRIM5α PRY/SPRY domain that reveals important features for viral capsid recognition. Both our EM and biochemical data demonstrate that rhTRIM5α PRY/SPRY (PRY/SPRY_rh) alone is able to recognize HIV-1 capsid tubes, whereas huTRIM5α PRY/SPRY (PRY/SPRY_hu) cannot. These findings provide a structural framework that enables us to begin understanding the capsid pattern-recognition mechanisms of TRIM5α.     

Understanding the kinetic mechanism of RNA single base pair formation

RNA functions are intrinsically tied to folding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model.RNAs perform critical cellular functions at the level of gene expression and regulation (1–4). RNA functions are determined not only by RNA structure or structure motifs [e.g., tetraloop hairpins (5, 6)] but also by conformational distributions and dynamics and kinetics of conformational changes. For example, riboswitches can adopt different conformations in response to specific conditions of the cellular environment (7, 8). Understanding the kinetics, such as the rate and pathways for the conformational changes, is critical for deciphering the mechanism of RNA function (9–19). Extensive experimental and theoretical studies on RNA folding kinetics have provided significant insights into the kinetic mechanism of RNA functions (19–36). However, due to the complexity of the RNA folding energy landscape (37–46) and the limitations of experimental tools (47–55), many fundamental problems, including single base flipping and base pair formation and fraying, remain unresolved. These unsolved fundamental problems have hampered our ability to resolve other important issues, such as RNA hairpin and larger structure folding kinetics. Several key questions remain unanswered, such as whether the hairpin folding is rate-limited by the conformational search of the native base pairs, whose formation leads to fast downhill folding of the whole structure, or by the breaking of misfolded base pairs before refolding to the native structure (18, 19, 54–73).Motivated by the need to understand the basic steps of nucleic acids folding, Hagan et al. (74) performed forty-three 200-ps unfolding trajectories at 400 K and identified both on- and off-pathway intermediates and two dominant unfolding pathways for a terminal C-G base pair in a DNA duplex. In one of the pathways, base pairing and stacking interactions are broken concomitantly, whereas in the other pathway, base stacking is broken after base pairing is disrupted. Furthermore, the unfolding requires that the Cyt diffuse away from the pairing Gua to a distance such that the C-G hydrogen bond cannot reform easily. More recently, Colizzi and Bussi (75) performed molecular dynamics (MD) pulling simulations for an RNA duplex and construct free energy landscape from the pulling simulation. The simulation showed that the base pair opening reaction starts with the unbinding of the 5′-base, followed by the unbinding of the 3′-base (i.e., the 5′-base is less stable than the 3′-base). These previous unfolding simulations offered significant insights into the pathways and transition states. However, as shown below, several important issues remain.One intriguing problem is the rate model for base pairing. There are currently three main types of models. In the first type of model, the barrier ΔG+‡ for closing a base pair is dominated by the entropic cost ΔS for positioning the nucleotides to the base-paired configuration and the barrier ΔG−‡ for opening a base pair is the enthalpic cost ΔH for disrupting the hydrogen bonds and base stacking interactions (18, 59, 60). In the second type of model, ΔG+‡ is the net free energy change for base pairing ΔG = ΔH ? TΔS and ΔG−‡ is zero (76, 77). In the third type of model, ΔG±‡=±ΔG/2 is used (78). In addition to the above three main types, other models, such as more sophisticated hybrid rate models, have been proposed (29).In this paper, we report a hybrid method (see Fig. 1) to investigate the single base pairing process. In contrast to the previous simulations for temperature- or force-induced unfolding reactions, we directly model the folding process here (i.e., the base pair closing process). Specifically, we use MD simulations to identify the conformational clusters. Based on the network of the conformational clusters as a reduced conformational ensemble, we apply kinetic Monte Carlo (KMC) and master equation (ME) methods to elucidate the detailed roles of base pairing and stacking interactions, as well as the roles of water and ions (79–82). The study reveals previously unidentified kinetics pathways, misfolded states, and rate-limiting steps. A clear understanding of the microscopic details of the elementary kinetic move is a prerequisite for further rigorous study of large-scale RNA kinetic studies. The method described here may provide a feasible way to develop a rate model for the base pair/stack-based kinetic move set. Furthermore, the mechanism of RNA single base folding may provide useful insights into many biologically significant processes, such as nucleotide flipping (83) in helicases and base pair fraying (84) (as the possible first step for nucleic duplex melting in nucleic acid enzymatic processes).Open in a separate windowFig. 1.(A) Folding of a single nucleotide (G1, red) from the unfolded (Left) to the native folded (Right) state. (B) Exhaustive sampling for the (discrete) conformations of the G1 nucleotide (Right) through enumeration of the torsion angles (formed by the blue bonds). (C) Schematic plot shows the trajectories on the energy landscape (depicted with two reaction coordinates for clarity) explored by the MD simulations. The lines, open circles, and hexagons denote the trajectories; the initial states; and the (centroid structures of the) clusters, respectively. (D) Conformational network based on six clusters. (E) The rmsds to the different clusters provide information about the structural changes in a MD trajectory.