ADP Release Research Articles

Intravascular hemolysis triggers ADP-mediated generation of platelet-rich thrombi in precapillary pulmonary arterioles.

Patients with hereditary or acquired hemolytic anemias have a high risk of developing in situ thrombosis of the pulmonary vasculature. While pulmonary thrombosis is a major morbidity associated with hemolytic disorders, the etiological mechanism underlying hemolysis-induced pulmonary thrombosis remains largely unknown. Here, we use intravital lung microscopy in mice to assess the pathogenesis of pulmonary thrombosis following deionized water-induced acute intravascular hemolysis. Acute hemolysis triggered the development of αIIbβ3-dependent platelet-rich thrombi in precapillary pulmonary arterioles, which led to the transient impairment of pulmonary blood flow. The hemolysis-induced pulmonary thrombosis was phenocopied with intravascular ADP- but not thrombin-triggered pulmonary thrombosis. Consistent with a mechanism involving ADP release from hemolyzing erythrocytes, the inhibition of platelet P2Y12 purinergic receptor signaling attenuated pulmonary thrombosis and rescued blood flow in the pulmonary arterioles of mice following intravascular hemolysis. These findings are the first in vivo studies to our knowledge to suggest that acute intravascular hemolysis promotes ADP-dependent platelet activation, leading to thrombosis in the precapillary pulmonary arterioles, and that thrombin generation most likely does not play a significant role in the pathogenesis of acute hemolysis-triggered pulmonary thrombosis.

Molecular movie of nucleotide binding to a motor protein

BackgroundThe SecA DEAD (Asp-Glu-Ala-Asp) motor protein uses binding and hydrolysis of adenosine triphosphate (ATP) to push secretory proteins across the plasma membrane of bacteria. The reaction coordinate of nucleotide exchange is unclear at the atomic level of detail. MethodsWe performed multiple atomistic computations of the DEAD motor domain of SecA with different occupancies of the nucleotide and magnesium ion sites, for a total of ~1.7 μs simulation time. To characterize dynamics at the active site we analyzed hydrogen-bond networks. ResultsATP and ADP can bind spontaneously at the interface between the nucleotide binding domains, albeit at an intermediate binding site distinct from the native site. Binding of the nucleotide is facilitated by the presence of a magnesium ion close to the glutamic group of the conserved DEAD motif. In the absence of the magnesium ion, protein interactions of the ADP molecule are perturbed. ConclusionsA protein hydrogen-bond network whose dynamics couples to the occupancy of the magnesium ion site helps guide the nucleotide along the nucleotide exchange path. In SecA, release of magnesium might be required to destabilize the ADP binding site prior to release of the nucleotide. General significanceWe identified dynamic hydrogen-bond networks that help control nucleotide exchange in SecA, and stabilize ADP at an intermediate site that could explain slow release. The reaction coordinate of the protein motor involves complex rearrangements of a hydrogen-bond network at the active site, with perturbation of the magnesium ion site likely occurring prior to the release of ADP.

Precisely Traceable Drug Delivery of Azoreductase-Responsive Prodrug for Colon Targeting via Multimodal Imaging.

We report the development of an azoreductase-responsive prodrug AP-N═N-Cy in which the precursor compound AP, a readily available podophyllotoxin derivative, is linked with a NIR fluorophore (Cy) via a multifunctional azobenzene group. This type of azo-based prodrug can serve as not only an azoreductase-responsive NIR probe to real-time tracking of the drug delivery process but also a delivery platform for an anticancer compound (AdP). We have shown that cleavage of the multifunctional azobenzene group in AP-N═N-Cy only occurred in the presence of azoreductase, which specifically secretes in the colon, resulting in direct release of AdP through an in situ modification of a phenylamino group on the precursor AP. Moreover, introduction of the azobenzene group endows the prodrug with an unique fluorescence "off-on" property and served as a switch to "turn on" the fluorescence of Cy as consequence of a self-elimination reaction with breakage of an azo bond. Such a prodrug can be administered orally and exhibit high stability and low toxicity before arriving at the colon. In view of the synchronism of drug release and the fluorescence turn-on process, the fluorescence imaging method was utilized to precisely trace drug delivery in vitro, ex vivo, and in vivo. Distinguishingly, the biodistribution of AdP and Cy in various tissues was further precisely mapped at the molecular level using imaging mass spectrometry. To the best of our knowledge, this is the first time that the in vivo real-time precise tracking of the colon-specific drug release and biodistribution was reported via a multimodal imaging method.

Conformational distributions of isolated myosin motor domains encode their mechanochemical properties.

Myosin motor domains perform an extraordinary diversity of biological functions despite sharing a common mechanochemical cycle. Motors are adapted to their function, in part, by tuning the thermodynamics and kinetics of steps in this cycle. However, it remains unclear how sequence encodes these differences, since biochemically distinct motors often have nearly indistinguishable crystal structures. We hypothesized that sequences produce distinct biochemical phenotypes by modulating the relative probabilities of an ensemble of conformations primed for different functional roles. To test this hypothesis, we modeled the distribution of conformations for 12 myosin motor domains by building Markov state models (MSMs) from an unprecedented two milliseconds of all-atom, explicit-solvent molecular dynamics simulations. Comparing motors reveals shifts in the balance between nucleotide-favorable and nucleotide-unfavorable P-loop conformations that predict experimentally measured duty ratios and ADP release rates better than sequence or individual structures. This result demonstrates the power of an ensemble perspective for interrogating sequence-function relationships.

Tracking the 3D Rotational Dynamics in Nanoscopic Biological Systems.

The rotation of an object cannot be fully tracked without understanding a set of three angles, namely, roll, pitch, and yaw. Tracking these angles as a three-degrees-of-freedom (3-DoF) rotation is a fundamental measurement, facilitating, for example, attitude control of a ship, image stabilization to reduce camera shake, and self-driving cars. Until now, however, there has been no method to track 3-DoF rotation to measure nanometer-scale dynamics in biomolecules and live cells. Here we show that 3-DoF rotation of biomolecules can be visualized via nitrogen-vacancy centers in a fluorescent nanodiamond using a tomographic vector magnetometry technique. We demonstrate application of the method to three different types of biological systems. First, we tracked the rotation of a single molecule of the motor protein F1-ATPase by attaching a nanodiamond to the γ-subunit. We visualized the 3-step rotation of the motor in 3D space and, moreover, a delay of ATP binding or ADP release step in the catalytic reaction. Second, we attached a nanodiamond to a membrane protein in live cells to report on cellular membrane dynamics, showing that 3D rotational motion of the membrane protein correlates with intracellular cytoskeletal density. Last, we used the method to track nonrandom motions in the intestine of Caenorhabditis elegans. Collectively, our findings show that the method can record nanoscale 3-DoF rotation in vitro, in cells, and even in vivo. 3-DoF rotation tracking introduces a new perspective on microscopic biological samples, revealing in greater detail the functional mechanisms due to nanoscale dynamics in molecules and cells.

Structural and kinetic basis for the regulation and potentiation of Hsp104 function

Hsp104 provides a valuable model for the many essential proteostatic functions performed by the AAA+ superfamily of protein molecular machines. We developed and used a powerful hydrogen exchange mass spectrometry (HX MS) analysis that can provide positionally resolved information on structure, dynamics, and energetics of the Hsp104 molecular machinery, even during functional cycling. HX MS reveals that the ATPase cycle is rate-limited by ADP release from nucleotide-binding domain 1 (NBD1). The middle domain (MD) serves to regulate Hsp104 activity by slowing ADP release. Mutational potentiation accelerates ADP release, thereby increasing ATPase activity. It reduces time in the open state, thereby decreasing substrate protein loss. During active cycling, Hsp104 transits repeatedly between whole hexamer closed and open states. Under diverse conditions, the shift of open/closed balance can lead to premature substrate loss, normal processing, or the generation of a strong pulling force. HX MS exposes the mechanisms of these functions at near-residue resolution.

Heat-stress induced expression of stress-inducible nucleotide exchange factor Fes1 in seagrass Zostera japonica.

Seagrass meadows are among the four most productive marine natural ecosystems in the world. Zostera japonica (Z. japonica) is the most widely distributed species of seagrass in China. Nucleotide exchange factors (NEFs) promote the release of ADP during heat stress, accelerating the rate-limiting step of Heat shock protein 70 (Hsp70). Although NEFs play an important role in abiotic stress tolerance of plants, NEFs in seagrass have not been studied. In this study, we cloned Fes1 from Z. japonica (ZjFes1) by rapid amplification of the cDNA ends using RACE, and full length ZjFes1 was 1171 bp. It contained an 81 bp 5'-terminal untranslated region (UTR), 109 bp 3'-UTR and 981 bp open reading frame (ORF). The ORF (ZjFes1) was predicted to encode a polypeptide of 326 amino acids with theoretical molecular weight (MW) of 36.10 kDa and pI of 5.22. ZjFes1 shared 89% amino acid identity with Fes1 from Zostera marina (Z. marina). The transcriptional levels of ZjFes1 increased significantly 1 h after heat treatment. ZjFes1 was localized to the cytoplasm. Taken together, we found that ZjFes1 was a stress-inducible gene that may be involved in heat stress response. This study lays the foundation for further studies on the role of ZjFes1 in heat resistance.

Tilianin Extracted From Dracocephalum moldavica L. Induces Intrinsic Apoptosis and Drives Inflammatory Microenvironment Response on Pharyngeal Squamous Carcinoma Cells via Regulating TLR4 Signaling Pathways.

Human pharyngeal squamous cell carcinoma is highly invasive and proliferative, and exhibits an extremely low 5-year survival rate due to poor understanding of the underlying pathogenic mechanisms, and lack of efficient treatment. It has been shown that the immunosuppressive microenvironment created by tumor cells impairs the immune response against tumor progression, thereby affecting the prognosis for tumor patients. Thus, to improve therapeutic efficacy, it is critical to identify novel drugs with immunoinflammatory modulatory properties to treat tumor immune evasion. Tilianin, the main ingredient of total flavonoids extracted from Dracocephalum moldavica L., has multiple biological functions, including cardiovascular protective effects, anti-tumor effects, and anti-inflammatory effects. In the present study, the suppressive effects of tilianin on human pharyngeal squamous cell carcinoma were investigated and the underlying mechanisms in regulating the tumor immunosuppressive microenvironment were explored. The cytotoxicity of tilianin on FaDu cells was determined by CCK-8 and clone formation assays. Moreover, the levels of toll-like receptor 4 (TLR4) signaling transduction and apoptotic pathways were determined by immunocytochemical, biochemical, and molecular biological technologies. In addition, the maturation of dendritic cells (DCs) that were co-cultured in supernatant of FaDu cells was evaluated by flow cytometry to investigate alterations in immune system function. For mechanistic exploration, TLR4 siRNA, p38 siRNA, c-Jun N-terminal kinase (JNK) siRNA, and p65 siRNA were used as loss-of-function target evaluation of tilianin therapy. Combined, these results showed that tilianin treatment increased cytotoxicity as well as the apoptotic population of FaDu cells in a dose-dependent manner. Furthermore, tilianin treatment decreased the level of anti-apoptotic markers Bcl-2 and Bcl-xL, increased the level of apoptotic factors Bad and Bax, and stimulated cytochrome c release, caspase-3 and poly ADP ribose polymerase (PARP) activation in FaDu cells. Furthermore, our findings indicated that tilianin treatment activated TLR4/p38/JNK/NF-κB signaling pathways and increased the release of inflammatory cytokines. This promoted the maturation of DCs to enhance immune system function in the tumor microenvironment. Moreover, the effects of tilianin on immune system function were abolished by TLR4 siRNA and p65 siRNA. In conclusion, these findings suggested that tilianin may be of immunotherapeutic value for inhibiting human pharyngeal squamous cell carcinoma.

The Rotary Motor of Life: Single-Molecule Imaging and Molecular Dynamics Simulation of F1-ATPase

F-ATPase is a rotary motor enzyme and its biological function is synthesis of ATP, the “fuel molecule” of cells. A subsystem of F-ATPase, the F1-ATPase, functions in reverse: it hydrolyses ATP into ADP and inorganic phosphate and during this process a rotation of its shaft-like rotor is driven by the chemical free energy which powers the organic nano-motor. Single-molecule imaging experiments revealed that during this rotation F1-ATPase proceeds through steps, i.e, the rotor fluctuates around specific dwell angles then jumps to the next dwell angle. There is a concerted behavior during this stepping rotation in which binding of ATP molecules in one subunit induce the release of ADP (product) molecules from another. Due to the size of the particle, as well as the minute time the process occurs over, it is extremely difficult to obtain accurate information about this sequence of states in the rotation. We examined it using two methods: by simulating molecular dynamics, and the analysis of single-molecule imaging experimental data. In the former case we examined a simulated molecule with triple-occupancy over a finite time increment with a Phosphate and ADP molecule in a binding pocket, and analyzed the simulation trajectory data. Preliminary results suggest a new finding that the Phosphate when doubly protonated can “escape” the pocket via a side door at the interface between a and b subunits. For the latter method, we examined a single molecule undergoing ATP synthesis using a 40 nm gold nano-probe to record measurements. Preliminary results suggest that the ATP binding dwell can be resolved and it is possible to focus the analysis on a hidden, short-lived intermediate step during the binding of ATP and release of ADP.

Exploring the State of the F1-ATPase after ATP Binding and before ADP Release: Effects of Conformational Changes on Phosphate Displacement

Single-molecule imaging experiments provide information that is not available from ensemble experiments. We are interested in the interpretation of dynamical studies imaging and manipulation in F₁-ATPase single-molecules. One key question that has arisen in single molecule stalling experiments is the erratic behavior a rotor angle of 55° between the binding and hydrolysis dwell angles of 0 and 80°, respectively. In our previous theoretical work, we used the elastic property of the rotor-stator structure to treat the experiments on controlled rotation. Our modeling suggests that there has to be a change in the bonding network, for example, of hydrogen bonds, as the system transitions between the two dwell points, perhaps at 55°, as indicated by an unusual stalling behavior around that angle. Therefore, in order to get further insights on these events, we have performed full-atomistic molecular dynamics (MD) simulations on the F₁-ATPase to explore the relationship between the conformational changes and the phosphate displacement. The pK_a values for phosphoric acid are 2.2, 7.2, and 12.3; although the effective pK_a for H₂PO₄⁻ within the binding site could be different. Hence, both protonation states (i.e., H₂PO₄⁻ and HPO₄²⁻) were assessed, performing molecular dynamics for 650 ns at 298.15K (Nose-Hoover thermostat) using the CHARMM36 force field. After the analysis of five MD trajectories, we found that the displacement of the phosphate was, to some extent, correlated with the dynamics of the protein. Moreover, one of the MD trajectories led to the complete release of the diprotic phosphate.

Fast States Revealed by Theory of Jumps in F1-ATPase Rotation Experiments

Single-molecule spectroscopies revealed the stepping rotation of an F1-ATPase enzyme in which a substep can have microsecond transition dynamics. Here we describe a method for analyzing fast single molecule rotation trajectories in F1-ATPase monitored by a 40 nanometer probe. This method focuses on the rotation jumps that occur in the transitions during the steps between dwells in single molecule trajectories. These jumps are related to the instantaneous rotation velocity and they exhibit a bimodal distribution at certain angles, indicating that the system produces both forward and a backward torques at the same angle, due to being in either of two states. Two states at the same angle is a key assumption used to extracts rate constants in stalling experiments, so by observing the bimodal distributions we provide support for this assumption. To calculate the distribution of jumps, we use a multi-state theory to describe the visco-elastic fluctuation of the imaging probe. The predicted jump distribution in the transitions yields a relaxation time which agrees with its value of 14 microseconds in the dwell fluctuations. Using a sequence of three states, the theoretical profile of angular jumps agrees with experiment for most of the angular range, but full agreement between theory and experiment is reached if a fourth, 10 microsecond lifetime state is assumed with an effective dwell half way through the 80 degree substep. It suggests that the ATP binding in one subunit and the ADP release from another subunit occurs via this transient. The ability to detect a state that is comparable or shorter than the instrumental relaxation time indicates that this jump distribution based method can be used to effectively increase the time resolution of the imaging apparatus.

Extracting the Hydrolysis Rate Constant from a Single-Molecule Controlled Rotation Experimental Data on a F1-ATPase

The chemo-mechanical energy transduction of the ATP Synthase is at the heart of the biological function, and it can be elucidated by studying the water soluble F1-ATPase, focusing on the individual sub-steps of the reactions in the enzyme. In controlled rotation experiment single F1-ATPase molecules deposited on a microscope slide are rotated by magnetic tweezers. Simultaneously the individual ATP binding and ADP release events are monitored by single molecule fluorescence imaging in real time. Our goal is to extract the ATP hydrolysis and synthesis rate constants as a function of rotor angle from the single molecule binding and release data. This work requires advanced analysis because these processes occurring in the binding pocket are “hidden”, and not directly monitored. Our approach is based on the treatment of composite events developed by Volkan-Kacso and Marcus, for a constant rotation rate. We will use high-quality single-molecule trajectories provided by our experimentalist collaborator K. Adachi, who pioneered the method of controlled rotation. Preliminary results indicate that fluorescent Cy3-ATP hydrolyses about 100 times slower than the wild species (ATP), at rotor angle of 240 degrees, its rate is about 1.7 X 10⁻³ per seconds. We plan to extract the hidden hydrolysis and synthesis rates for the complete 360 degrees cycle.

Myosin Motors’ Kinetic Diversity is Encoded by the Conformational Dynamics of the Motor Domain

Myosin molecular motors are involved in a range of diverse cellular processes, including muscle contraction, mechanosensation, intracellular transport, and membrane remodeling. All myosin isoforms share a conserved chemomechanical cycle that couples ATP hydrolysis to mechanical work on an actin filament. The kinetic and thermodynamic parameters that define this cycle, including duty ratio (the fraction of its cycle spent attached to actin) and ADP release rate (which controls actin detachment rate), can vary by orders of magnitude between isoforms, thus enabling this diversity of function. High resolution structures of the motor domains of many myosin isoforms have revealed surprising structural conservation between these biochemically diverse motors, raising the question as to how these motors are structurally tuned to such an array of molecular functions. We hypothesized that variability in the kinetic and thermodynamic parameters between isoforms results from alterations to the distribution of conformations a myosin adopts, and hence cannot be captured by any single structural model. To test this hypothesis, we performed over two milliseconds of explicit-solvent, all-atom molecular dynamics simulations for eight human myosin isoforms and constructed Markov state models (MSMs) of their free energy landscapes. These models capture significant conformational heterogeneity not observed in existing crystal models. We find that the relative populations of the two most probable conformational states predict biochemically measured duty ratios. Furthermore, the rates of transitioning between these populations predict ADP release rates measured in biochemical experiments. Finally, we investigate the role of allostery in modulating these crucial thermodynamic and kinetic parameters and suggest how perturbations at distant sites may be transmitted to and integrated at the active site. Taken together, our results shed light on myosin motors’ remarkable ability to provide vast physiologic diversity with the same structural fold.

Impact of Regulatory Light Chain Mutation (K104E) on the Atpase and Motor Properties of Human Cardiac Myosin

Mutations in the cardiac myosin heavy chain (MYH7) as well as the essential (MYL3) and regulatory (MYL2) light chains are known to lead to cardiomyopathies with variable phenotypes, including hypertrophic (HCM) and dilated (DCM) cardiomyopathy. We investigated the impact of the HCM-associated regulatory light chain (RLC) mutation (K104E) on myosin ATPase kinetics and in vitro motility. We expressed and purified human cardiac myosin subfragment 1 (M2B-S1) and exchanged on the human ventricular RLC, with the mutant or WT RLC. A mouse model of K104E was found to cause cardiac hypertrophy, fibrosis, and diastolic dysfunction in older animals suggesting a slow development of HCM, while variable penetrance of the mutation in human populations suggest the impact of K104E may be subtle. We found that maximum actin-activated ATPase activity and the actin-dependence of ATPase were similar in K104E and WT. Actin gliding velocities in the in vitro motility assay were slightly increased in K104E. We found that the ADP release rate constant in the presence of actin also was slightly increased in K104E compared to WT. Our results suggest that HCM-associated RLC mutations may only have a minor impact on unloaded ATPase kinetics and in vitro actin gliding, which may suggest other factors could be altered by the mutation such as mechanical load and myosin regulation. Future experiments will address the impact of the mutation in mouse cardiac vs. human cardiac myosin as well as the impact of the mutation on load-dependent motility and the off state (super-relaxed state) of cardiac myosin.

Role of substrate-product frustration on enzyme functional dynamics.

Natural enzymes often have enormous catalytic power developed by evolution. Revealing the underlying physical strategy used by enzymes to achieve high catalysis efficiency is one of the central focuses in the field of biological physics. Our recent work demonstrated that multisubstrate enzymes can utilize steric frustration encountered in the substrate-product cobound complex to overcome the bottleneck of the enzymatic cycle [W. Li et al., Phys. Rev. Lett. 122, 238102 (2019)10.1103/PhysRevLett.122.238102]. However, the key atomic-level interactions by which the steric frustration contributes to the enzymatic cycle remain elusive. In this work we study the microscopic mechanism for the role of the substrate-product frustration on the key physical steps in the enzymatic cycle of adenylate kinase (AdK), a multisubstrate enzyme catalyzing the reversible phosphoryl transfer reaction ATP+AMP⇋ADP+ADP. By using atomistic molecular dynamics simulations with enhanced sampling, we showed that the competitive interactions from the phosphate groups of the substrate ATP and product ADP in the ATP-ADP cobound complex of the AdK lead to local frustration in the binding pockets. Such local frustration disrupts the hydrogen bond network around the binding pockets, which causes lowered barrier height for the opening of the enzyme conformations and expedited release of the bottleneck product ADP. Our results directly demonstrated from the atomistic level that the local frustration in the active sites of the enzyme can be utilized to facilitate the key physical steps of the enzymatic cycle, providing numerical evidence to the predictions of the previous theoretical work.

Single-molecule analysis reveals rotational substeps and chemo-mechanical coupling scheme of Enterococcus hirae V1-ATPase

V1-ATPase (V1), the catalytic domain of an ion-pumping V-ATPase, is a molecular motor that converts ATP hydrolysis–derived chemical energy into rotation. Here, using a gold nanoparticle probe, we directly observed rotation of V1 from the pathogen Enterococcus hirae (EhV1). We found that 120° steps in each ATP hydrolysis event are divided into 40 and 80° substeps. In the main pause before the 40° substep and at low ATP concentration ([ATP]), the time constant was inversely proportional to [ATP], indicating that ATP binds during the main pause with a rate constant of 1.0 × 107 m−1 s−1. At high [ATP], we observed two [ATP]-independent time constants (0.5 and 0.7 ms). One of two time constants was prolonged (144 ms) in a rotation driven by slowly hydrolyzable ATPγS, indicating that ATP is cleaved during the main pause. In another subpause before the 80° substep, we noted an [ATP]-independent time constant (2.5 ms). Furthermore, in an ATP-driven rotation of an arginine-finger mutant in the presence of ADP, −80 and −40° backward steps were observed. The time constants of the pauses before −80° backward and +40° recovery steps were inversely proportional to [ADP] and [ATP], respectively, indicating that ADP- and ATP-binding events trigger these steps. Assuming that backward steps are reverse reactions, we conclude that 40 and 80° substeps are triggered by ATP binding and ADP release, respectively, and that the remaining time constant in the main pause represents phosphate release. We propose a chemo-mechanical coupling scheme of EhV1, including substeps largely different from those of F1-ATPases.

Dilated cardiomyopathy mutation in the converter domain of human cardiac myosin alters motor activity and response to omecamtiv mecarbil

We investigated a dilated cardiomyopathy (DCM) mutation (F764L) in human β-cardiac myosin by determining its motor properties in the presence and absence of the heart failure drug omecamtive mecarbil (OM). The mutation is located in the converter domain, a key region of communication between the catalytic motor and lever arm in myosins, and is nearby but not directly in the OM-binding site. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing the F764L mutation, and compared it to WT with in vitro motility as well as steady-state and transient kinetics measurements. In the absence of OM we demonstrate that the F764L mutation does not significantly change maximum actin-activated ATPase activity but slows actin sliding velocity (15%) and the actomyosin ADP release rate constant (25%). The transient kinetic analysis without OM demonstrates that F764L has a similar duty ratio as WT in unloaded conditions. OM is known to enhance force generation in cardiac muscle while it inhibits the myosin power stroke and enhances actin-attachment duration. We found that OM has a reduced impact on F764L ATPase and sliding velocity compared with WT. Specifically, the EC50 for OM induced inhibition of in vitro motility was 3-fold weaker in F764L. Also, OM reduces maximum actin-activated ATPase 2-fold in F764L, compared with 4-fold with WT. Overall, our results suggest that F764L attenuates the impact of OM on actin-attachment duration and/or the power stroke. Our work highlights the importance of mutation-specific considerations when pursuing small molecule therapies for cardiomyopathies.

A novel mode of DnaA-DnaA interaction promotes ADP dissociation for reactivation of replication initiation activity.

ATP-DnaA is temporally increased to initiate replication during the cell cycle. Two chromosomal loci, DARS (DnaA-reactivating sequences) 1 and 2, promote ATP-DnaA production by nucleotide exchange of ADP-DnaA for timely initiation. ADP-DnaA complexes are constructed on DARS1 and DARS2, bearing a cluster of three DnaA-binding sequences (DnaA boxes I−III), promoting ADP dissociation. Although DnaA has an AAA+ domain, which ordinarily directs construction of oligomers in a head-to-tail manner, DnaA boxes I and II are oriented oppositely. In this study, we constructed a structural model of a head-to-head dimer of DnaA AAA+ domains, and analyzed residues residing on the interface of the model dimer. Gln208 was specifically required for DARS-dependent ADP dissociation in vitro, and in vivo analysis yielded consistent results. Additionally, ADP release from DnaA protomers bound to DnaA boxes I and II was dependent on Gln208 of the DnaA protomers, and DnaA box III-bound DnaA did not release ADP nor require Gln208 for ADP dissociation by DARS–DnaA complexes. Based on these and other findings, we propose a model for DARS–DnaA complex dynamics during ADP dissociation, and provide novel insight into the regulatory mechanisms of DnaA and the interaction modes of AAA+ domains.

Structural and functional characterisation of human RNA helicase DHX8 provides insights into the mechanism of RNA-stimulated ADP release.

DHX8 is a crucial DEAH-box RNA helicase involved in splicing and required for the release of mature mRNA from the spliceosome. Here, we report the biochemical characterisation of full-length human DHX8 and the catalytically active helicase core DHX8Δ547, alongside crystal structures of DHX8Δ547 bound to ADP and a structure of DHX8Δ547 bound to poly(A)6 single-strand RNA. Our results reveal that DHX8 has an in vitro binding preference for adenine-rich RNA and that RNA binding triggers the release of ADP through significant conformational flexibility in the conserved DEAH-, P-loop and hook-turn motifs. We demonstrate the importance of R620 and both the hook-turn and hook-loop regions for DHX8 helicase activity and propose that the hook-turn acts as a gatekeeper to regulate the directional movement of the 3' end of RNA through the RNA-binding channel. This study provides an in-depth understanding of the activity of DHX8 and contributes insights into the RNA-unwinding mechanisms of the DEAH-box helicase family.

The ATPase cycle of human muscle myosin II isoforms: Adaptation of a single mechanochemical cycle for different physiological roles

Striated muscle myosins are encoded by a large gene family in all mammals, including humans. These isoforms define several of the key characteristics of the different striated muscle fiber types, including maximum shortening velocity. We have previously used recombinant isoforms of the motor domains of seven different human myosin isoforms to define the actin·myosin cross-bridge cycle in solution. Here, we present data on an eighth isoform, the perinatal, which has not previously been characterized. The perinatal is distinct from the embryonic isoform, appearing to have features in common with the adult fast-muscle isoforms, including weak affinity of ADP for actin·myosin and fast ADP release. We go on to use a recently developed modeling approach, MUSICO, to explore how well the experimentally defined cross-bridge cycles for each isoform in solution can predict the characteristics of muscle fiber contraction, including duty ratio, shortening velocity, ATP economy, and load dependence of these parameters. The work shows that the parameters of the cross-bridge cycle predict many of the major characteristics of each muscle fiber type and raises the question of what sequence changes are responsible for these characteristics.