Pi Release Research Articles

Structural basis of actin filament assembly and aging

The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state1–3. It remains unclear how F-actin hydrolyses ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here we present cryo-electron microscopy structures of F-actin in all nucleotide states, polymerized in the presence of Mg2+ or Ca2+ at approximately 2.2 Å resolution. The structures show that actin polymerization induces the relocation of water molecules in the nucleotide-binding pocket, activating one of them for the nucleophilic attack of ATP. Unexpectedly, the back door for the subsequent release of inorganic phosphate (Pi) is closed in all structures, indicating that Pi release occurs transiently. The small changes in the nucleotide-binding pocket after ATP hydrolysis and Pi release are sensed by a key amino acid, amplified and transmitted to the filament periphery. Furthermore, differences in the positions of water molecules in the nucleotide-binding pocket explain why Ca2+-actin shows slower polymerization rates than Mg2+-actin. Our work elucidates the solvent-driven rearrangements that govern actin filament assembly and aging and lays the foundation for the rational design of drugs and small molecules for imaging and therapeutic applications.

Structures and mechanisms of actin ATP hydrolysis

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG-O3B bond cleavage; 3) four concomitant events: W1-PO3- formation, OH- and proton cleavage, nucleophilic attack by the OH- against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi-bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Structural Significance of His73 in F-Actin Dynamics: Insights from Ab Initio Study.

F-actin dynamics (polymerization and depolymerization) are associated with nucleotide exchange, providing the driving forces for dynamic cellular activities. As an important residue in the nucleotide state-sensing region in actin, His73 is often found to be methylated in natural actin and directly participates in F-actin dynamics by regulating nucleotide exchange. The interaction between His73 and its neighboring residue, Gly158, has significance for F-actin dynamics. However, this weak chemical interaction is difficult to characterize using classic molecular modeling methods. In this study, ab initio modeling was employed to explore the binding energy between His73 and Gly158. The results confirm that the methyl group on the His73 side chain contributes to the structural stability of atomistic networks in the nucleotide state-sensing region of actin monomers and confines the material exchange (Pi release) pathway within F-actin dynamics. Further binding energy analyses of actin structures under different nucleotide states showed that the potential model of His73/Gly158 hydrogen bond breaking in the material exchange mechanism is not obligatory within F-actin dynamics.

Advances in Plant Lipid Metabolism Responses to Phosphate Scarcity.

Low phosphate (Pi) availability in soils severely limits crop growth and production. Plants have evolved to have numerous physiological and molecular adaptive mechanisms to cope with Pi starvation. The release of Pi from membrane phospholipids is considered to improve plant phosphorus (P) utilization efficiency in response to Pi starvation and accompanies membrane lipid remodeling. In this review, we summarize recent discoveries related to this topic and the molecular basis of membrane phospholipid alteration and triacylglycerol metabolism in response to Pi depletion in plants at different subcellular levels. These findings will help to further elucidate the molecular mechanisms underlying plant adaptation to Pi starvation and thus help to develop crop cultivars with high P utilization efficiency.

The effect of gender and obesity in modulating cross-bridge function in cardiac muscle fibers.

The effect of obesity on cross-bridge (CB) function was investigated in mice lacking functional Melanocortin-4 Receptor (MC4R-/-), the loss of which causes dilated cardiomyopathy (DCM) in humans and mice. Skinned cardiac muscle fibers from male and female mice were used, and activated in the presence of Ca2+. To characterize CB kinetics, we changed the length of fibers in sinewaves (15 frequencies: 1‒187Hz) at a small amplitude (0.2%L0), studied concomitant tension transients, and deduced the kinetic constants of the CB cycle from the ATP and Pi effects. In males, active tension and stiffness during full activation and rigor were ~ 1.5X in WT compared to MC4R-/- mice. This effect was not observed in females. We also observed that ATP binding and subsequent CB detachment steps were not altered by the mutation/gender. The equilibrium constant of the force generation step (K4) and Pi release step (association constant: K5) were not affected by the mutation, but there was a gender difference in WT mice: K4 and K5 were ~ 2.2X in males than in females. Concomitantly, the forward rate constant (r4) and backward rate constant (r-4) of the force generation step were 1.5-2.5X in muscles from female MC4R-/- mice relative to male MC4R-/- mice. However, these effects did not cause a significant difference in CB distributions among six CB states. In both genders, Ca2+ sensitivity decreased slightly (0.12pCa unit) in mutants. We conclude that the CB functions are differentially affected both by obesity induced in the absence of functional MC4R-/- and gender.

Compact IF2 allows initiator tRNA accommodation into the P site and gates the ribosome to elongation

During translation initiation, initiation factor 2 (IF2) holds initiator transfer RNA (fMet-tRNAifMet) in a specific orientation in the peptidyl (P) site of the ribosome. Upon subunit joining IF2 hydrolyzes GTP and, concomitant with inorganic phosphate (Pi) release, changes conformation facilitating fMet-tRNAifMet accommodation into the P site and transition of the 70 S ribosome initiation complex (70S-IC) to an elongation-competent ribosome. The mechanism by which IF2 separates from initiator tRNA at the end of translation initiation remains elusive. Here, we report cryo-electron microscopy (cryo-EM) structures of the 70S-IC from Pseudomonas aeruginosa bound to compact IF2-GDP and initiator tRNA. Relative to GTP-bound IF2, rotation of the switch 2 α-helix in the G-domain bound to GDP unlocks a cascade of large-domain movements in IF2 that propagate to the distal tRNA-binding domain C2. The C2-domain relocates 35 angstroms away from tRNA, explaining how IF2 makes way for fMet-tRNAifMet accommodation into the P site. Our findings provide the basis by which IF2 gates the ribosome to the elongation phase.

The nucleoporin Gle1 activates DEAD-box protein 5 (Dbp5) by promoting ATP binding and accelerating rate limiting phosphate release.

The DEAD-box protein Dbp5 is essential for RNA export, which involves regulation by the nucleoporins Gle1 and Nup159 at the cytoplasmic face of the nuclear pore complex (NPC). Mechanistic understanding of how these nucleoporins regulate RNA export requires analyses of the intrinsic and activated Dbp5 ATPase cycle. Here, kinetic and equilibrium analyses of the Saccharomyces cerevisiae Gle1-activated Dbp5 ATPase cycle are presented, indicating that Gle1 and ATP, but not ADP-Pi or ADP, binding to Dbp5 are thermodynamically coupled. As a result, Gle1 binds Dbp5-ATP > 100-fold more tightly than Dbp5 in other nucleotide states and Gle1 equilibrium binding of ATP to Dbp5 increases >150-fold via slowed ATP dissociation. Second, Gle1 accelerated Dbp5 ATPase activity by increasing the rate-limiting Pi release rate constant ∼20-fold, which remains rate limiting. These data show that Gle1 activates Dbp5 by modulating ATP binding and Pi release. These Gle1 activities are expected to facilitate ATPase cycling, ensuring a pool of ATP bound Dbp5 at NPCs to engage RNA during export. This work provides a mechanism of Gle1-activation of Dbp5 and a framework to understand the joint roles of Gle1, Nup159, and other nucleoporins in regulating Dbp5 to mediate RNA export and other Dbp5 functions in gene expression.

Structural snapshots of V/A-ATPase reveal the rotary catalytic mechanism of rotary ATPases

V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase. When powered by ATP hydrolysis, the V1 domain rotates the central rotor against the A3B3 hexamer, composed of three catalytic AB dimers adopting different conformations (ABopen, ABsemi, and ABclosed). Here, we report the atomic models of 18 catalytic intermediates of the V1 domain of V/A-ATPase under different reaction conditions, determined by single particle cryo-EM. The models reveal that the rotor does not rotate immediately after binding of ATP to the V1. Instead, three events proceed simultaneously with the 120˚ rotation of the shaft: hydrolysis of ATP in ABsemi, zipper movement in ABopen by the binding ATP, and unzipper movement in ABclosed with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in ABsemi, rather than the power stroke model proposed previously for F1-ATPase.

Molecular dynamics simulations of inorganic phosphate and ADP release from F1-ATPase

Single-molecule imaging has produced many images of biomolecular events including the binding and release of ADP and inorganic phosphate (Pi) from hydrolysis in F1-ATPase. However, the video quality of single-molecule imaging is often inadequate for robust research. In the past two decades, molecular dynamics (MD) computer simulations have been able to access the microsecond timescales associated with the release of ADP and Pi. Using MD simulations, large trajectory files have been developed to describe large domain motion of F1-ATPase subunits.

A mutation in the switch 1 region of myosin VA alters the load-dependence of phosphate rebinding

The load-dependence of reversible product release in myosins is key to understanding how it converts chemical energy into mechanical force. Following the powerstroke and phosphate (Pi) release, the rebinding of Pi to the actomyosin⋅ADP complex is highly load-dependent, but the underlying mechanisms/structures involved remain unclear. Therefore, we introduced a mutation (S217A) into the switch I region of the active site to examine its impact on the load dependence of Pi rebinding to single-headed myosin Va (MV) in a mini-ensemble (∼10 heads) three-bead laser trap assay at 100 uM ATP.

Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP

During translation, a conserved GTPase elongation factor—EF-G in bacteria or eEF2 in eukaryotes—translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosome•EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by ~20 Å. An additional 4-Å translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation.

Structural mechanism of GTPase-powered ribosome-tRNA movement

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

Recent insights into the relative timing of myosin's powerstroke and release of phosphate.

Myosin is a motor enzyme that converts the chemical energy in ATP into mechanical work to drive a myriad of intracellular processes, from muscle contraction to vesicular transport. Key steps in the transduction of energy are the force-generating powerstroke, and the release of phosphate (Pi ) from the nucleotide-binding site. Both events occur rapidly after binding to actin, making it difficult to determine which event occurs first. Early efforts suggested that these events occur simultaneously; however, recent findings indicate that they are separate and distinct events that occur at different rates. High-resolution crystal structures of myosin captured in intermediate states of the ATPase cycle suggest that when Pi is in the active site it prevents the powerstroke from occurring, leading to the hypothesis that Pi -release precedes the powerstroke. However, advances in functional assays, enabling sub-millisecond temporal and nanometer spatial resolution, are challenging this hypothesis. For example, Föster Resonance Energy Transfer (FRET) based assays, as well as single molecule laser trap assays, suggest the opposite; that the powerstroke occurs prior to the release of Pi from myosin's active site. This review provides some historical context and then highlights recent reports that reveal exciting new insight into this fundamental mechanism of energy transduction by this prototypical motor enzyme.

Myosin's powerstroke occurs prior to the release of phosphate from the active site.

Myosins are a family of motor proteins responsible for various forms of cellular motility, including muscle contraction and vesicular transport. The most fundamental aspect of myosin is its ability to transduce the chemical energy from the hydrolysis of ATP into mechanical work, in the form of force and/or motion. A key unanswered question of the transduction process is the timing of the force-generating powerstroke relative to the release of phosphate (Pi ) from the active site. We examined the ability of single-headed myosin Va to generate a powerstroke in a single molecule laser trap assay while maintaining Pi in its active site, by either elevating Pi in solution or by introducing a mutation in myosin's active site (S217A) to slow Pi -release from the active site. Upon binding to the actin filament, WT myosin generated a powerstoke rapidly (≥500 s-1 ) and without a detectable delay, both in the absence and presence of 30 mM Pi . The elevated levels of Pi did, however, affect event lifetime, eliminating the longest 25% of binding events, confirming that Pi rebound to myosin's active site and accelerated detachment. The S217A construct also generated a powerstroke similar in size and rate upon binding to actin despite the slower Pi release rate. These findings provide direct evidence that myosin Va generates a powerstroke with Pi still in its active site. Therefore, the findings are most consistent with a model in which the powerstroke occurs prior to the release of Pi from the active site.

Organic acid regulation of inorganic phosphorus release from Mollisols with different organic matter contents

AbstractPhosphorus (P) is used to enhance agricultural productivity. Organic acids increase the release of inorganic phosphorus (Pi) into soil solutions and thereby improve phosphorus utilization in soils; however, the effects of both organic acids and the soil organic matter contents on Pi release are poorly understood. To assess the importance of these factors in determining phosphorus availability, the release of soil Pi fractions (i.e. aluminium‐bound phosphorus (Al‐P), iron‐bound phosphorus (Fe‐P), occluded phosphorus (O‐P) and calcium‐bound phosphorus (Ca‐P)) was evaluated on addition of organic acids (citric, oxalic and malic acids) to soils from Mollisols region that had different organic matter contents and received phosphorus fertilizer. Biochemically produced humic acid was used to modify soil organic matter contents. Among Pi fractions, the Fe‐P fraction had the highest concentration in all the soils with different organic matter contents. In the presence of organic acids, the release of Pi fractions decreased as the soil organic matter increased. Among the fractions, Al‐P had the highest release, followed by Fe‐P. Comparison among various organic acids revealed that oxalic acid was the most effective for Pi release. Path analysis showed that Al‐P release had larger direct‐path and indirect‐path coefficients, which implied that Al‐P release had the largest contribution to total Pi (TPi) release. We concluded that the effectiveness of organic acids in improving soil phosphorus availability was most pronounced in soils with low organic matter contents, but further field‐scale research is needed to confirm this. Al‐P was the most effective source of inorganic phosphorus in Mollisols.

Compressive and Tensile Deformations Alter ATP Hydrolysis and Phosphate Release Rates in Actin Filaments.

Actin filament networks in eukaryotic cells are constantly remodeled through nucleotide state controlled interactions with actin binding proteins, leading to macroscopic structures such as bundled filaments, branched filaments, and so on. The nucleotide (ATP) hydrolysis, phosphate release, and polymerization/depolymerization reactions that lead to the formation of these structures are correlated with the conformational fluctuations of the actin subunits at the molecular scale. The resulting structures generate and experience varying levels of force and impart cells with several functionalities such as their ability to move, divide, transport cargo, etc. Models that explicitly connect the structure to reactions are essential to elucidate a fundamental level of understanding of these processes. In this regard, a bottom-up Ultra-Coarse-Grained (UCG) model of actin filaments that can simulate ATP hydrolysis, inorganic phosphate release (Pi), and depolymerization reactions is presented in this work. In this model, actin subunits are represented using coarse-grained particles that evolve in time and undergo reactions depending on the conformations sampled. The reactions are represented through state transitions, with each state represented by a unique effective coarse-grained potential. Effects of compressive and tensile strains on the rates of reactions are then analyzed. Compressive strains tend to unflatten the actin subunits, reduce the rate of ATP hydrolysis, and increase the Pi release rate. On the other hand, tensile strain flattens subunits, increases the rate of ATP hydrolysis, and decrease the Pi release rate. Incorporating these predictions into a Markov State Model highlighted that strains alter the steady-state distribution of subunits with ADPPi and ADP nucleotide, thus identifying possible additional factors underlying the cooperative binding of regulatory proteins to actin filaments.

Phosphate has dual roles in cross-bridge kinetics in rabbit psoas single myofibrils.

In this study, we aimed to study the role of inorganic phosphate (Pi) in the production of oscillatory work and cross-bridge (CB) kinetics of striated muscle. We applied small-amplitude sinusoidal length oscillations to rabbit psoas single myofibrils and muscle fibers, and the resulting force responses were analyzed during maximal Ca2+ activation (pCa 4.65) at 15°C. Three exponential processes, A, B, and C, were identified from the tension transients, which were studied as functions of Pi concentration ([Pi]). In myofibrils, we found that process C, corresponding to phase 2 of step analysis during isometric contraction, is almost a perfect single exponential function compared with skinned fibers, which exhibit distributed rate constants, as described previously. The [Pi] dependence of the apparent rate constants 2πb and 2πc, and that of isometric tension, was studied to characterize the force generation and Pi release steps in the CB cycle, as well as the inhibitory effect of Pi. In contrast to skinned fibers, Pi does not accumulate in the core of myofibrils, allowing sinusoidal analysis to be performed nearly at [Pi] = 0. Process B disappeared as [Pi] approached 0 mM in myofibrils, indicating the significance of the role of Pi rebinding to CBs in the production of oscillatory work (process B). Our results also suggest that Pi competitively inhibits ATP binding to CBs, with an inhibitory dissociation constant of ∼2.6 mM. Finally, we found that the sinusoidal waveform of tension is mostly distorted by second harmonics and that this distortion is closely correlated with production of oscillatory work, indicating that the mechanism of generating force is intrinsically nonlinear. A nonlinear force generation mechanism suggests that the length-dependent intrinsic rate constant is asymmetric upon stretch and release and that there may be a ratchet mechanism involved in the CB cycle.

Myosin'S Powerstroke Occurs with Phosphate Still in the Active Site

A fundamental property of myosin is its ability to harness the power of ATP hydrolysis to generate force. A key unanswered question is the timing of the force-generating powerstroke relative to phosphate (Pi) -release from its active site. We examined the ability of single-headed myosin Va (MV) construct to bind to actin and generate a powerstroke in a single molecule laser trap assay in the presence of elevated Pi. We examined both WT MV and the impact of a mutation (S217A) in the conserved switch I region of the active site which slows actin-activated Pi-release. Upon binding to actin WT myosin rapidly (>500/s) generated a 7±0.5 nm powerstroke. In the presence of 30mM Pi, no decrease in the size (7±0.4 nm) or delay in the rate of the development of the powerstroke (>500/s) was observed, however the average duration of strong actomyosin binding (ton) was reduced in the presence of Pi (240±20 vs 113± ms, p<0.05). The reduced ton is consistent with Pi rebinding to the active site and accelerating detachment from actin. Therefore, this suggests myosin generates the powerstroke with Pi still in the active site. S217A generated a powerstroke with a similar rate (>500/s) and size (9±0.9 nm) to the WT, but ton was shorter, but not significantly different, than WT (143±20 ms, p=0.22 and 62±5 ms, p<0.05 for S217A in the absence and presence of Pi, respectively). Since the S217A mutant slows actin-activated Pi-release from the active site our results fit best to a model in which the powerstroke occurs with Pi still in the active site. Together these findings suggest that actin-binding, and not the release of Pi from the active site, trigger the conformational changes that lead to the powerstroke, and thus force-generation by myosin.

Omecamtiv Mecarbil Modulation of Force Generation in Single Myofibrils from Human Cardiac Muscle

Omecamtiv mecarbil (OM) is a small molecule proved to enhance the function of the beta-myosin motor and presently used for the treatment of heart failure. Despite the positive outcomes of clinical trials, the mechanistic basis of its action is controversial and little is known of its functional impact in human cardiac sarcomeres. Here myofibrils from human donor left ventricle (frozen samples) have been used to study the effects of μmolar [OM] on isometric force in relaxing conditions (pCa 9) and at maximal (pCa 4.5) or half-maximal (pCa 5.75) calcium activation, both in control conditions (15°C, equimolar DMSO, contaminant [Pi] ∼ 170µM) or in presence of 5 mM [Pi]. In all conditions tested, OM increased isometric force with a complex dose-dependent effect peaking (about 40% increase) at 0.5 µM and never decreasing below control values. As already reported in human cardiac muscle (Swenson et al. 2017), OM strongly depressed the kinetics of force development up to 90% at 10 µM OM. Of note, OM was found to decrease the inhibition of force operated by Pi, in agreement with observations in rabbit slow-muscle (Governali et al. 2020), but the effect was modest and occurred at low (1-2 mM) [Pi]. At pCa 9 OM induced calcium-independent force development which approached the level of calcium activated tension at 10 µM OM. The kinetics of the calcium-independent force development at pCa 9 was very slow and dose-independent. The results support a role of OM in operating a complex perturbation of the thin/thick filament regulatory state as a result of its binding to myosin allosteric sites coupled to Pi release that favours the formation of strongly actin-bound motors. SilicoFCM EU H2020 grant agreement n. 777204.

Step-Wise Hydration of Magnesium by Four Water Molecules Precedes Phosphate Release in a Myosin Motor.

Molecular motors, such as myosin, kinesin, and dynein, convert the energy released by the hydrolysis of ATP into mechanical work, thus allowing them to undergo directional motion on cytoskeletal tracks. A pivotal step in the chemomechanical transduction in myosin motors occurs after they bind to the actin filament, which triggers the release of phosphate (Pi, product of ATP hydrolysis) and the rotation of the lever arm. Here, we investigate the mechanism of phosphate release in myosin VI using extensive molecular dynamics simulations involving multiple trajectories of several μs. Because the escape of phosphate is expected to occur on time-scales on the order of milliseconds or more in myosin VI, we observed Pi release only if the trajectories were initiated with a rotated phosphate inside the nucleotide binding pocket. We discovered that although Pi populates the traditional "back door" route, phosphate exits through various other gateways, thus establishing the heterogeneity in the escape routes. Remarkably, we observed that the release of phosphate is preceded by a stepwise hydration of the ADP-bound magnesium ion. The release of the anion occurred only after four water molecules hydrated the cation (Mg2+). By performing comparative structural analyses, we show that hydration of magnesium is the key step in the phosphate release in a number of ATPases and GTPases. Nature may have evolved hydration of Mg2+ as a general molecular switch for Pi release, which is a universal step in the catalytic cycle of many machines that share little sequence or structural similarity.