Search

Myosin-IC (myo1c) is a class-I myosin that supports transport and remodeling of the plasma membrane and membrane-bound vesicles. Like other members of the myosin family, its biochemical kinetics are altered in response to changes in mechanical loads that resist the power stroke. However, myo1c is unique in that the primary force-sensitive kinetic transition is the isomerization that follows ATP binding, not ADP release as in other slow myosins. Myo1c also powers actin gliding along curved paths, propelling actin filaments in leftward circles. To understand the origins of this unique force-sensing and motile behavior, we solved actin-bound myo1c cryo-EM structures in the presence and absence of ADP. Our structures reveal that in contrast with other myosins, the myo1c lever arm swing is skewed, partly due to a different actin interface that reorients the motor domain on actin. The structures also reveal unique nucleotide-dependent behavior of both the nucleotide pocket as well as an element called the N-terminal extension (NTE). We incorporate these observations into a model that explains why force primarily regulates ATP binding in myo1c, rather than ADP release as in other myosins. Integrating our cryo-EM data with available crystallography structures allows the modeling of full-length myo1c during force generation, supplying insights into its role in membrane remodeling. These results highlight how relatively minor sequence differences in members of the myosin superfamily can significantly alter power stroke geometry and force-sensing properties, with important implications for biological function., Competing Interests: Competing interests statement:The authors declare no competing interest.

Bacterial RNA helicase ρ is a genome sentinel that terminates the synthesis of damaged and junk RNAs that are not translated by the ribosome. It is unclear how ρ is regulated during dormancy or stress, when translation is inefficient and RNAs are vulnerable to ρ-mediated release. We use cryogenic electron microscopy, biochemical, and genetic approaches to show that substitutions of residues in the connector between two ρ domains or ADP promote the formation of extended Escherichia coli ρ filaments. By contrast, (p)ppGpp induces the formation of transient ρ dodecamers. Our results demonstrate that ADP and (p)ppGpp nucleotides bound at subunit interfaces inhibit ρ ring closure that underpins the hexamer activation, thus favoring the assembly of inactive higher-order oligomers. Connector substitutions and antibiotics that inhibit RNA and protein syntheses trigger ρ aggregation in the cell. These and other recent data implicate aggregation as a widespread strategy to tune ρ activity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Microglial activation and pyroptosis are central to neuroinflammation and significantly contribute to cognitive decline associated with neurodegenerative diseases. Alpha protein kinase 1 (ALPK1) is recently identified as a critical mediator of inflammatory responses in response to ADP-heptose (a pathogen-associated molecular pattern). However, its specific role in microglial pyroptosis and cognitive dysfunction remains unclear. In this study, we investigated the effects of ALPK1 on cognitive function and pyroptosis in wild-type (WT) and ALPK1 KO mice by intracerebroventricular administration of ADP-heptose to induce neuroinflammation. Cognitive performance was evaluated using behavioral tests (the Y-Maze, Morris Water Maze, and step-down passive avoidance), while Western blot, immunofluorescence, transmission electron microscopy, and enzyme-linked immunosorbent assay were used to evaluate the expression of pyroptosis markers such as NLRP3, Caspase-1, and gasdermin D (GSDMD) in vivo and in vitro. Our results reveal that the absence of ALPK1 significantly attenuated ADP-heptose-induced cognitive deficits and neuronal injury, and inhibited the NLRP3/Caspase-1/GSDMD pathway of pyroptosis and the secretion of pro-inflammatory cytokines IL-1β and IL-18. Notably, ADP-heptose-stimulated conditioned media from primary microglial cells of ALPK1 KO mice significantly enhanced neuronal cell viability, suggesting a protective role for ALPK1 deficiency in supporting neuronal health. These findings suggest the pivotal role of ALPK1 in ADP-heptose-induced microglial pyroptosis and cognitive impairment, thereby highlighting its potential as a therapeutic target in neuroinflammatory disorders., (© 2025 Federation of American Societies for Experimental Biology.)

The cause and consequences of inosine monophosphate (IMP) formation when adenosine triphosphate (ATP) declines during muscular contractions in vivo are not fully understood. The purpose of this study was to examine the role of IMP formation in the maintenance of the Gibbs free energy for ATP hydrolysis (ΔG ATP ) during dynamic contractions of increasing workload and the implications of ATP loss in vivo. Eight males (median 27.5, 25-35 yr range) completed an 8-min incremental protocol [2-min stages of isotonic knee extensions (0.5 Hz)] in a 3-T magnetic resonance (MR) system. Phosphorus MR spectra were obtained from the knee extensor muscles at rest and during contractions and recovery. Although the ATP demand during contractions was met primarily by oxidative phosphorylation, [ATP] decreased from 8.2 mM to 7.5 (range 6.4-8.0) mM and [IMP] increased from 0 mM to 0.6 (0.1-1.7) mM. Modeling showed that, in the absence of IMP formation, excess adenosine diphosphate (ADP) would result in a less favorable ΔG ATP ( P < 0.001). Neither [ATP] nor [IMP] had returned to baseline following 10 min of recovery ( P < 0.001). Notably, Δ[ATP] was linearly related to the post-contraction reduction in muscle oxidative capacity ( r = 0.74, P = 0.037). Our results highlight the importance of IMP formation in preserving cellular energy status by avoiding increases in ADP above that necessary to stimulate energy production pathways. However, the consequence of IMP formation was an incomplete recovery of [ATP], which in turn was related to decreased muscle oxidative capacity following contractions. These results likely have implications for the capacity to generate adequate energy during repeated bouts of muscular work. NEW & NOTEWORTHY An ∼9% decline in [ATP] led to the formation of inosine monophosphate (IMP) during submaximal muscular contractions. Modeling revealed IMP formed to preserve a favorable energy state (ΔG ATP ) by minimizing large increases in [ADP], whereas the loss of [ATP] did not alter ΔG ATP . [ATP] did not recover by 10 min, and the loss of [ATP] was associated with a reduced oxidative capacity, providing a new link between [ATP] loss and an impaired energetic capacity in vivo.

Thrombosis is one of the most prevalent and serious health issues amongst humans. A key component of thrombotic events is the activation and aggregation of platelets, of which the P2Y 1 and P2Y 12 receptors play a crucial role in this process. Despite a breadth of knowledge on thrombosis and its mechanisms and treatment in various disorders in humans, there is less of an understanding of the expression and exact role of these receptors in companion animals such as dogs and cats. Therefore, this study aimed to investigate P2Y 1 and P2Y 12 receptors on dog and cat platelets in platelet-rich plasma and compare them to human platelets. Immunoblotting revealed the presence of P2Y 1 and P2Y 12 receptor proteins on dog and cat platelets, although relative amounts of each receptor appeared to contrast those of human platelets, with increased amounts of P2Y 1 compared to P2Y 12 receptors in dogs and cats. Using a modified 384-well plate aggregation assay, designed for use with small volumes, the human P2Y 1 and P2Y 12 receptor agonists adenosine 5'-diphosphate and 2-methylthio-adenosine 5'-diphosphate caused aggregation of dog and cat platelets. This aggregation was near-completely inhibited by the selective P2Y 12 antagonist ticagrelor. Aggregation of dog and cat platelets was partly inhibited by the human P2Y 1 receptor antagonist MRS2179. The agonist and antagonist responses in dog and cat platelets were like those of human platelets. In contrast, the aggregation of dog platelets in the absence of added nucleotides was two-fold greater than that of cats and humans. This study indicates that platelets of cats and dogs possess functional P2Y 1 and P2Y 12 receptors that can be inhibited by human antagonists. The data presented suggest differing roles or responses of the platelet P2Y receptors in dogs and cats compared to humans but also highlight the potential of using currently available P2Y 1 or P2Y 12 antiplatelet drugs such as ticagrelor for the treatment of thrombosis in these companion animals.

We hypothesized that a strategy employing tissue-specific endothelial cells (EC) might facilitate the identification of tissue- or organ-specific vascular functions of ubiquitous metabolites. An unbiased approach was employed to identify water-soluble small molecules with mitogenic activity on choroidal EC. We identified adenosine diphosphate (ADP) as a candidate, following biochemical purification from mouse EL4 lymphoma extracts. ADP stimulated the growth of bovine choroidal EC (BCEC) and other bovine or human eye-derived EC. ADP induced rapid phosphorylation of extracellular signal-regulated kinase in a dose- and time-dependent manner. ADP-induced BCEC proliferation could be blocked by pretreatment with specific antagonists of the purinergic receptor P2Y1 but not with a vascular endothelial growth factor (VEGF) inhibitor, indicating that the EC mitogenic effects of ADP are not mediated by stimulation of the VEGF pathway. Intravitreal administration of ADP expanded the neovascular area in a mouse model of choroidal neovascularization. Single-cell transcriptomics from human choroidal datasets show the expression of P2RY1, but not other ADP receptors, in EC with a pattern similar to VEGFR2. Although ADP has been reported to be a growth inhibitor for vascular EC, here we describe its growth-stimulating effects for BCEC and other eye-derived EC., Competing Interests: Competing interests statement:The authors declare no competing interest.

The DdmDE antiplasmid system, consisting of the helicase-nuclease DdmD and the prokaryotic Argonaute (pAgo) protein DdmE, plays a crucial role in defending Vibrio cholerae against plasmids. Guided by DNA, DdmE specifically targets plasmids, disassembles the DdmD dimer, and forms a DdmD-DdmE handover complex to facilitate plasmid degradation. However, the precise ATP-dependent DNA translocation mechanism of DdmD has remained unclear. Here, we present cryo-EM structures of DdmD bound to single-stranded DNA (ssDNA) in nucleotide-free, ATPγS-bound, and ADP-bound states. These structures, combined with biochemical analysis, reveal a unique "gate-clamp" mechanism for ssDNA translocation by DdmD. Upon ATP binding, arginine finger residues R855 and R858 reorient to interact with the γ-phosphate, triggering HD2 domain movement. This shift repositions the gate residue Q781, causing a flip of the 3' flank base, which is then clamped by residue F639. After ATP hydrolysis, the arginine finger releases the nucleotide, inducing HD2 to return to its open state. This conformational change enables DdmD to translocate along ssDNA by one nucleotide in the 5' to 3' direction. This study provides new insights into the ATP-dependent translocation of DdmD and contributes to understanding the mechanistic diversity within SF2 helicases., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Helicase is a nucleic acid motor that catalyses the unwinding of double-stranded (ds) RNA and DNA via ATP hydrolysis. Helicases can act either as a nucleic acid motor that unwinds its ds substrates or as a chaperone that alters the stability of its substrates, but the two activities have not yet been reported to act simultaneously. Here, we used single-molecule techniques to unravel the synergistic coordination of helicase and chaperone activities, and found that the severe acute respiratory syndrome coronavirus helicase (nsp13) is capable of two modes of action: (i) binding of nsp13 in tandem with the fork junction of the substrate mechanically unwinds the substrate by an ATP-driven synchronous power stroke; and (ii) free nsp13, which is not bound to the substrate but complexed with ADP in solution, destabilizes the substrate through collisions between transient binding and unbinding events with unprecedented melting capability. Our findings provide new insights into how the same enzyme works via two modes on different parts of the substrate and synergistically catalyses the unwinding reaction, utilizing ATP and recycling its by-product ADP as an energy source., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Here we present the first use of principal component analysis of the full spectrum of a single europium complex to differentiate between structurally-similar analytes. We demonstrate that it can be used to distinguish between the nucleoside phosphate guests AMP, ADP, and ATP.

Oscillation of the active form of the initiator protein DnaA (ATP-DnaA) allows for the timely regulation for chromosome replication. After initiation, DnaA-bound ATP is hydrolyzed, producing inactive ADP-DnaA. For the next round of initiation, ADP-DnaA interacts with the chromosomal locus DARS2 bearing binding sites for DnaA, a DNA-bending protein IHF, and a transcription activator Fis. The IHF binding site is about equidistant between the DnaA and Fis binding sites within DARS2. The DARS2-IHF-Fis complex promotes ADP dissociation from DnaA and furnishes ATP-DnaA at the pre-initiation stage, which dissociates Fis in a negative-feedback manner. However, regulation for IHF binding as well as mechanistic roles of Fis and specific DNA structure at DARS2 remain largely unknown. We have discovered that negative DNA supercoiling of DARS2 is required for stimulating IHF binding and ADP dissociation from DnaA in vitro. Consistent with these, novobiocin, a DNA gyrase inhibitor, inhibits DARS2 function in vivo. Fis Gln68, an RNA polymerase-interaction site, is suggested to be required for interaction with DnaA and full DARS2 activation. Based on these and other results, we propose that DNA supercoiling activates DARS2 function by stimulating stable IHF binding and DNA loop formation, thereby directing specific Fis-DnaA interaction., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Eukaryotic cells not only sense carbon nutrients directly, but also sense them indirectly by monitoring the levels of cellular adenine nucleotides produced by their catabolism. Starvation for a key carbon source may cause increases in ADP:ATP ratios that are amplified by the adenylate kinase reaction (2ADP ↔ ATP + AMP) into even larger increases in AMP:ATP ratios. The major cellular sensor of such changes in nucleotide levels is the AMP-activated protein kinase (AMPK) which, once activated, acts to restore energy balance within cells. Increases in the AMP:ATP ratio result in conformational changes in AMPK that: (1) cause allosteric activation; (2) promote phosphorylation at a critical threonine residue (Thr172) in the kinase domain of AMPK-α subunits by the upstream kinase LKB1; (3) protect against Thr172 dephosphorylation by protein phosphatases. Together, these three mechanisms of activation can cause up to a 1000-fold increase in AMPK activity, and this chapter will describe assays to monitor these different activation mechanisms in cell-free assays. Adenine nucleotides (AMP/ADP/ATP) bind to sites formed by sequence repeats termed CBS motifs in the AMPK-γ subunits. Several pathogenic mutations in these motifs have been identified that either reduce or eliminate the sensitivity of AMPK to changes in AMP and other nucleotides. We also describe the use of cells expressing such mutants to determine whether agents that activate AMPK do so by disturbing adenine nucleotide levels., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

ATP synthase is a rotary motor enzyme that drives the formation of ATP from ADP and P and uses multiple electrical forces to do this. This chapter outlines the exquisite use of these electrical forces to generate the high energy phosphates on which all our lives depend. Vacuolar ATPases and the ADP/ATP carrier also are explored., (© 2025. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

CD39 is one of the important nucleotidases to adjust extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) concentration. However, the enzyme mimics to simulate the activity of CD39 still remains to be explored. Herein nitrogen-rich molybdenum nitride (Mo 5 N 6 ) nanosheets are explored to possess CD39-like activity, which are able to catalyze the hydrolysis of the high-energy phosphate bonds (HEPBs) in ATP and ADP but not the common phosphate bonds in adenosine monophosphate (AMP). The catalytic hydrolysis of the phosphate bond over Mo 5 N 6 -700 nanosheets is first investigated using para-nitrophenyl phosphate as the model substrate and then the CD39-like activity is further explored and verified by 31 p NMR spectroscopy. Mo 4+ on the surface of Mo 5 N 6 -700 nanosheets are the catalytic active sites. Using ATP as the model substrate, the K m and V max values of CD39-like activity at optimal pH 9.0 are 3.2 µmol L -1 and 18.5 µmol L -1 h -1 , respectively. The CD39-like activity of Mo 5 N 6 -700 nanosheets enabled the down-regulation of intracellular ATP concentration to a larger degree for cancer cells than normal cells, which makes Mo 5 N 6 -700 nanosheets a potential therapeutic reagent for cancers., (© 2024 Wiley‐VCH GmbH.)

Living systems depend on continuous energy input for growth, replication and information processing. Cells use membrane proteins as nanomachines to convert light or chemical energy of nutrients into other forms of energy, such as ion gradients or adenosine triphosphate (ATP). However, engineering sustained fuel supply and metabolic energy conversion in synthetic systems is challenging. Here, inspired by endosymbionts that rely on the host cell for their nutrients, we introduce the concept of cross-feeding to exchange ATP and ADP between lipid-based compartments hundreds of nanometres in size. One population of vesicles enzymatically produces ATP in the mM concentration range and exports it. A second population of vesicles takes up this ATP to fuel internal reactions. The produced ADP feeds back to the first vesicles, and ATP-dependent reactions can be fuelled sustainably for up to at least 24 h. The vesicles are a platform technology to fuel ATP-dependent processes in a sustained fashion, with potential applications in synthetic cells and nanoreactors. Fundamentally, the vesicles enable studying non-equilibrium processes in an energy-controlled environment and promote the development and understanding of constructing life-like metabolic systems on the nanoscale., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The hexameric AAA+ disaggregase, Hsp104, collaborates with Hsp70 and Hsp40 via its autoregulatory middle domain (MD) to solubilize aggregated proteins. However, how ATP- or ADP-specific MD configurations regulate Hsp104 hexamers remains poorly understood. Here, we define an ATP-specific network of interprotomer contacts between nucleotide-binding domain 1 (NBD1) and MD helix L1, which tunes Hsp70 collaboration. Manipulating this network can (1) reduce Hsp70 collaboration without enhancing activity, (2) generate Hsp104 hypomorphs that collaborate selectively with class B Hsp40s, (3) produce Hsp70-independent potentiated variants, or (4) create species barriers between Hsp104 and Hsp70. Conversely, ADP-specific intraprotomer contacts between MD helix L2 and NBD1 restrict activity, and their perturbation frequently potentiates Hsp104. Importantly, adjusting an NBD1:MD helix L1 rheostat via rational design enables finely tuned collaboration with Hsp70 to safely potentiate Hsp104, minimize off-target toxicity, and counteract FUS and TDP-43 proteinopathies in human cells. Thus, we establish design principles to tailor Hsp104 therapeutics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The PII signaling proteins are ubiquitous in prokaryotes serving as crucial metabolic hubs in different metabolic pathways because of their ability to sense and integrate signals of the cellular nitrogen, carbon, and energy levels. In this study, we used ligand fishing assays to identify the ribonucleotide monophosphatase UmpH enzyme as a novel target of the PII signaling protein GlnK in Escherichia coli. In vitro analyses showed that UmpH interacts specifically with the PII protein GlnK but not with its paralog protein GlnB. The UmpH-GlnK complex is modulated by the GlnK uridylylation status and by the levels of the GlnK allosteric effectors ATP, ADP, and 2-oxoglutarate. Upon engaging interaction with GlnK, UmpH becomes less active toward its substrate uridine 5'-monophosphate. We suggest a model where GlnK will physically interact to reduce the UmpH activity during the transition from N-starvation to N-sufficient conditions. Such a mechanism may help the cells to reprogram the fate of uridine 5'-monophosphate from catabolism to anabolism avoiding futile cycling of key nutrients., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Myosins are ATP-powered, force-generating motor proteins involved in cardiac and muscle contraction. The external load experienced by the myosins modulates and coordinates their function in vivo. Here, this study investigates the tension-sensing mechanisms of rabbit native β-cardiac myosin (βM-II) and slow skeletal myosins (SolM-II) that perform in different physiological settings. Using mobile optical tweezers with a square wave-scanning mode, a range of external assisting and resisting loads from 0 to 15 pN is exerted on single myosin molecules as they interact with the actin filament. Influenced of load on specific strongly-bound states in the cross-bridge cycle is examined by adjusting the [ATP]. The results implies that the detachment kinetics of actomyosin ADP.Pi strongly-bound force-generating state are load sensitive. Low assisting load accelerates, while the resisting load hinders the actomyosin detachment, presumably, by slowing both the Pi and ADP release. However, under both high assisting and resisting load, the rate of actomyosin dissociation decelerates. The transition from actomyosin ADP.Pi to ADP state appears to occur with a higher probability for βM-II than SolM-II. This study interpret that dissociation of at least three strongly-bound actomyosin states are load-sensitive and may contribute to functional diversity among different myosins., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Enzymatic mechanisms are typically inferred from structural data. However, understanding enzymes require unravelling the intricate dynamic interplay between dynamics, conformational substates, and multiple protein structures. Here, we use single-molecule nanopore analysis to investigate the catalytic conformational changes of adenylate kinase (AK), an enzyme that catalyzes the interconversion of various adenosine phosphates (ATP, ADP, and AMP). Kinetic analysis validated by hidden Markov models unravels the details of domain motions during catalysis. Our findings reveal that allosteric interactions between ligands and cofactor enable converting binding energies into directional conformational changes of the two catalytic domains of AK. These coordinated motions emerged to control the exact sequence of ligand binding and the affinity for the three different substrates, thereby guiding the reactants along the reaction coordinates. Interestingly, we find that about 10% of enzymes show altered allosteric regulation and ligand affinities, indicating that a subset of enzymes folds in alternative catalytically active forms. Since molecules or proteins might be able to selectively stabilize one of the folds, this observation suggests an evolutionary path for allostery in enzymes. In AK, this complex catalytic framework has likely emerged to prevent futile ATP/ADP hydrolysis and to regulate the enzyme for different energy needs of the cell., Competing Interests: Competing interests: G.M. is founder, director, and shareholder in Portal Biotech Limited, a nanopore company. This work was not supported by Portal Biotech Limited. The remaining authors declare no competing interests’., (© 2024. The Author(s).)

DEAD-box helicases use ATP to unwind short double-stranded RNA (dsRNA). The helicase core consists of two discrete domains, termed RecA&#95;N and RecA&#95;C. The nucleotide binding site is harbored in RecA&#95;N, while both RecA&#95;N and RecA&#95;C are involved in RNA recognition and ATP hydrolysis. In the absence of nucleotides or RNA, RecA&#95;N and RecA&#95;C do not interact ("open" form of the enzyme). In the presence of both RNA and ATP the two domains come together ("closed" form), building the composite RNA binding site and stimulating ATP hydrolysis. Because of the different roles and thermodynamic properties of the ADP-bound and ATP-bound states in the catalytic cycle, the conformations of DEAD-box helicases in complex with ATP and ADP are assumed to be different. However, the available crystal structures do not recapitulate these supposed differences and show identical conformations of DEAD-box helicases independent of the identity of the bound nucleotide. Here, we use NMR to demonstrate that the conformations of the ATP- and ADP-bound forms of the DEAD-box helicase Vasa are indeed different, contrary to the results from x-ray crystallography. These differences do not relate to the populations of the open and closed forms, but are intrinsic to the RecA&#95;N domain. NMR chemical shift analysis reveals the regions of RecA&#95;N where the average conformations of Vasa-ADP and Vasa-ATP are most different and indicates that these differences may contribute to modulating the affinity of the two nucleotide-bound complexes for RNA substrates., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In recent years, a convenient phosphatase-coupled sulfotransferase assay method has been proven to be applicable to most sulfotransferases. The central principle of the method is that phosphatase specifically degrades 3'-phosphoadenosine-5'-phosphate (PAP) and leaves 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Our group previously acquired a yeast 3',5'-bisphosphate nucleotidase (YND), which showed a higher catalytic activity for PAP than PAPS and could be a potential phosphatase for the sulfotransferase assay. Here, we obtained a beneficial mutant of YND with markedly improved substrate specificity towards PAP via rational design. Of 9 chosen mutation sites in the active site pocket, the mutation G236D showed the best specificity for PAP. After optimization of the reaction conditions, the mutant YND G236D displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YND G236D to the assay of human SULT1A1 and SULT1A3 with their known substrate 1-naphthol, indicating that the mutant could be used to evaluate sulfotransferase activity by colorimetry. Analysis of the MD simulation results revealed that the improved substrate specificity of the mutant towards PAP may stem from a more stable protein conformation and the changed flexibility of key residues in the entrance of the substrate tunnel. This research will provide a valuable reference for the development of efficient sulfotransferase activity assays.

The mitochondrial adenosine 5'-diphosphate (ADP)/adenosine 5'-triphosphate (ATP) carrier imports ADP into the mitochondrion and exports ATP to the cell. Here, we demonstrate that 3.3 positive charges are translocated with the negatively charged substrate in each transport step. They can be assigned to three positively charged residues of the central substrate-binding site and two asparagine/arginine pairs. In this way, the membrane potential stimulates not only the ATP 4- export step, as a net -0.7 charge is transported, but also the ADP 3- import step, as a net +0.3 charge is transported with the electric field. These positive charge movements also inhibit the import of ATP and export of ADP in the presence of a membrane potential, allowing these nucleotides to be maintained at high concentrations in the cytosol and mitochondrial matrix to drive the hydrolysis and synthesis of ATP, respectively. Thus, this is the mechanism by which the membrane potential drives adenine nucleotide exchange with high directional fluxes to fuel the cellular processes.

Human phosphoglycerate kinase 1(hPGK1) is a key glycolytic enzyme that regulates the balance between ADP and ATP concentrations inside the cell. Phosphorylation of hPGK1 at S203 and S256 has been associated with enzyme import from the cytosol to the mitochondria and the nucleus respectively. These changes in subcellular locations drive tumorigenesis and are likely associated with site-specific changes in protein stability. In this work, we investigate the effects of site-specific phosphorylation on thermal and kinetic stability and protein structural dynamics by hydrogen-deuterium exchange (HDX) and molecular dynamics (MD) simulations. We also investigate the binding of 3-phosphoglycerate and Mg-ADP using these approaches. We show that the phosphomimetic mutation S256D reduces hPGK1 kinetic stability by 50-fold, with no effect of the mutation S203D. Calorimetric studies of ligand binding show a large decrease in affinity for Mg-ADP in the S256D variant, whereas Mg-ADP binding to the WT and S203D can be accurately investigated using protein kinetic stability and binding thermodynamic models. HDX and MD simulations confirmed the destabilization caused by the mutation S256D (with some long-range effects on stability) and its reduced affinity for Mg-ADP due to the strong destabilization of its binding site (particularly in the apo-state). Our research provides evidence suggesting that modifications in protein stability could potentially enhance the translocation of hPGK1 to the nucleus in cancer. While the structural and energetic basis of its mitochondrial import remain unknown., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Human nucleotide exchange factors GRPEL1 and GRPEL2 play pivotal roles in the ADP-ATP exchange within the protein folding cycle of mitochondrial HSP70 (mtHSP70), a crucial chaperone facilitating protein import into the mitochondrial matrix. Studies in human cells and mice have indicated that while GRPEL1 serves as an essential co-chaperone for mtHSP70, GRPEL2 has a role regulated by stress. However, the precise structural and biochemical mechanisms underlying the distinct functions of the GRPEL proteins have remained elusive. In our study, we present evidence revealing that ADP-bound mtHSP70 exhibits remarkably higher affinity for GRPEL1 compared to GRPEL2, with the latter experiencing a notable decrease in affinity upon ADP binding. Additionally, Pi assay showed that GRPEL1, but not GRPEL2, enhanced the ATPase activity of mtHSP70. Utilizing Alphafold modeling, we propose that the interaction between GRPEL1 and mtHSP70 can induce the opening of the nucleotide binding cleft of the chaperone, thereby facilitating the release of ADP, whereas GRPEL2 lacks this capability. Additionally, our findings suggest that the redox-regulated Cys87 residue in GRPEL2 does not play a role in dimerization but rather reduces its affinity for mtHSP70. Our findings on the structural and functional disparities between GRPEL1 and GRPEL2 may have implications for mitochondrial protein folding and import processes under varying cellular conditions., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Intracellular molecules are transported by motor proteins or move by diffusion resulting from random molecular motion. Cardiomyocytes are packed with structures that are crucial for function, but also confine the diffusional spaces, providing cells with a means to control diffusion. They form compartments in which local concentrations are different from the overall, average concentrations. For example, calcium and cyclic AMP are highly compartmentalized, allowing these versatile second messengers to send different signals depending on their location. In energetic compartmentalization, the ratios of AMP and ADP to ATP are different from the average ratios. This is important for the performance of ATPases fuelling cardiac excitation-contraction coupling and mechanical work. A recent study suggested that compartmentalization modulates the activity of creatine kinase and adenylate kinase in situ. This could have implications for energetic signaling through, for example, AMP-activated kinase. It highlights the importance of taking compartmentalization into account in our interpretation of cellular physiology and developing methods to assess local concentrations of AMP and ADP to enhance our understanding of compartmentalization in different cell types., (© 2024 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Depending on the light conditions, photosynthetic organisms switch between carbohydrate synthesis or breakdown, for which the reversibility of carbohydrate metabolism, including glycolysis, is essential. Kinetic regulation of phosphofructokinase (PFK), a key-control point in glycolysis, was studied in the cyanobacterium Synechocystis sp. PCC 6803. The two PFK iso-enzymes (PFK- A1, PFK-A2), were found to use ADP instead of ATP, and have similar kinetic characteristics, but differ in their allosteric regulation. PFK-A1 is inhibited by 3-phosphoglycerate, a product of the Calvin-Benson-Bassham cycle, while PFK-A2 is inhibited by ATP, which is provided by photosynthesis. This regulation enables cyanobacteria to switch PFK off in light, and on in darkness. ADP dependence has not been reported before for the PFK-A enzyme family and was thought to be restricted to the PFK-B ribokinase superfamily. Phosphate donor specificity within the PFK-A family could be related to specific binding motifs in ATP-, ADP-, and PPi-dependent PFK-As. Phylogenetic analysis revealed a distribution of ADP-PFK-As in cyanobacteria and in a few alphaproteobacteria, which was confirmed in enzymatic studies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Red blood cells (RBCs) have been hypothesized to support hemostasis by facilitating platelet margination and releasing platelet-activating factors such as adenosine 5'-diphosphate (ADP). Significant knowledge gaps remain regarding how RBCs influence platelet function, especially in (patho)physiologically relevant hemodynamic conditions. Here, we present results showing how RBCs affect platelet function and hemostasis in conditions of anemia, thrombocytopenia, and pancytopenia and how the biochemical and biophysical properties of RBCs regulate platelet function at the blood and vessel wall interface and in the fluid phase under flow conditions. We found that RBCs promoted platelet deposition to collagen under flow conditions in moderate (50 × 103/μL) but not severe (10 × 103/μL) thrombocytopenia in vitro. Reduction in hematocrit by 45% increased bleeding in mice with hemolytic anemia. In contrast, bleeding diathesis was observed in mice with a 90% but not with a 60% reduction in platelet counts. RBC transfusion improved hemostasis by enhancing fibrin clot formation at the site of vascular injury in mice with severe pancytopenia induced by total body irradiation. Altering membrane deformability changed the ability of RBCs to promote shear-induced platelet aggregation. RBC-derived ADP contributed to platelet activation and aggregation in vitro under pathologically high shear stresses, as observed in patients supported by left ventricular assist devices. These findings demonstrate that RBCs support platelet function and hemostasis through multiple mechanisms, both at the blood and vessel wall interface and in the fluidic phase of circulation., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

The omega-3 polyunsaturated fatty acids (PUFAs) Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) exert multiple cardioprotective effects, influencing inflammation, platelet activation, endothelial function and lipid metabolism, besides their well-established triglyceride lowering properties. It is not uncommon for omega-3 PUFAs to be prescribed for hypertriglyceridemia, alongside antiplatelet therapy in cardiovascular disease (CVD) patients. In this regard, we studied the effect of EPA and DHA, in combination with antiplatelet drugs, in platelet aggregation and P-selectin and α IIb β 3 membrane expression. The antiplatelet drugs aspirin and triflusal, inhibitors of cyclooxygenase-1 (COX-1); ticagrelor, an inhibitor of the receptor P2Y 12 ; vorapaxar, an inhibitor of the PAR-1 receptor, were combined with DHA or EPA and evaluated against in vitro platelet aggregation induced by agonists arachidonic acid (AA), adenosine diphosphate (ADP) and TRAP-6. We further investigated procaspase-activating compound 1 (PAC-1) binding and P-selectin membrane expression in platelets stimulated with ADP and TRAP-6. Both DHA and EPA displayed a dose-dependent inhibitory effect on platelet aggregation induced by AA, ADP and TRAP-6. In platelet aggregation induced by AA, DHA significantly improved acetylsalicylic acid (ASA) and triflusal's inhibitory activity, while EPA enhanced the inhibitory effect of ASA. In combination with EPA, ASA and ticagrelor expressed an increased inhibitory effect towards ADP-induced platelet activation. Both fatty acids could not improve the inhibitory effect of vorapaxar on AA- and ADP-induced platelet aggregation. In the presence of EPA, all antiplatelet drugs displayed a stronger inhibitory effect towards TRAP-6-induced platelet activation. Both omega-3 PUFAs inhibited the membrane expression of α IIb β 3 , though they had no effect on P-selectin expression induced by ADP or TRAP-6. The antiplatelet drugs exhibited heterogeneity regarding their effect on P-selectin and α IIb β 3 membrane expression, while both omega-3 PUFAs inhibited the membrane expression of α IIb β 3 , though had no effect on P-selectin expression induced by ADP or TRAP-6. The combinatory effect of DHA and EPA with the antiplatelet drugs did not result in enhanced inhibitory activity compared to the sum of the individual effects of each component.

Experiments now support theoretical suggestions that coenzymes mediated key metabolic reactions before the emergence of enzymes. Three coenzymes believed essential to the core metabolism of the last universal common ancestor to extant life (pyridoxal phosphate, adenosine diphosphate, and nicotinamide adenine dinucleotide) were recently found to be active in their corresponding metabolic reactions in the absence of enzymes. These findings suggest an earlier contribution of coenzymes to abiogenesis, ultimately yielding insights into the prebiotic origins of metabolism.

Magnesium (Mg 2+ ) is the most abundant divalent cation in the cell and is essential to nearly every biochemical reaction involving adenosine triphosphate (ATP) and its lower energy counterpart, adenosine diphosphate (ADP). In this work, we examine the solution dynamics of ADP at different concentrations and record the changes thereof due to the presence of Mg 2+ ions. Relaxation and diffusion experiments were performed on a range of ADP solutions with increasing magnesium concentration. The most significant changes of both relaxation and diffusion behaviors are observed when adding Mg 2+ up to 0.5 ADP equivalent (eq), with most of the changes complete at 1 eq. Molecular dynamics simulations also show a significant structure introduced by Mg 2+ with very stable pyramidal coordination with the phosphate oxygens. A more extended structure found in the presence of Mg 2+ is consistent with the experimental slowing of diffusion and an increase in the spin-lattice relaxation rate. We do not observe direct evidence of aggregation in solution, although translational diffusion is slowed down significantly at higher concentrations (while solvent diffusion remains constant).

A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process., (© 2024. The Author(s).)

Nucleoside diphosphate kinases (NDPKs) are encoded by nme genes and exist in various isoforms. Based on interactions with other proteins, they are involved in signal transduction, development and pathological processes such as tumorigenesis, metastasis and heart failure. In this study, we report a 1.25 Å resolution structure of human homohexameric NDPK-C bound to ADP and describe the yet unknown complexes formed with GDP, UDP and cAMP, all obtained at a high resolution via X-ray crystallography. Each nucleotide represents a distinct group of mono- or diphosphate purine or pyrimidine bases. We analyzed different NDPK-C nucleotide complexes in the presence and absence of Mg 2+ and explain how this ion plays an essential role in NDPKs' phosphotransferase activity. By analyzing a nucleotide-depleted NDPK-C structure, we detected conformational changes upon substrate binding and identify flexible regions in the substrate binding site. A comparison of NDPK-C with other human isoforms revealed a strong similarity in the overall composition with regard to the 3D structure, but significant differences in the charge and hydrophobicity of the isoforms' surfaces. This may play a role in isoform-specific NDPK interactions with ligands and/or important complex partners like other NDPK isoforms, as well as monomeric and heterotrimeric G proteins. Considering the recently discovered role of NDPK-C in different pathologies, these high-resolution structures thus might provide a basis for interaction studies with other proteins or small ligands, like activators or inhibitors.

Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cells depend on a continuous supply of adenosine triphosphate (ATP), the universal energy currency. In mitochondria, ATP is produced by a series of redox reactions, whereby an electrochemical gradient is established across the inner mitochondrial membrane. The ATP synthase harnesses the energy of the gradient to generate ATP from adenosine diphosphate (ADP) and inorganic phosphate. We determined the structure of ATP synthase within mitochondria of the unicellular flagellate Polytomella by electron cryo-tomography and subtomogram averaging at up to 4.2-angstrom resolution, revealing six rotary positions of the central stalk, subclassified into 21 substates of the F 1 head. The Polytomella ATP synthase forms helical arrays with multiple adjacent rows defining the cristae ridges. The structure of ATP synthase under native operating conditions in the presence of a membrane potential represents a pivotal step toward the analysis of membrane protein complexes in situ.

Background: Endocytosis is the process by which platelets incorporate extracellular molecules into their secretory granules. Endocytosis is mediated by the actin cytoskeleton in nucleated cells; however, the endocytic mechanisms in platelets are undefined., Objectives: To better understand platelet endocytosis, we studied gelsolin (Gsn), an actin-severing protein that promotes actin assembly., Methods: Mouse platelets from Gsn-null (Gsn -/- ) and wild-type (WT) controls were used. The uptake of fluorescent cargo molecules was compared as a measure of their endocytic efficiency. Receptor-mediated endocytosis was measured by the uptake of fibrinogen and transferrin; fluid-phase endocytosis was monitored by the uptake of fluorescent dextrans., Results: Adenosine diphosphate (ADP)-stimulated WT platelets readily internalized both receptor-mediated and fluid-phase cargoes. In contrast, Gsn -/- platelets showed a severe defect in the endocytosis of both types of cargo. The treatment of WT platelets with the actin-disrupting drugs cytochalasin D and jasplakinolide also reduced endocytosis. Notably, the individual and combined effects of Gsn deletion and drug treatment were similar for both receptor-mediated and fluid-phase endocytosis, indicating that Gsn mediates endocytosis via its action on the actin cytoskeleton., Conclusion: Our study demonstrates that Gsn plays a key role in the uptake of bioactive mediators by platelets., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

The HSPA5 protein (BiP/Grp78) serves as a pivotal chaperone in maintaining cellular protein quality control. As a member of the human HSP70 family, HSPA5 comprises two distinct domains: a nucleotide-binding domain (NBD) and a peptide-binding domain (PBD). In this study, we investigated the interdomain interactions of HSPA5, aiming to elucidate how these domains regulate its function as a chaperone. Our findings revealed that HSPA5-FL, HSPA5-T, and HSPA5-N exhibit varying affinities for ATP and ADP, with a noticeable dependency on Mg 2+ for optimal interactions. Interestingly, in ADP assays, the presence of the metal ion seems to enhance NBD binding only for HSPA5-FL and HSPA5-T. Moreover, while the truncation of the C-terminus does not significantly impact the thermal stability of HSPA5, experiments involving MgATP underscore its essential role in mediating interactions and nucleotide hydrolysis. Thermal stability assays further suggested that the NBD-PBD interface enhances the stability of the NBD, more pronounced for HSPA5 than for the orthologous HSPA1A, and prevents self-aggregation through interdomain coupling. Enzymatic analyses indicated that the presence of PBD enhances NBD ATPase activity and augments its nucleotide affinity. Notably, the intrinsic chaperone activity of the PBD is dependent on the presence of the NBD, potentially due to the propensity of the PBD for self-oligomerization. Collectively, our data highlight the pivotal role of allosteric mechanisms in modulating thermal stability, nucleotide interaction, and ATPase activity of HSPA5, underscoring its significance in protein quality control within cellular environments., Competing Interests: Declaration of competing interest The authors affirm that they do not have any known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Leukocyte-platelet aggregates comprise a pathogenic link between hemostasis and immunity, but the prerequisites and mechanisms of their formation remain not understood., Aims: To quantify the formation, composition, and morphology of leukocyte-platelet aggregates in vitro under the influence of various cellular activators., Methods: Phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), thrombin receptor-activating peptide (TRAP-6), and adenosine diphosphate (ADP) were used as cellular activators. Flow cytometry was utilized to identify and quantify aggregates in whole human blood and platelet-rich plasma. Cell types and cellular aggregates were identified using fluorescently labeled antibodies against the appropriate cellular markers, and cell activation was assessed by the expression of appropriate surface markers. For confocal fluorescent microscopy, cell membranes and nuclei were labeled. Neutrophil-platelet aggregates were studied using scanning electron microscopy., Results: In the presence of PMA, ADP or TRAP-6, about 17-38 % of neutrophils and 61-77 % of monocytes formed aggregates with platelets in whole blood, whereas LPS did not induce platelet aggregation with either neutrophils or monocytes due the inability to activate platelets. Similar results were obtained when isolated neutrophils were added to platelet-rich plasma. All the cell types involved in the heterotypic aggregation expressed molecular markers of activation. Fluorescent and electron microscopy of the aggregates showed that the predominant platelet/leukocyte ratios were 1:1 and 2:1., Conclusions: Formation of leukocyte-platelet aggregates depends on the nature of the cellular activator and the spectrum of its cell-activating ability. An indispensable condition for formation of leukocyte-platelet aggregates is activation of all cell types including platelets, which is the restrictive step., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cell-to-cell mitochondrial transfer has recently been shown to play a role in maintaining physiological functions of cell. We previously illustrated that mitochondrial transfer within osteocyte dendritic network regulates bone tissue homeostasis. However, the mechanism of triggering this process has not been explored. Here, we showed that stressed osteocytes in mice release adenosine diphosphate (ADP), resulting in triggering mitochondrial transfer from healthy osteocytes to restore the oxygen consumption rate (OCR) and to alleviate reactive oxygen species accumulation. Furthermore, we identified that P2Y2 and P2Y6 transduced the ADP signal to regulate osteocyte mitochondrial transfer. We showed that mitochondrial metabolism is impaired in aged osteocytes, and there were more extracellular nucleotides release into the matrix in aged cortical bone due to compromised membrane integrity. Conditioned medium from aged osteocytes triggered mitochondrial transfer between osteocytes to enhance the energy metabolism. Together, using osteocyte as an example, this study showed new insights into how extracellular ADP triggers healthy cells to rescue energy metabolism crisis in stressed cells via mitochondrial transfer in tissue homeostasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Hsp70 belongs to a family of molecular chaperones ubiquitous through organisms that assist client protein folding and prevent aggregation. It works through a tightly ATP-regulated allosteric cycle mechanism, which organizes its two NBD and SBD into alternate open and closed arrangements that facilitate loading and unloading of client proteins. The two cytosolic human isoforms Hsc70 and HspA1 are relevant targets for neurodegenerative diseases and cancer. Illuminating the molecular details of Hsp70 functional dynamics is essential to rationalize differences among the well-characterized bacterial homologue DnaK and the less explored human forms and develop subtype- or species-selective allosteric drugs. We present here a molecular dynamics-based analysis of the conformational dynamics of HspA1. By using an "allosterically impaired" mutant for comparison, we can reconstruct the impact of the ADP-ATP swap on interdomain contacts and dynamic coordination in full-length HspA1, supporting previous predictions that were, however, limited to the NBD. We model the initial onset of the conformational cycle by proposing a sequence of structural steps, which reveal the role of a specific human sequence insertion at the linker, and a modulation of the angle formed by the two NBD lobes during the progression of docking. Our findings pinpoint functionally relevant conformations and set the basis for a selective structure-based drug discovery approach targeting allosteric sites in human Hsp70.

Investigating a long-standing conceptual question in bacterial physiology, we examine why DnaA, the bacterial master replication initiator protein, exists in both ATP and ADP forms, despite only the ATP form being essential for initiation. We engineered the Δ4 Escherichia coli strain, devoid of all known external elements facilitating the DnaA-ATP/ADP conversion and found that these cells display nearly wild-type behaviors under nonoverlapping replication cycles. However, during rapid growth with overlapping cycles, Δ4 cells exhibit initiation instability. This aligns with our model predictions, suggesting that the intrinsic ATPase activity of DnaA alone is sufficient for robust initiation control in E. coli and the DnaA-ATP/ADP conversion regulatory elements extend the robustness to multifork replication, indicating an evolutionary adaptation. Moreover, our experiments revealed constant DnaA concentrations during steady-state cell elongation in both wild-type and Δ4 cells. These insights not only advance our understanding of bacterial cell-cycle regulation and DnaA but also highlight a fundamental divergence from eukaryotic cell-cycle controls, emphasizing protein copy-number sensing in bacteria versus programmed protein concentration oscillations in eukaryotes., Competing Interests: Competing interests statement:The authors declare no competing interest.

Phosphoryl transfer is a fundamental reaction in cellular signaling and metabolism that requires Mg 2+ as an essential cofactor. While the primary function of Mg 2+ is electrostatic activation of substrates, such as ATP, the full spectrum of catalytic mechanisms exerted by Mg 2+ is not known. In this study, we integrate structural biology methods, molecular dynamic (MD) simulations, phylogeny, and enzymology assays to provide molecular insights into Mg 2+ -dependent structural reorganization in the active site of the metabolic enzyme adenylate kinase. Our results demonstrate that Mg 2+ induces a conformational rearrangement of the substrates (ATP and ADP), resulting in a 30° adjustment of the angle essential for reversible phosphoryl transfer, thereby optimizing it for catalysis. MD simulations revealed transitions between conformational substates that link the fluctuation of the angle to large-scale enzyme dynamics. The findings contribute detailed insight into Mg 2+ activation of enzymes and may be relevant for reversible and irreversible phosphoryl transfer reactions.

The ClC-3 chloride/proton exchanger is both physiologically and pathologically critical, as it is potentiated by ATP to detect metabolic energy level and point mutations in ClC-3 lead to severe neurodegenerative diseases in human. However, why this exchanger is differentially modulated by ATP, ADP or AMP and how mutations caused gain-of-function remains largely unknow. Here we determine the high-resolution structures of dimeric wildtype ClC-3 in the apo state and in complex with ATP, ADP and AMP, and the disease-causing I607T mutant in the apo and ATP-bounded state by cryo-electron microscopy. In combination with patch-clamp recordings and molecular dynamic simulations, we reveal how the adenine nucleotides binds to ClC-3 and changes in ion occupancy between apo and ATP-bounded state. We further observe I607T mutation induced conformational changes and augments in current. Therefore, our study not only lays the structural basis of adenine nucleotides regulation in ClC-3, but also clearly indicates the target region for drug discovery against ClC-3 mediated neurodegenerative diseases., (© 2024. The Author(s).)

Understanding muscle contraction mechanisms is a standing challenge, and one of the approaches has been to create models of the sarcomere-the basic contractile unit of striated muscle. While these models have been successful in elucidating many aspects of muscle contraction, they fall short in explaining the energetics of functional phenomena, such as rigor, and in particular, their dependence on the concentrations of the biomolecules involved in the cross-bridge cycle. Our hypothesis posits that the stochastic time delay between ATP adsorption and ADP/Pi release in the cross-bridge cycle necessitates a modeling approach where the rates of these two reaction steps are controlled by two independent parts of the total free energy change of the hydrolysis reaction. To test this hypothesis, we built a two-filament, stochastic-mechanical half-sarcomere model that separates the energetic roles of ATP and ADP/Pi in the cross-bridge cycle's free energy landscape. Our results clearly demonstrate that there is a nontrivial dependence of the cross-bridge cycle's kinetics on the independent concentrations of ATP, ADP, and Pi. The simplicity of the proposed model allows for analytical solutions of the more basic systems, which provide novel insight into the dominant mechanisms driving some of the experimentally observed contractile phenomena., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Schmidt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Acetylcholine is the main neurotransmitter at the vertebrate neuromuscular junctions (NMJs). ACh exocytosis is precisely modulated by co-transmitter ATP and its metabolites. It is assumed that ATP/ADP effects on ACh release rely on activation of presynaptic G i protein-coupled P 2 Y 13 receptors. However, downstream signaling mechanism of ATP/ADP-mediated modulation of neuromuscular transmission remains elusive. Using microelectrode recording and fluorescent indicators, the mechanism underlying purinergic regulation was studied in the mouse diaphragm NMJs. Pharmacological stimulation of purinoceptors with ADP decreased synaptic vesicle exocytosis evoked by both low and higher frequency stimulation. This inhibitory action was suppressed by antagonists of P 2 Y 13 receptors (MRS 2211), Ca 2+ mobilization (TMB8), protein kinase C (chelerythrine) and NADPH oxidase (VAS2870) as well as antioxidants. This suggests the participation of Ca 2+ and reactive oxygen species (ROS) in the ADP-triggered signaling. Indeed, ADP caused an increase in cytosolic Ca 2+ with subsequent elevation of ROS levels. The elevation of [Ca 2+ ] in was blocked by MRS 2211 and TMB8, whereas upregulation of ROS was prevented by pertussis toxin (inhibitor of G i protein) and VAS2870. Targeting the main components of lipid rafts, cholesterol and sphingomyelin, suppressed P 2 Y 13 receptor-dependent attenuation of exocytosis and ADP-induced enhancement of ROS production. Inhibition of P 2 Y 13 receptors decreased ROS production and increased the rate of exocytosis during intense activity. Thus, suppression of neuromuscular transmission by exogenous ADP or endogenous ATP can rely on P 2 Y 13 receptor/G i protein/Ca 2+ /protein kinase C/NADPH oxidase/ROS signaling, which is coordinated in a lipid raft-dependent manner., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Precise modulation of host-guest interactions between programmable Ln-MOFs (lanthanide metal-organic frameworks) and phosphate analytes holds immense promise for enabling novel functionalities in biosensing. However, the intricate relationship between these functionalities and structures remains largely elusive. Understanding this correlation is crucial for advancing the rational design of fluorescent biosensor technology. Presently, there exists a large research gap concerning the utilization of Ln-MOFsto monitor the conversion of ATP to ADP, which poses a limitation for kinase detection. In this work, we delve into the potential of Ln-MOFs to amplify the fluorescence response during the kinase-mediated ATP-to-ADP conversion. Six Eu-MOFs were synthesized and Eu-TPTC ([1,1':4',1″]-terphenyl-3,3'',5,5''-tetracarboxylic acid) was selected as a ratiometric fluorescent probe, which is most suitable for high-precision detection of creatine kinase activity through the differential response from ATP to ADP. The molecular -level mechanism was confirmed by density functional theory. Furthermore, a simple paper chip-based platform was constructed to realize the fast (20 min) and sensitive (limit of detection is 0.34 U/L) creatine kinase activity detection in biological samples. Ln-MOF-phosphate interactions offer promising avenues for kinase activity assays and hold the potential for precise customization of analytical chemistry.

The Orthoflavivirus NS3 helicase (NS3h) is crucial in virus replication, representing a potential drug target for pathogenesis. NS3h utilizes nucleotide triphosphate (ATP) for hydrolysis energy to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. Intermediate states along the ATP hydrolysis cycle and conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. Extensive molecular dynamics simulations of West Nile virus NS3h+ssRNA in the apo, ATP, ADP+Pi and ADP bound states were used to model the conformational ensembles along this cycle. Energetic and structural clustering analyses depict a clear trend of differential enthalpic affinity of NS3h with ADP, demonstrating a probable mechanism of hydrolysis turnover regulated by the motif-VI loop (MVIL). Based on these results, MVIL mutants (D471L, D471N and D471E) were found to have a substantial reduction in ATPase activity and RNA replication compared to the wild-type. Simulations of the mutants in the apo state indicate a shift in MVIL populations favoring either a closed or open 'valve' conformation, affecting ATP entry or stabilization, respectively. Combining our molecular modeling with experimental evidence highlights a conformation-dependent role for MVIL as a 'valve' for the ATP-pocket, presenting a promising target for antiviral development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ATP-binding cassette (ABC) transporter, MsbA, plays a pivotal role in lipopolysaccharide (LPS) biogenesis by facilitating the transport of the LPS precursor lipooligosaccharide (LOS) from the cytoplasmic to the periplasmic leaflet of the inner membrane. Despite multiple studies shedding light on MsbA, the role of lipids in modulating MsbA-nucleotide interactions remains poorly understood. Here we use native mass spectrometry (MS) to investigate and resolve nucleotide and lipid binding to MsbA, demonstrating that the transporter has a higher affinity for adenosine 5'-diphosphate (ADP). Moreover, native MS shows the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) 2 -lipid A (KDL) can tune the selectivity of MsbA for adenosine 5'-triphosphate (ATP) over ADP. Guided by these studies, four open, inward-facing structures of MsbA are determined that vary in their openness. We also report a 2.7 Å-resolution structure of MsbA in an open, outward-facing conformation that is not only bound to KDL at the exterior site, but with the nucleotide binding domains (NBDs) adopting a distinct nucleotide-free structure. The results obtained from this study offer valuable insight and snapshots of MsbA during the transport cycle., (© 2024. The Author(s).)

Given its presence in a wide spectrum of soils relevant to food process hygiene, the biological metabolite adenosine triphosphate (ATP) is used as a target for surface hygiene assessments in food processing facilities. Yet, ample evidence demonstrates that ATP is depleted into adenosine di- (ADP) and monophosphate (AMP) homologs resulting in a loss of sensitivity for ATP-based hygiene assays. Yet, there are few studies that denote the degree of these shifts under routine processing conditions such as those encountered during various meat processing steps that may likely alter redox potential and adenosine profiles (e.g., tissue/cellular disruption, application of reducing additives, fermentation, or thermal treatment steps). In this study, meat samples were collected from homogenized beef tissue treated with nonmeat ingredients (sodium chloride, sodium nitrite, sodium erythorbate, natural smoke condensate, and sodium acid pyrophosphate) during manufacture at predetermined steps, and from retail meat products purchased from local markets. Concentrations of ATP, ADP, AMP, and AXP (sum concentration of all homologs) in a lab setting and in situ meat processing venues were determined and compared. Greater differences in AXP were seen during manufacture, where ADP generally comprised ∼90% as a mole fraction of AXP across all treatments, with the exception of the final cook step where AMP predominated. ATP concentrations averaged 2 log values lower than ADP and AMP. Adenosine profiles in retail samples followed similar trends with minimal ATP concentrations with ADP predominant in uncooked samples and AMP predominant in cooked samples. Resultingly, meat processing steps during product manufacture will alter AXP-reliant test sensitivities which should be considered when such technologies are utilized for hygiene verification in meat processing., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Scott A. Rankin reports financial support was provided by Kikkoman USA R&D Laboratory, Inc. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Isopentenyl phosphate kinases (IPKs) have recently garnered attention for their central role in biocatalytic "isoprenol pathways," which seek to reduce the synthesis of the isoprenoid precursors to two enzymatic steps. Furthermore, the natural promiscuity of IPKs toward non-natural alkyl-monophosphates (alkyl-Ps) as substrates has hinted at the isoprenol pathways' potential to access novel isoprenoids with potentially useful activities. However, only a handful of IPK crystal structures have been solved to date, and even fewer of these contain non-natural substrates bound in the active site. The current study sought to elucidate additional ternary complexes bound to non-natural substrates using the IPK homolog from Thermococcus paralvinellae (TcpIPK). Four such structures were solved, each bound to a different non-natural alkyl-P and the phosphoryl donor substrate/product adenosine triphosphate (ATP)/adenosine diphosphate (ADP). As expected, the quaternary, tertiary, and secondary structures of TcpIPK closely resembled those of IPKs published previously, and kinetic analysis of a novel alkyl-P substrate highlighted the potentially dramatic effects of altering the core scaffold of the natural substrate. Even more interesting, though, was the discovery of a trend correlating the position of two α helices in the active site with the magnitude of an IPK homolog's reaction rate for the natural reaction. Overall, the current structures of TcpIPK highlight the importance of continued structural analysis of the IPKs to better understand and optimize their activity with both natural and non-natural substrates., (© 2024 Wiley Periodicals LLC.)

Background: Lactobacillus plantarum has been found to play a significant role in maintaining the balance of intestinal flora in the human gut. However, it is sensitive to commonly used antibiotics and is often incidentally killed during treatment. We attempted to identify a means to protect L. plantarum ATCC14917 from the metabolic changes caused by two commonly used antibiotics, ampicillin, and doxycycline. We examined the metabolic changes under ampicillin and doxycycline treatment and assessed the protective effects of adding key exogenous metabolites., Results: Using metabolomics, we found that under the stress of ampicillin or doxycycline, L. plantarum ATCC14917 exhibited reduced metabolic activity, with purine metabolism a key metabolic pathway involved in this change. We then screened the key biomarkers in this metabolic pathway, guanine and adenosine diphosphate (ADP). The exogenous addition of each of these two metabolites significantly reduced the lethality of ampicillin and doxycycline on L. plantarum ATCC14917. Because purine metabolism is closely related to the production of reactive oxygen species (ROS), the results showed that the addition of guanine or ADP reduced intracellular ROS levels in L. plantarum ATCC14917. Moreover, the killing effects of ampicillin and doxycycline on L. plantarum ATCC14917 were restored by the addition of a ROS accelerator in the presence of guanine or ADP., Conclusions: The metabolic changes of L. plantarum ATCC14917 under antibiotic treatments were determined. Moreover, the metabolome information that was elucidated can be used to help L. plantarum cope with adverse stress, which will help probiotics become less vulnerable to antibiotics during clinical treatment., (© 2024. The Author(s).)

The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements., Competing Interests: Declaration of interests Cornell University has filed patent applications with J.E.P. and M.T.P. as inventors involving CRISPR-Cas systems associated with transposons that are not related to this work., (Copyright © 2024 Elsevier Inc. All rights reserved.)