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 F1 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.

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.

Phosphoryl transfer is a fundamental reaction in cellular signaling and metabolism that requires Mg2+ as an essential cofactor. While the primary function of Mg2+ is electrostatic activation of substrates, such as ATP, the full spectrum of catalytic mechanisms exerted by Mg2+ is not known. In this study, we integrate structural biology methods, molecular dynamic (MD) simulations, phylogeny, and enzymology assays to provide molecular insights into Mg2+-dependent structural reorganization in the active site of the metabolic enzyme adenylate kinase. Our results demonstrate that Mg2+ 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 Mg2+ 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.

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.

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.

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.

BACKGROUND: Leukocyte-platelet aggregates comprise a pathogenic link between hemostasis and immunity, but the prerequisites and mechanisms of their formation remain not understood.
OBJECTIVE: 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.

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.

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 Mg2+ 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.