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p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo–electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)–bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5′-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.

Activation of G protein-coupled receptors upon agonist binding is a critical step in the signaling cascade for this family of cell surface proteins. We report the crystal structure of the A(2A) adenosine receptor (A(2A)AR) bound to an agonist UK-432097 at 2.7 angstrom resolution. Relative to inactive, antagonist-bound A(2A)AR, the agonist-bound structure displays an outward tilt and rotation of the cytoplasmic half of helix VI, a movement of helix V, and an axial shift of helix III, resembling the changes associated with the active-state opsin structure. Additionally, a seesaw movement of helix VII and a shift of extracellular loop 3 are likely specific to A(2A)AR and its ligand. The results define the molecule UK-432097 as a \"conformationally selective agonist\" capable of receptor stabilization in a specific active-state configuration.

Design of artificial systems, which can respond to fluctuations in concentration of adenosine phosphates (APs), can be useful in understanding various biological processes. Helical assemblies of chromophores, which dynamically respond to such changes, can provide real-time chiroptical readout of various chemical transformations. Towards this concept, here we present a supramolecular helix of achiral chromophores, which shows chiral APs responsive tunable handedness along with dynamically switchable helicity. This system, composing of naphthalenediimides with phosphate recognition unit, shows opposite handedness on binding with ATP compared with ADP or AMP, which is comprehensively analysed with molecular dynamic simulations. Such differential signalling along with stimuli-dependent fast stereomutations have been capitalized to probe the reaction kinetics of enzymatic ATP hydrolysis. Detailed chiroptical analyses provide mechanistic insights into the enzymatic hydrolysis and various intermediate steps. Thus, a unique dynamic helical assembly to monitor the real-time reaction processes via its stimuli-responsive chiroptical signalling is conceptualized. Interesting changes in physical and optical properties can result from the binding of small molecules to supramolecular polymers. Here, the authors present an ATP assay, using a supramolecular helix to switch between left- and right-handed conformations on binding different adenosine phosphates.

The ability to sense chemical gradients and respond with directional motility and chemical activity is a defining feature of complex living systems. There is a strong interest among scientists to design synthetic systems that emulate these properties. Here, we realize and control such behaviors in a synthetic system by tailoring multivalent interactions of adenosine nucleotides with catalytic microbeads. We first show that multivalent interactions of the bead with gradients of adenosine mono-, di- and trinucleotides (AM/D/TP) control both the phoretic motion and a proton-transfer catalytic reaction, and find that both effects are diminished greatly with increasing valence of phosphates. We exploit this behavior by using enzymatic hydrolysis of ATP to AMP, which downregulates multivalent interactivity in situ. This produces a sudden increase in transport of the catalytic microbeads (a phoretic jump), which is accompanied by increased catalytic activity. Finally, we show how this enzymatic activity can be systematically tuned, leading to simultaneous in situ spatial and temporal control of the location of the microbeads, as well as the products of the reaction that they catalyze. These findings open up new avenues for utilizing multivalent interaction-mediated programming of complex chemo-mechanical behaviors into active systems. The design of synthetic systems that can sense chemical gradients and respond with directional motility and chemical activity is of interest. Here, the authors realize and control such behaviors in a synthetic system by tailoring multivalent interactions of adenosine nucleotides with catalytic microbeads.

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. Single molecule nanopore analysis is used to reveal adenylate kinase global dynamics in real time. It was found that allosteric interactions guide domain motions and substrate affinity, with 10% of enzymes displaying alternative active forms, suggesting evolutionary paths for enzyme regulation.

Adenosine nucleotides and polyphosphates play a significant role in biochemistry, from participating in the formation of genetic material to serving as metabolic energy currency. In this study, we examine the stability and decomposition rates of adenosine phosphates—5′-AMP, 5′-ADP, and 5′-ATP (mentioned simply as AMP, ADP and ATP hereafter)—at temperatures of 22–25 °C, 50–55 °C, 70–75 °C, and 85–90 °C, at a pH of 4, over periods of 2 and 4 days, in both saltwater and ultrapure water, under unsealed and completely dried down conditions. We found that adenosine phosphates degrade rapidly under heat and dehydration, particularly at temperatures above 25 °C. Among the three compounds, AMP is the most stable, maintaining its integrity between 22 and 55 °C, whereas ATP begins to degrade at 22–25 °C and ADP is completely decomposed at temperatures above this range. Decomposition rates were analyzed using quantitative 31P-NMR, based on the detection of various phosphorus-containing species. AMP primarily hydrolyzed into phosphate, pyrophosphate and even formed 2′,3′-cAMP. In contrast, the condensed adenosine phosphates (ADP and ATP) hydrolyzed to AMP, phosphate, pyrophosphate, triphosphate, 5′-AMP and, in some cases, 2′,3′-cyclic adenosine monophosphate (2′,3′-cAMP). We also investigated the formation of these compounds in the presence of N-containing additives such as thiourea, urea, imidazole, and cyanamide at temperatures between 65 and 70 °C. Among these, cyanamide and urea were particularly effective in promoting the synthesis of adenosine monophosphates (AMPs) from phosphate and adenosine. The major products observed were 2′,3′,5′-AMPs and cyclic 2′,3′-AMPs. In some experiments, adenosine diphosphate (ADP) and dimeric nucleotide species were also detected. These findings suggest that moderately heated evaporating pools could facilitate the abiotic formation of AMPs. However, such environments would likely have been unsuitable for the long-term accumulation of these compounds due to continued degradation, though there would exist some level of these nucleotides at steady state.

Adenosine receptors (ARs: A AR, A AR, A AR, and A AR) are crucial therapeutic targets; however, developing selective, efficacious drugs for them remains a significant challenge. Here, we present high-resolution cryo-electron microscopy (cryo-EM) structures of the human A AR in three distinct functional states: bound to the endogenous agonist adenosine, the clinically relevant agonist Piclidenoson, and the covalent antagonist LUF7602. These structures, complemented by mutagenesis and pharmacological studies, reveal an A AR activation mechanism that involves an extensive hydrogen bond network from the extracellular surface down to the orthosteric binding site. In addition, we identify a cryptic pocket that accommodates the N -iodobenzyl group of Piclidenoson through a ligand-dependent conformational change of M174 . Our comprehensive structural and functional characterisation of A AR advances our understanding of adenosine receptor pharmacology and establishes a foundation for developing more selective therapeutics for various disorders, including inflammatory diseases, cancer, and glaucoma.

Mutations in p97, a major cytosolic AAA (ATPases associated with a variety of cellular activities) chaperone, cause inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD). IBMPFD mutants have single amino‐acid substitutions at the interface between the N‐terminal domain (N‐domain) and the adjacent AAA domain (D1), resulting in a reduced affinity for ADP. The structures of p97 N–D1 fragments bearing IBMPFD mutations adopt an atypical N‐domain conformation in the presence of Mg 2+ ·ATPγS, which is reversible by ADP, showing for the first time the nucleotide‐dependent conformational change of the N‐domain. The transition from the ADP‐ to the ATPγS‐bound state is accompanied by a loop‐to‐helix conversion in the N–D1 linker and by an apparent re‐ordering in the N‐terminal region of p97. X‐ray scattering experiments suggest that wild‐type p97 subunits undergo a similar nucleotide‐dependent N‐domain conformational change. We propose that IBMPFD mutations alter the timing of the transition between nucleotide states by destabilizing the ADP‐bound form and consequently interfere with the interactions between the N‐domains and their substrates.

Significance Pex1 and Pex6 are members of the AAA family of ATPases, which contain two ATPase domains in a single polypeptide chain and form hexameric double rings. These two Pex proteins are involved in the biogenesis of peroxisomes, and mutations in them frequently cause diseases. Here, we determined structures of the Pex1/Pex6 complex by cryo-electron microscopy. Novel computational modeling methods allowed placement of Pex1/Pex6 domains into subnanometer density maps. Our results show that the peroxisomal Pex1/Pex6 ATPases form a unique double-ring structure in which the two proteins alternate around the ring. Our data shed light on the mechanism and function of this ATPase and suggest a role in peroxisomal protein import similar to that of p97 in ER-associated protein degradation. Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the Pex1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. Pex1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of Pex1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between Pex1/Pex6 and p97, supporting the hypothesis that the Pex1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.

Subunits in multimeric ring-shaped motors must coordinate their activities to ensure correct and efficient performance of their mechanical tasks. Here, we study WT and arginine finger mutants of the pentameric bacteriophage φ29 DNA packaging motor. Our results reveal the molecular interactions necessary for the coordination of ADP–ATP exchange and ATP hydrolysis of the motor’s biphasic mechanochemical cycle. We show that two distinct regulatory mechanisms determine this coordination. In the first mechanism, the DNA up-regulates a single subunit’s catalytic activity, transforming it into a global regulator that initiates the nucleotide exchange phase and the hydrolysis phase. In the second, an arginine finger in each subunit promotes ADP–ATP exchange and ATP hydrolysis of its neighbor. Accordingly, we suggest that the subunits perform the roles described for GDP exchange factors and GTPase-activating proteins observed in small GTPases. We propose that these mechanisms are fundamental to intersubunit coordination and are likely present in other ring ATPases.