Inhibition of ATP synthase reverse activity restores energy homeostasis in mitochondrial pathologies.

The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.

Road-blocker HSP disease mutation disrupts pre-organization for ATP hydrolysis in kinesin through a second sphere control.

Kinesin motor proteins perform several essential cellular functions powered by the adenosine triphosphate (ATP) hydrolysis reaction. Several single-point mutations in the kinesin motor protein KIF5A have been implicated to hereditary spastic paraplegia disease (HSP), a lethal neurodegenerative disease in humans. In earlier studies, we have shown that a series of HSP-related mutations can impair the kinesin's long-distance displacement or processivity by modulating the order-disorder transition of the linker connecting the heads to the coiled coil. On the other hand, the reduction of kinesin's ATP hydrolysis reaction rate by a distal asparagine-to-serine mutation is also known to cause HSP disease. However, the molecular mechanism of the ATP hydrolysis reaction in kinesin by this distal mutation is still not fully understood. Using classical molecular dynamics simulations combined with quantum mechanics/molecular mechanics calculations, the pre-organization geometry required for optimal hydrolysis in kinesin motor bound to α/ß-tubulin is determined. This optimal geometry has only a single salt-bridge (of the possible two) between Arg203-Glu236, putting a reactive water molecule at a perfect position for hydrolysis. Such geometry is also needed to create the appropriate configuration for proton translocation during ATP hydrolysis. The distal asparagine-to-serine mutation is found to disrupt this optimal geometry. Therefore, the current study along with our previous one demonstrates how two different effects on kinesin dynamics (processivity and ATP hydrolysis), caused by a different set of genotypes, can give rise to the same phenotype leading to HSP disease.

Structures and mechanisms of actin ATP hydrolysis.

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

Structure and function of ClpXP, a AAA+ proteolytic machine powered by probabilistic ATP hydrolysis.

ClpXP is an archetypical AAA+ protease, consisting of ClpX and ClpP. ClpX is an ATP-dependent protein unfoldase and polypeptide translocase, whereas ClpP is a self-compartmentalized peptidase. ClpXP is currently the only AAA+ protease for which high-resolution structures exist, the molecular basis of recognition for a protein substrate is understood, extensive biochemical and genetic analysis have been performed, and single-molecule optical trapping has allowed direct visualization of the kinetics of substrate unfolding and translocation. In this review, we discuss our current understanding of ClpXP structure and function, evaluate competing sequential and probabilistic mechanisms of ATP hydrolysis, and highlight open questions for future exploration.

The cilia and flagella associated protein CFAP52 orchestrated with CFAP45 is required for sperm motility in mice.

Asthenozoospermia characterized by decreased sperm motility is a major cause of male infertility, but the majority of the etiology remains unknown. Here, we showed that the cilia and flagella associated protein 52 (Cfap52) gene was predominantly expressed in testis and its deletion in a Cfap52 knockout mouse model resulted in decreased sperm motility and male infertility. Cfap52 knockout also led to the disorganization of the midpiece-principal piece junction of the sperm tail but had no effect on the axoneme ultrastructure in spermatozoa. Furthermore, we found that CFAP52 interacted with the cilia and flagella associated protein 45 (CFAP45) and knockout of Cfap52 decreased the expression level of CFAP45 in sperm flagellum, which further disrupted the microtubule sliding produced by dynein ATPase. Together, our studies demonstrate that CFAP52 plays an essential role in sperm motility by interacting with CFAP45 in sperm flagellum, providing insights into the potential pathogenesis of the infertility of the human CFAP52 mutations.

Phosphate toxicity and SERCA2a dysfunction in sudden cardiac arrest.

Almost half of the people who die from sudden cardiac arrest have no detectable heart disease. Among children and young adults, the cause of approximately one-third of deaths from sudden cardiac arrest remains unexplained after thorough examination. Sudden cardiac arrest and related sudden cardiac death are attributed to dysfunctional cardiac ion-channels. The present perspective paper proposes a pathophysiological mechanism by which phosphate toxicity from cellular accumulation of dysregulated inorganic phosphate interferes with normal calcium handling in the heart, leading to sudden cardiac arrest. During cardiac muscle relaxation following contraction, SERCA2a pumps actively transport calcium ions into the sarcoplasmic reticulum, powered by ATP hydrolysis that produces ADP and inorganic phosphate end products. Reviewed evidence supports the proposal that end-product inhibition of SERCA2a occurs as increasing levels of inorganic phosphate drive up phosphate toxicity and bring cardiac function to a sudden and unexpected halt. The paper concludes that end-product inhibition from ATP hydrolysis is the mediating factor in the association of sudden cardiac arrest with phosphate toxicity. However, current technology lacks the ability to directly measure this pathophysiological mechanism in active myocardium, and further research is needed to confirm phosphate toxicity as a risk factor in individuals with sudden cardiac arrest. Moreover, phosphate toxicity may be reduced through modification of dietary phosphate intake, with potential for employing low-phosphate dietary interventions to reduce the risk of sudden cardiac arrest.

Impaired ATP hydrolysis in blood plasma contributes to age-related neutrophil dysfunction.

BACKGROUND: The function of polymorphonuclear neutrophils (PMNs) decreases with age, which results in infectious and inflammatory complications in older individuals. The underlying causes are not fully understood. ATP release and autocrine stimulation of purinergic receptors help PMNs combat microbial invaders. Excessive extracellular ATP interferes with these mechanisms and promotes inflammatory PMN responses. Here, we studied whether dysregulated purinergic signaling in PMNs contributes to their dysfunction in older individuals. RESULTS: Bacterial infection of C57BL/6 mice resulted in exaggerated PMN activation that was significantly greater in old mice (64 weeks) than in young animals (10 weeks). In contrast to young animals, old mice were unable to prevent the systemic spread of bacteria, resulting in lethal sepsis and significantly greater mortality in old mice than in their younger counterparts. We found that the ATP levels in the plasma of mice increased with age and that, along with the extracellular accumulation of ATP, the PMNs of old mice became increasingly primed. Stimulation of the formyl peptide receptors of those primed PMNs triggered inflammatory responses that were significantly more pronounced in old mice than in young animals. However, bacterial phagocytosis and killing by PMNs of old mice were significantly lower than that of young mice. These age-dependent PMN dysfunctions correlated with a decrease in the enzymatic activity of plasma ATPases that convert extracellular ATP to adenosine. ATPases depend on divalent metal ions, including Ca2+, Mg2+, and Zn2+, and we found that depletion of these ions blocked the hydrolysis of ATP and the formation of adenosine in human blood, resulting in ATP accumulation and dysregulation of PMN functions equivalent to those observed in response to aging. CONCLUSIONS: Our findings suggest that impaired hydrolysis of plasma ATP dysregulates PMN function in older individuals. We conclude that strategies aimed at restoring plasma ATPase activity may offer novel therapeutic opportunities to reduce immune dysfunction, inflammation, and infectious complications in older patients.

Study of the Myosin Relay Helix Peptide by Molecular Dynamics Simulations, Pump-Probe and 2D Infrared Spectroscopy.

The Davydov model was conjectured to describe how an amide I excitation created during ATP hydrolysis in myosin might be significant in providing energy to drive myosin's chemomechanical cycle. The free energy surfaces of the myosin relay helix peptide dissolved in 2,2,2-trifluoroethanol (TFE), determined by metadynamics simulations, demonstrate local minima differing in free energy by only ~2 kT, corresponding to broken and stabilized hydrogen bonds, respectively. Experimental pump-probe and 2D infrared spectroscopy were performed on the peptide dissolved in TFE. The relative heights of two peaks seen in the pump-probe data and the corresponding relative volumes of diagonal peaks seen in the 2D-IR spectra at time delays between 0.5 ps and 1 ps differ noticeably from what is seen at earlier or later time delays or in the linear spectrum, indicating that a vibrational excitation may influence the conformational state of this helix. Thus, it is possible that the presence of an amide I excitation may be a direct factor in the conformational state taken on by the myosin relay helix following ATP hydrolysis in myosin.

Betasatellite-encoded ßC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of geminivirus-encoded replication initiator protein.

Geminivirus-betasatellite disease complexes are an epidemic threat to the majority of economically important crops across the world. Plant virus satellites including betasatellites are maintained by their associated helper virus. Geminivirus-betasatellites influence viral pathogenesis by substantially increasing or decreasing their helper virus accumulation. In the present study, we attempted to understand the mechanistic details of the geminivirus-betasatellite interaction. Here, we used tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl Patna betasatellite (ToLCPaB) as a model system. This study reveals that ToLCGV can efficiently trans-replicate ToLCPaB in Nicotiana benthamiana plants, but ToLCPaB greatly reduced the accumulation of its helper virus DNA. For the first time, we have identified that the ToLCPaB-encoded ßC1 protein is able to interact with ToLCGV-encoded replication initiator protein (Rep). In addition, we demonstrate that the C-terminal region of ßC1 interacts with the C-terminus of Rep (RepC) protein. Our previous study had established that ßC1 proteins encoded by diverse betasatellites possess a novel ATP hydrolysis activity and the conserved lysine/arginine residues at positions 49 and 91 are necessary for this function. Here, we show that mutating lysine at positions 49 to alanine of ßC1 (ßC1K49A) protein did not affect its ability to interact with RepC protein. Biochemical studies performed with ATP hydrolysis activity-deficient K49A mutated ßC1 (ßC1K49A) and RepC proteins revealed that Rep-ßC1 interaction interferes with the ATP hydrolysis activity of Rep protein. Further, we demonstrate that ßC1 protein is able to interact with D227A and D289A mutated RepC proteins but not with D262A, K272A or D286A mutated RepC proteins, suggesting that the ßC1-interacting region of Rep protein encompasses its Walker-B and B' motifs. The results of docking studies supported that the ßC1-interacting region of Rep protein encompasses its motifs associated with ATP binding and ATP hydrolysis activities. Docking studies also provided evidence that the Rep-ßC1 interaction interferes with the ATP binding activity of Rep protein. Together, our findings suggest that ßC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of helper virus Rep protein.

ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy.

The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of k 1 = 0.49 ± 0.28 s-1 and k 2 = 0.014 ± 0.003 s-1, respectively. We further verified these rates with photocaged NPEcgAppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis (k 2 < 2 × 10-4 s-1). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA.

The G-patch protein Spp2 couples the spliceosome-stimulated ATPase activity of the DEAH-box protein Prp2 to catalytic activation of the spliceosome.

Structural rearrangement of the activated spliceosome (B(act)) to yield a catalytically active complex (B*) is mediated by the DEAH-box NTPase Prp2 in cooperation with the G-patch protein Spp2. However, how the energy of ATP hydrolysis by Prp2 is coupled to mechanical work and what role Spp2 plays in this process are unclear. Using a purified splicing system, we demonstrate that Spp2 is not required to recruit Prp2 to its bona fide binding site in the B(act) spliceosome. In the absence of Spp2, the B(act) spliceosome efficiently triggers Prp2's NTPase activity, but NTP hydrolysis is not coupled to ribonucleoprotein (RNP) rearrangements leading to catalytic activation of the spliceosome. Transformation of the B(act) to the B* spliceosome occurs only when Spp2 is present and is accompanied by dissociation of Prp2 and a reduction in its NTPase activity. In the absence of spliceosomes, Spp2 enhances Prp2's RNA-dependent ATPase activity without affecting its RNA affinity. Our data suggest that Spp2 plays a major role in coupling Prp2's ATPase activity to remodeling of the spliceosome into a catalytically active machine.

Mechanism of ADP-Inhibited ATP Hydrolysis in Single Proton-Pumping FoF1-ATP Synthase Trapped in Solution.

FoF1-ATP synthases in mitochondria, in chloroplasts, and in most bacteria are proton-driven membrane enzymes that supply the cells with ATP made from ADP and phosphate. Different control mechanisms exist to monitor and prevent the enzymes' reverse chemical reaction of fast wasteful ATP hydrolysis, including mechanical or redox-based blockade of catalysis and ADP inhibition. In general, product inhibition is expected to slow down the mean catalytic turnover. Biochemical assays are ensemble measurements and cannot discriminate between a mechanism affecting all enzymes equally or individually. For example, all enzymes could work more slowly at a decreasing substrate/product ratio, or an increasing number of individual enzymes could be completely blocked. Here, we examined the effect of increasing amounts of ADP on ATP hydrolysis of single Escherichia coli FoF1-ATP synthases in liposomes. We observed the individual catalytic turnover of the enzymes one after another by monitoring the internal subunit rotation using single-molecule Förster resonance energy transfer (smFRET). Observation times of single FRET-labeled FoF1-ATP synthases in solution were extended up to several seconds using a confocal anti-Brownian electrokinetic trap (ABEL trap). By counting active versus inhibited enzymes, we revealed that ADP inhibition did not decrease the catalytic turnover of all FoF1-ATP synthases equally. Instead, increasing ADP in the ADP/ATP mixture reduced the number of remaining active enzymes that operated at similar catalytic rates for varying substrate/product ratios.

Molecular Mechanisms of Deregulation of Muscle Contractility Caused by the R168H Mutation in TPM3 and Its Attenuation by Therapeutic Agents.

The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction.

Phosphorus Chemistry at the Roots of Bioenergetics: Ligand Permutation as the Molecular Basis of the Mechanism of ATP Synthesis/Hydrolysis by FOF1-ATP Synthase.

The integration of phosphorus chemistry with the mechanism of ATP synthesis/hydrolysis requires dynamical information during ATP turnover and catalysis. Oxygen exchange reactions occurring at ß-catalytic sites of the FOF1-ATP synthase/F1-ATPase imprint a unique record of molecular events during the catalytic cycle of ATP synthesis/hydrolysis. They have been shown to provide valuable time-resolved information on enzyme catalysis during ATP synthesis and ATP hydrolysis. The present work conducts new experiments on oxygen exchange catalyzed by submitochondrial particles designed to (i) measure the relative rates of Pi-ATP, Pi-HOH, and ATP-HOH isotope exchanges; (ii) probe the effect of ADP removal on the extent of inhibition of the exchanges, and (iii) test their uncoupler sensitivity/resistance. The objectives have been realized based on new experiments on submitochondrial particles, which show that both the Pi-HOH and ATP-HOH exchanges occur at a considerably higher rate relative to the Pi-ATP exchange, an observation that cannot be explained by previous mechanisms. A unifying explanation of the kinetic data that rationalizes these observations is given. The experimental results in (ii) show that ADP removal does not inhibit the intermediate Pi-HOH exchange when ATP and submitochondrial particles are incubated, and that the nucleotide requirement of the intermediate Pi-HOH exchange is adequately met by ATP, but not by ADP. These results contradicts the central postulate in Boyer's binding change mechanism of reversible catalysis at a F1 catalytic site with Keq~1 that predicts an absolute requirement of ADP for the occurrence of the Pi-HOH exchange. The prominent intermediate Pi-HOH exchange occurring under hydrolytic conditions is shown to be best explained by Nath's torsional mechanism of energy transduction and ATP synthesis/hydrolysis, which postulates an essentially irreversible cleavage of ATP by mitochondria/particles, independent from a reversible formation of ATP from ADP and Pi. The explanation within the torsional mechanism is also shown to rationalize the relative insensitivity of the intermediate Pi-HOH exchange to uncouplers observed in the experiments in (iii) compared to the Pi-ATP and ATP-HOH exchanges. This is shown to lead to new concepts and perspectives based on ligand displacement/substitution and ligand permutation for the elucidation of the oxygen exchange reactions within the framework of fundamental phosphorus chemistry. Fast mechanisms that realize the rotation/twist, tilt, permutation and switch of ligands, as well as inversion at the γ-phosphorus synchronously and simultaneously and in a concerted manner, have been proposed, and their stereochemical consequences have been analyzed. These considerations take us beyond the binding change mechanism of ATP synthesis/hydrolysis in bioenergetics.

Molecular basis for ATPase-powered substrate translocation by the Lon AAA+ protease.

The Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of a substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state at 3.6 Å resolution. Our data indicate that substrate interactions are mediated by the dual pore loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states. However, a closed AAA+ ring is maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. This structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding.

Sisyphus observed: Unraveling the high ATP usage of an RNA chaperone.

DEAD-box proteins are nonprocessive RNA helicases that can function as RNA chaperones by coupling ATP binding and hydrolysis to structural reorganization of RNA. Here, Jarmoskaite et al. quantify the ATP utilization of an RNA chaperone during refolding of a misfolded ribozyme substrate. Strikingly, 100 ATP hydrolysis events are needed per successfully refolded ribozyme, suggesting that each round of unfolding requires ten ATP molecules, since 90% of substrate unfolding cycles only lead back to the kinetically favored misfolded state. This near-Sisyphean effort reveals a potentially conserved model for RNA reorganization by RNA chaperones.

Geminivirus Betasatellite-Encoded ßC1 Protein Exhibits Novel ATP Hydrolysis Activity That Influences Its DNA-Binding Activity and Viral Pathogenesis.

Plant virus satellites are maintained by their associated helper viruses, and satellites influence viral pathogenesis. Diseases caused by geminivirus-betasatellite complexes can become epidemics and therefore have become a threat to economically important crops across the world. Here, we identified a novel molecular function of the betasatellite-encoded pathogenicity determinant ßC1. The tomato leaf curl Patna betasatellite (ToLCPaB)-encoded ßC1 protein was found to exhibit novel ATPase activity in the presence of the divalent metal ion cofactor MgCl2. Moreover, ATPase activity was confirmed to be ubiquitously displayed by ßC1 proteins encoded by diverse betasatellites. Mutational and sequence analysis showed that conserved lysine/arginine residues at positions 49/50 and 91 of ßC1 proteins are essential for their ATPase activity. Biochemical studies revealed that the DNA-binding activity of the ßC1 protein was interfered with by the binding of ATP to the protein. Mutating arginine 91 of ßC1 to alanine reduced its DNA-binding activity. The results of docking studies provided evidence for an overlap of the ATP-binding and DNA-binding regions of ßC1 and for the importance of arginine 91 for both ATP-binding and DNA-binding activities. A mutant betasatellite with a specifically ßC1-ATPase dominant negative mutation was found to induce symptoms on Nicotiana benthamiana plants similar to those induced by wild-type betasatellite infection. The ATPase function of ßC1 was found to be negatively associated with geminivirus-betasatellite DNA accumulation, despite the positive influence of this ATPase function on the accumulation of replication-associated protein (Rep) and ßC1 transcripts. IMPORTANCE Most satellites influence the pathogenesis of their helper viruses. Here, we characterized the novel molecular function of ßC1, a nonstructural pathogenicity determinant protein encoded by a betasatellite. We demonstrated the display of ATPase activity by this ßC1 protein. Additionally, we confirmed the ubiquitous display of ATPase activity by ßC1 proteins encoded by diverse betasatellites. The lysine/arginine residues conserved at positions 49 and 91 of ßC1 were found to be crucial for its ATPase function. DNA-binding activity of ßC1 was found to be reduced in the presence of ATP. Inhibition of ATPase activity of ßC1 in the presence of an excess concentration of cold ATP, GTP, CTP, or UTP suggested that the purified ßC1 can also hydrolyze other cellular nucleoside triphosphates (NTPs) besides ATP in vitro. These results established the importance of the ATPase and DNA-binding activities of the ßC1 protein in regulating geminivirus-betasatellite DNA accumulation in the infected plant cell.

Extrinsic conditions influence the self-association and structure of IF1, the regulatory protein of mitochondrial ATP synthase.

The endogenous inhibitor of ATP synthase in mitochondria, called IF1, conserves cellular energy when the proton-motive force collapses by inhibiting ATP hydrolysis. Around neutrality, the 84-amino-acid bovine IF1 is thought to self-assemble into active dimers and, under alkaline conditions, into inactive tetramers and higher oligomers. Dimerization is mediated by formation of an antiparallel α-helical coiled-coil involving residues 44-84. The inhibitory region of each monomer from residues 1-46 is largely α-helical in crystals, but disordered in solution. The formation of the inhibited enzyme complex requires the hydrolysis of two ATP molecules, and in the complex the disordered region from residues 8-13 is extended and is followed by an α-helix from residues 14-18 and a longer α-helix from residue 21, which continues unbroken into the coiled-coil region. From residues 21-46, the long α-helix binds to other α-helices in the C-terminal region of predominantly one of the ß-subunits in the most closed of the three catalytic interfaces. The definition of the factors that influence the self-association of IF1 is a key to understanding the regulation of its inhibitory properties. Therefore, we investigated the influence of pH and salt-types on the self-association of bovine IF1 and the folding of its unfolded region. We identified the equilibrium between dimers and tetramers as a potential central factor in the in vivo modulation of the inhibitory activity and suggest that the intrinsically disordered region makes its inhibitory potency exquisitely sensitive and responsive to physiological changes that influence the capability of mitochondria to make ATP.

Structural insights into RNA recognition by the Chikungunya virus nsP2 helicase.

Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural protein 2 (nsP2) exhibits the protease and RNA helicase activities that are required for viral RNA replication and transcription. Unlike for the C-terminal protease, the structure of the N-terminal RNA helicase (nsP2h) has not been determined. Here, we report the crystal structure of the nsP2h bound to the conserved 3'-end 14 nucleotides of the CHIKV genome and the nonhydrolyzable transition-state nucleotide analog ADP-AlF4 Overall, the structural analysis revealed that nsP2h adopts a uniquely folded N-terminal domain followed by a superfamily 1 RNA helicase fold. The conserved helicase motifs establish polar contacts with the RNA backbone. There are three hydrophobic residues (Y161, F164, and F287) which form stacking interactions with RNA bases and thereby bend the RNA backbone. An F287A substitution that disrupted these stacking interactions increased the basal ATPase activity but decreased the RNA binding affinity. Furthermore, the F287A substitution reduced viral infectivity by attenuating subgenomic RNA synthesis. Replication of the mutant virus was restored by pseudoreversion (A287V) or adaptive mutations in the RecA2 helicase domain (T358S or V410I). Y161A and/or F164A substitutions, which were designed to disrupt the interactions with the RNA molecule, did not affect the ATPase activity but completely abolished the replication and transcription of viral RNA and the infectivity of CHIKV. Our study sheds light on the roles of the RNA helicase region in viral replication and provides insights that might be applicable to alphaviruses and other RNA viruses in general.

Functional stability of CFTR depends on tight binding of ATP at its degenerate ATP-binding site.

Opening of the cystic fibrosis transmembrane conductance regulator (CFTR) channel is coupled to the motion of its two nucleotide-binding domains: they form a heterodimer sandwiching two functionally distinct ATP-binding sites (sites 1 and 2). While active ATP hydrolysis in site 2 triggers rapid channel closure, the functional role of stable ATP binding in the catalysis-incompetent (or degenerate) site 1, a feature conserved in many other ATP-binding cassette (ABC) transporter proteins, remains elusive. Here, we found that CFTR loses its prompt responsiveness to ATP after the channel is devoid of ATP for tens to hundreds of seconds. Mutants with weakened ATP binding in site 1 and the most prevalent disease-causing mutation, F508del, are more vulnerable to ATP depletion. In contrast, strengthening ligand binding in site 1 with N6 -(2-phenylethyl)-ATP, a high-affinity ATP analogue, or abolishing ATP hydrolysis in site 2 by the mutation D1370N, helps sustain a durable function of the otherwise unstable mutant channels. Thus, tight binding of ATP in the degenerate ATP-binding site is crucial to the functional stability of CFTR. Small molecules targeting site 1 may bear therapeutic potential to overcome the membrane instability of F508del-CFTR. KEY POINTS: During evolution, many ATP-binding cassette transporters - including the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, whose dysfunction causes cystic fibrosis (CF) - lose the ability to hydrolyse ATP in one of the two ATP-binding sites. Here we show that tight ATP binding at this degenerate site in CFTR is central for maintaining the stable, robust function of normal CFTR. We also demonstrate that membrane instability of the most common CF-causing mutant, F508del-CFTR, can be rescued by strengthening ATP binding at CFTR's degenerate site. Our data thus explain an evolutionary puzzle and offer a potential therapeutic strategy for CF.