Nonhydrolyzable ATP Analog Research Articles

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis

Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed.

Novel translocation intermediate allows re-evaluation of roles of ATP, proton motive force and SecG at the late stage of preprotein translocation.

Presecretory proteins such as pOmpA are translocated across the inner membrane of Escherichia coli by Sec translocase powered by ATP and proton motive force (PMF). Translocation activity has been determined by protease protection assaying invitro. We identified a new translocation intermediate at a late stage, which was protected by proteinase K (PK), but became PK sensitive upon urea extraction. At a late stage of pOmpA translocation driven by PMF in the presence of a nonhydrolyzable ATP analogue, the PK-protected materials arose, but were pulled back upon urea extraction, indicating that completion of translocation requires ATP hydrolysis. When inverted membrane vesicles prepared from secG-null strain (ΔSecG IMV) were used in the absence of PMF, the translocation intermediate was accumulated. When the ATP concentration was low in the absence of PMF, the translocation intermediate was also accumulated. Imposition of PMF in the presence of a low ATP concentration caused recovery of pOmpA translocation and resistance to urea extraction for SecG+ IMV, but not for ΔSecG IMV. Thus, analysis of the late translocation intermediate showed that two of three constituents, physiological concentration of ATP, PMF and SecG, are required for the catalytic cycle of preprotein translocation, that is, completion and subsequent initiation of translocation.

Nucleotide dependence of the dimerization of ATP binding cassette nucleotide binding domains

ATP-binding cassette proteins are ubiquitously present throughout all known genomes. Their basic functional unit possesses two transmembrane domains and two nucleotide-binding domains. The nucleotide-binding domains are responsible for ATP binding and hydrolysis, and their 3-dimensional structure is conserved across ATP-binding cassette proteins. Binding of ATP produces nucleotide-binding domain dimerization, a step necessary for hydrolysis. However, the possibility that nucleotide-binding domains bind and/or hydrolyze nucleotide triphosphates different from ATP has not been explored in detail. Here, we studied that possibility using M. jannaschii MJ0796, a prototypical ATP-binding cassette nucleotide-binding domain. We found that nucleotide-binding domain dimerization occurs as a result of binding to the natural nucleotide triphosphates ATP, GTP, CTP and UTP, and also to the analog ATP-γ-S. All the natural nucleotide triphosphates are hydrolyzed at similar rates, whereas ATP-γ-S is not hydrolyzed. We also found that the non-hydrolyzable ATP analog AMP-PNP, frequently assumed to produce the nucleotide-bound conformation, failed to elicit nucleotide-binding domain dimerization. Our results raise the possibility that not all the nucleotide binding sites of nucleotide-binding domains are occupied by ATP under physiological conditions, and that ATP is not always the nucleotide hydrolyzed to dissociate the nucleotide-binding domain dimers.

Role of the σ54 Activator Interacting Domain in Bacterial Transcription Initiation

Bacterial sigma factors are subunits of RNA polymerase that direct the holoenzyme to specific sets of promoters in the genome and are a central element of regulating transcription. Most polymerase holoenzymes open the promoter and initiate transcription rapidly after binding. However, polymerase containing the members of the σ54 family must be acted on by a transcriptional activator before DNA opening and initiation occur. A key domain in these transcriptional activators forms a hexameric AAA+ ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and σ54 are primarily made through an N-terminal σ54 activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the σ54 AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in σ54 alone. We identified a minimal construct of the Aquifex aeolicus σ54 AID that consists of two predicted helices and retains native-like binding affinity for the transcriptional activator NtrC1. Using the NtrC1 ATPase domain, bound with the non-hydrolyzable ATP analog ADP-beryllium fluoride, we studied the NtrC1–σ54 AID complex using NMR spectroscopy. We show that the σ54 AID becomes structured after associating with the core loops of the transcriptional activators in their ATP state and that the primary site of the interaction is the first predicted helix. Understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of σ54 bacterial transcription initiation.

Biophysical characterization of drug-resistant mutants of fibroblast growth factor receptor 1.

Over-expression and aberrant activation of tyrosine kinases occur frequently in human cancers. Various tyrosine kinase inhibitors (TKIs) are under clinical use, but acquisition of resistance to these drugs is a major problem. Here, we studied the interaction between two drug-resistant mutants of fibroblast growth factor receptor 1 (FGFR1), N546K and V561M, and four ATP-competitive inhibitors, ponatinib, dovitinib, PD173074 and BGJ-398. Among these protein-drug systems, the only marked reduction in affinity was that of PD173074 for the V561M mutant. We also examined the interaction of these FGFR1 variants to AMP-PNP, a nonhydrolyzable analogue of ATP, and showed that N546K showed increased affinity for the ATP analogue as compared with the wild type. These findings will help to clarify the mechanism of drug resistance in mutant tyrosine kinases.

Mouse MORC3 is a GHKL ATPase that localizes to H3K4me3 marked chromatin

Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) ATPases that function in gene regulation in multiple organisms. Animal MORCs also contain CW-type zinc finger domains, which are known to bind to modified histones. We solved the crystal structure of the murine MORC3 ATPase-CW domain bound to the nucleotide analog AMPPNP (phosphoaminophosphonic acid-adenylate ester) and in complex with a trimethylated histone H3 lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. We used native mass spectrometry to show that dimerization is ATP-dependent, and that dimer formation is enhanced in the presence of nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage to bind trimethylated Lys4 and forms extensive hydrogen bonds with the H3 tail. We found that MORC3 localizes to promoters marked by H3K4me3 throughout the genome, consistent with its binding to H3K4me3 in vitro. Our work sheds light on aspects of the molecular dynamics and function of MORC3.

Cooperativity between verapamil and ATP bound to the efflux transporter P-glycoprotein

The P-glycoprotein (Pgp) transporter plays a central role in drug disposition by effluxing a chemically diverse range of drugs from cells through conformational changes and ATP hydrolysis. A number of drugs are known to activate ATP hydrolysis of Pgp, but coupling between ATP and drug binding is not well understood. The cardiovascular drug verapamil is one of the most widely studied Pgp substrates and therefore, represents an ideal drug to investigate the drug-induced ATPase activation of Pgp. As previously noted, verapamil-induced Pgp-mediated ATP hydrolysis kinetics was biphasic at saturating ATP concentrations. However, at subsaturating ATP concentrations, verapamil-induced ATPase activation kinetics became monophasic. To further understand this switch in kinetic behavior, the Pgp-coupled ATPase activity kinetics was checked with a panel of verapamil and ATP concentrations and fit with the substrate inhibition equation and the kinetic fitting software COPASI. The fits suggested that cooperativity between ATP and verapamil switched between low and high verapamil concentration. Fluorescence spectroscopy of Pgp revealed that cooperativity between verapamil and a non-hydrolyzable ATP analog leads to distinct global conformational changes of Pgp. NMR of Pgp reconstituted in liposomes showed that cooperativity between verapamil and the non-hydrolyzable ATP analog modulate each other’s interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis.

Monitoring conformational heterogeneity of the lid of DnaK substrate-binding domain during its chaperone cycle.

DnaK or Hsp70 of Escherichia coli is a master regulator of the bacterial proteostasis network. Allosteric communication between the two functional domains of DnaK, the N-terminal nucleotide-binding domain (NBD) and the C-terminal substrate- or peptide-binding domain (SBD) regulate its activity. X-ray crystallography and NMR studies have provided snapshots of distinct conformations of Hsp70 proteins in various physiological states; however, the conformational heterogeneity and dynamics of allostery-driven Hsp70 activity remains underexplored. In this work, we employed single-molecule Förster resonance energy transfer (sm-FRET) measurements to capture distinct intradomain conformational states of a region within the DnaK-SBD known as the lid. Our data conclusively demonstrate prominent conformational heterogeneity of the DnaK lid in ADP-bound states; in contrast, the ATP-bound open conformations are homogeneous. Interestingly, a nonhydrolysable ATP analogue, AMP-PNP, imparts heterogeneity to the lid conformations mimicking the ADP-bound state. The cochaperone DnaJ confers ADP-like heterogeneous lid conformations to DnaK, although the presence of the cochaperone accelerates the substrate-binding rate by a hitherto unknown mechanism. Irrespective of the presence of DnaJ, binding of a peptide substrate to the DnaK-SBD leads to prominent lid closure. Lid closure is only partial upon binding to molten globule-like authentic cellular substrates, probably to accommodate non-native substrate proteins of varied structures.

ATP-sensitive K(+) channels (Kir6.1/SUR1) regulate gap junctional coupling in cochlear-supporting cells.

Using the double whole-cell patch-clamp technique, we found that the absence of intracellular ATP led to gap junction uncoupling in cochlear-supporting Hensen cells. The uncoupling was observed as a progressive reduction of the gap junctional electrical conductance from a starting value of approximately 40nS to less than 0.04nS within 10-20min. The conductance rundown was partly avoided by at least 3mM ATP and completely suppressed by 5mM ATP or 5'-adenylyl-imidodiphosphate (AMP-PNP), the non-hydrolysable ATP analog, in the pipette filling solution, suggesting that ATP was needed as ligand and not as a hydrolysable energy supplier or substrate for enzymatic reactions. The effect of intracellular ATP was mimicked by the external application of barium, a nonselective blocker of inwardly rectifying K(+) (Kir) channels, and glibenclamide, an inhibitor of the ATP-sensitive Kir channels (KATP). Moreover a Ba(2+)-sensitive whole-cell inward current was observed in absence of internal ATP. We propose that the internal ATP kept the KATP channels in a closed state, thereby maintaining the gap junction coupling of Hensen cells. The immunostaining of guinea pig cochlear tissue revealed for the first time the expression of the KATP channel subunits Kir6.1 and SUR1 in Hensen cells and supported the proposed hypothesis. The results suggest that KATP channels, as regulator of the gap junction coupling in Hensen cells, could be the physiological link between the metabolic state of the supporting cells and K(+) recycling in the organ of Corti.

Chitosan-clodronate nanoparticles loaded in poloxamer gel for intra-articular administration

This work was based on the study of an intra-articular delivery system constituted by a poloxamer gel vehiculating clodronate in chitosan nanoparticles. This system has been conceived to obtain a specific and controlled release of clodronate in the joints to reduce the arthritis rheumatoid degenerative effect. Clodronate (CLO) is a first-generation bisphosphonate with anti-inflammatory properties, inhibiting the cytokine and NO secretion from macrophages, therefore causing apoptosis in these cells. This is related to its ability to be metabolized by cells and converted into a cytotoxic intermediate as a non-hydrolysable analogue of ATP. Chitosan (CHI) was used to develop nanosystems, by ionotropic gelation induced by clodronate itself. A fractional factorial experimental design allowed us to obtain nanoparticles, the diameter of which ranged from 200 to 300nm. Glutaraldehyde was used to increase nanoparticle stability and modify the drug release profile. The zeta potential value of crosslinked nanopaparticles was 21.0mV±1.3, while drug loading was 31.0%±5.4 w/w; nanoparticle yield was 18.2%±1.8 w/w, the encapsulation efficiency was 48.8%±9.9 w/w. Nanoparticles were homogenously loaded in a poloxamer sol, and the drug delivery system is produced in-situ after local administration, when sol become gel at physiological temperature. The properties of poloxamer gels containing CHI-CLO nanoparticles, such as viscosity, gelation temperature and drug release properties, were evaluated. In vitro studies were conducted to evaluate the effects of these nanoparticles on a human monocytic cell line (THP1). The results showed that this drug delivery system is more efficient, with respect to the free drug, to counteract the inflammatory process characteristic of several degenerative diseases.

Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A

The canonical function of kinases is to transfer a phosphoryl group to substrates, initiating a signaling cascade; while their non-canonical role is to bind other kinases or substrates, acting as scaffolds, competitors, and signal integrators. Here, we show how to uncouple kinases' dual function by tuning the binding cooperativity between nucleotide (or inhibitors) and substrate allosterically. We demonstrate this new concept for the C subunit of protein kinase A (PKA-C). Using thermocalorimetry and nuclear magnetic resonance, we found a linear correlation between the degree of cooperativity and the population of the closed state of PKA-C. The non-hydrolyzable ATP analog (ATPγC) does not follow this correlation, suggesting that changing the chemical groups around the phosphoester bond can uncouple kinases' dual function. Remarkably, this uncoupling was also found for two ATP-competitive inhibitors, H89 and balanol. Since the mechanism for allosteric cooperativity is not conserved in different kinases, these results may suggest new approaches for designing selective kinase inhibitors.

Functional expression of P-glycoprotein in the hepatic canalicular membrane of developing rats

□ P-glycoprotein (P-gp), the multidrug resistance gene product, is expressed in a normal liver exclusively on the canalicular membrane of the hepatocyte. The objective of this study was to examine the effect of age on the P-gp transport system using canalicular membrane (cLPM) vesicles isolated from the liver of developing (22 days old) and adult rats. No differences in protein yield, intravesicular volumes, and enrichments of cLPM enzymes or enzymes representing contamination of subcellular organelles were found for vesicles isolated from both groups, demonstrating the isolation of similar cLPM vesicle preparations. The transport of daunomycin (DNM), a P-gp substrate, was used to study age-related functional differences in P-gp. DNM uptake in the presence of ATP was greater than uptake in the absence of ATP in both young and adult cLPM vesicles, showing that P-gp is functional in both groups. In young and adult groups only ATP was a potent stimulator of transport when compared with ATP degradation products and a nonhydrolyzable ATP analogue. Although ATP-dependent uptake tended to be greater in the adult compared to the young, there was no statistically significant difference in DNM kinetics (Vmax, km, γ) between groups. Canalicular membrane from the young rats showed decreased fluidity, as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene; however there was no significant difference between groups. Examination of P-gp expression using the monoclonal antibody C219 revealed similar levels of expression in the young as in the adult. Our results suggest that P-gp in the bile canaliculus of developing rats is functional with similar levels of function and expression as observed in the adult.

Cloning, expression, purification, crystallization and initial crystallographic analysis of FleN from Pseudomonas aeruginosa.

The assembly of bacterial flagella requires the coordinated expression of a large number of genes in a hierarchical manner. These genes code for structural components of flagella, regulatory components and components that are required for chemotaxis. Stringent spatial and numerical control of flagella biosynthesis is essential for promoting motility and pathogenesis in bacteria. These genes are regulated at the level of transcription. FleN, a P-loop-containing ATPase, plays an important role in maintaining flagellar number in Pseudomonas aeruginosa. FleN exhibits anti-activator activity against FleQ, the global transcriptional regulator of flagellar genes. In order to gain insights into the regulatory mechanism of flagella synthesis, full-length FleN was crystallized in complex with the nonhydrolyzable ATP analogue β,γ-imidoadenosine 5'-triphosphate (AMPPNP) in space group C2221, with unit-cell parameters a = 49.1, b = 206.9, c = 103.3 Å. The Matthews coefficient is 2.19 Å(3) Da(-1) assuming the presence of two molecules in the asymmetric unit, and the corresponding solvent content is 43.7%. X-ray diffraction data were collected to a minimum Bragg spacing of 2.21 Å and crystals of FleN-AMPPNP were prepared with selenomethionine-labelled FleN for ab initio phasing.

The new antihypertensive drug iptakalim activates ATP-sensitive potassium channels in the endothelium of resistance blood vessels.

To investigate the mechanisms underlying the activation of ATP-sensitive potassium channels (K(ATP)) by iptakalim in cultured rat mesenteric microvascular endothelial cells (MVECs). Whole-cell KATP currents were recorded in MVECs using automated patch clamp devices. Nucleotides (ATP, ADP and UDP) were added to the internal perfusion system, whereas other drugs were added to the cell suspension on NPC-1 borosilicate glass chips. Application of iptakalim (10 and 100 μmol/L) significantly increased the whole-cell K(ATP) currents, which were prevented by the specific K(ATP) blocker glibenclamide (1.0 μmol/L). The opening of K(ATP) channels by iptakalim depended upon the intracellular concentrations of ATP or NDPs: iptakalim activated K(ATP) channels when the intracellular ATP or NDPs were at 100 or 1000 μmol/L, and was ineffective when the non-hydrolysable ATP analogue ATPγS (1000 μmol/L) was infused into the cells. In contrast, the K(ATP) opener pinacidil activated K(ATP) channels when the intracellular concentrations of ATP or NDPs ranged from 10 to 5000 μmol/L, and even ATPγS (1000 μmol/L) was infused into the cells. Iptakalim activates K(ATP) channels in the endothelial cells of resistance blood vessels with a low metabolic status, and this activation is dependent on both ATP hydrolysis and ATP ligands.

Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase.

Would calcium or potassium channels be responsible for cardiac arrest produced by adenosine and ATP in the right atria of Wistar rats?

Autonomic nerves release ATP, which is processed into adenosine in the synaptic cleft. Adenosine and ATP exert a negative chronotropic effect in the heart. This study aims to evaluate adenosine and P2 receptors and cellular signalling in cardiac arrest produced by purines in the heart. Right atria of adult Wistar rats were used to evaluate the effects of adenosine, ATP and CPA (an adenosine A1 receptor agonist), in the presence and absence of DPCPX, an adenosine A1 receptor antagonist. Effects of adenosine A2 and A3 receptors agonists and antagonists were also investigated. Finally, involvement of calcium and potassium channels in these responses was assessed using BayK 8644 and 4-Aminopyridine. Cumulative concentration–effect curves of adenosine and CPA resulted in a negative chronotropic effect culminating in cardiac arrest at 1000μM (adenosine) and 1µM (CPA). Furthermore, ATP produced a negative chronotropic effect at 1–300µM and cardiac arrest at 1000μM in the right atrium. ATPγS (a non-hydrolysable analogue of ATP) reduced chronotropism only. The effects of adenosine, CPA and ATP were inhibited by DPCPX, a selective adenosine A1 receptor antagonist. The selective adenosine A2 and A3 receptors antagonists did not alter the chronotropic response of adenosine. 4-Aminopyridine, a blocker of potassium channels at 10mM, prevented the cardiac arrest produced by adenosine and ATP, while BayK 8644, activator of calcium channels, did not prevent cardiac arrest. Adenosine A1 receptor activation by adenosine and ATP produces cardiac arrest in the right atrium of Wistar rats predominantly through activation of potassium channels.

Conformational Transitions that Enable Histidine Kinase Autophosphorylation and Receptor Array Integration

During bacterial chemotaxis, transmembrane chemoreceptor arrays regulate autophosphorylation of the dimeric histidine kinase CheA. The five domains of CheA (P1–P5) each play a specific role in coupling receptor stimulation to CheA activity. Biochemical and X-ray scattering studies of thermostable CheA from Thermotoga maritima determine that the His-containing substrate domain (P1) is sequestered by interactions that depend upon P1 of the adjacent subunit. Non-hydrolyzable ATP analogs (but not ATP or ADP) release P1 from the protein core (domains P3P4P5) and increase its mobility. Detachment of both P1 domains or removal of one within a dimer increases net autophosphorylation substantially at physiological temperature (55°C). However, nearly all activity is lost without the dimerization domain (P3). The linker length between P1 and P3 dictates intersubunit (trans) versus intrasubunit (cis) autophosphorylation, with the trans reaction requiring a minimum length of 47 residues. A new crystal structure of the most active dimerization-plus-kinase unit (P3P4) reveals trans directing interactions between the tether connecting P3 to P2–P1 and the adjacent ATP-binding (P4) domain. The orientation of P4 relative to P3 in the P3P4 structure supports a planar CheA conformation that is required by membrane array models, and it suggests that the ATP lid of CheA may be poised to interact with receptors and coupling proteins. Collectively, these data suggest that the P1 domains are restrained in the off-state as a result of cross-subunit interactions. Perturbations at the nucleotide-binding pocket increase P1 mobility and access of the substrate His to P4-bound ATP.

Structural and Biochemical Studies on the Reaction Mechanism of Uridine–Cytidine Kinase

Uridine-cytidine kinase catalyzes phosphorylation of the pyrimidine nucleosides uridine and cytidine and plays an important role in nucleotide metabolism. However, the detailed molecular mechanism of these reactions remains to be elucidated. Here, we determined the structure of the ternary complex of Uridine-cytidine kinase from Thermus thermophilus HB8 with both cytidine and β,γ-methyleneadenosine 5'-triphosphate, a non-hydrolysable ATP analogue. Substrate binding is accompanied by substantial domain movement that allows the substrate-binding cleft to close. The terminal phosphodiester bond of the ATP analogue is in an ideal location for an inline attack of the 5'-hydroxyl group of cytidine. Asp40 is located near the 5'-hydroxyl group of cytidine. Mutation of this conserved residue to Asn or Ala resulted in a complete loss of enzyme activity, which is consistent with the notion that Asp40 acts as a general base that activates the 5'-hydroxyl group of cytidine. The pH profile of the activity showed an apparent pK a value of 7.4. Based on this structure, a likely mechanism of the catalytic step is discussed.

Two-subunit DNA escort mechanism and inactive subunit bypass in an ultra-fast ring ATPase.

SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in Bacillus subtilis. Here, we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base pair, we deduce that SpoIIIE's step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism that is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such a flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell.

Recapitulating the Structural Evolution of Redox Regulation in Adenosine 5′-Phosphosulfate Kinase from Cyanobacteria to Plants

In plants, adenosine 5'-phosphosulfate (APS) kinase (APSK) is required for reproductive viability and the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfur donor in specialized metabolism. Previous studies of the APSK from Arabidopsis thaliana (AtAPSK) identified a regulatory disulfide bond formed between the N-terminal domain (NTD) and a cysteine on the core scaffold. This thiol switch is unique to mosses, gymnosperms, and angiosperms. To understand the structural evolution of redox control of APSK, we investigated the redox-insensitive APSK from the cyanobacterium Synechocystis sp. PCC 6803 (SynAPSK). Crystallographic analysis of SynAPSK in complex with either APS and a non-hydrolyzable ATP analog or APS and sulfate revealed the overall structure of the enzyme, which lacks the NTD found in homologs from mosses and plants. A series of engineered SynAPSK variants reconstructed the structural evolution of the plant APSK. Biochemical analyses of SynAPSK, SynAPSK H23C mutant, SynAPSK fused to the AtAPSK NTD, and the fusion protein with the H23C mutation showed that the addition of the NTD and cysteines recapitulated thiol-based regulation. These results reveal the molecular basis for structural changes leading to the evolution of redox control of APSK in the green lineage from cyanobacteria to plants.