Absence Of ATP Research Articles

A recombinase paralog from the hyperthermophilic crenarchaeon Sulfolobus solfataricus enhances SsoRadA ssDNA binding and strand displacement

Homologous recombination (HR) is a major pathway for the repair of double-strand DNA breaks, a highly deleterious form of DNA damage. The main catalytic protein in HR is the essential RecA-family recombinase, which is conserved across all three domains of life. Eukaryotes and archaea encode varying numbers of proteins paralogous to their main recombinase. Although there is increasing evidence for the functions of some of these paralog proteins, overall their mechanism of action remains largely unclear. Here we present the first biochemical characterization of one of the paralog proteins, SsoRal3, from the crenarchaeaon Sulfolobus solfataricus. The SsoRal3 protein is a ssDNA-dependent ATPase that can catalyze strand invasion at both saturating and subsaturating concentrations. It can bind both ssDNA and dsDNA, but its binding preference is altered by the presence or absence of ATP. Addition of SsoRal3 to SsoRadA nucleoprotein filaments reduces total ATPase activity. Subsaturating concentrations of SsoRal3 increase the ssDNA binding activity of SsoRadA approximately 9-fold and also increase the persistence of SsoRadA catalyzed strand invasion products. Overall, these results suggest that SsoRal3 functions to stabilize the SsoRadA presynaptic filament.

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6')-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6')-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k(cat)/K(m) = 10(5)-10(7) M(-1) s(-1)). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.

Numerical simulation of nucleotide transport in the human thoracic aorta

Extracellular adenine nucleotides ATP and ADP on vascular endothelial cells may play a role in the localization of atherogenesis by regulating the release of nitric oxide from endothelial cells and modulating intracellular calcium levels. To quantitatively investigate the concentration distribution of nucleotides on the luminal surface of the human thoracic aorta, we numerically simulated the transport of nucleotides using an aorta model constructed based on MRI images and analyzed the effects of different factors on nucleotide transport, such as ATP release rate (SATP), pulsatile flow and the absence of ATP in the main blood stream. The numerical results revealed that the combined concentration of ATP and ADP (cw−ATP+ADP) on the aortic surface varied from place to place, being relatively low in disturbed flow regions. In addition, cw−ATP+ADP was significantly affected by SATP. For relatively slow SATP, such as the moderate sigmoidal release model, cw−ATP+ADP was very low in certain flow regions with low wall shear stress. However, for very rapid SATP, such as the rapid linear release model, cw−ATP+ADP was relatively high in these same regions. The results also demonstrated that for relatively slow SATP, pulsatile blood flow enhanced cw−ATP+ADP. However, for very rapid SATP, pulsatile blood flow would reduce cw−ATP+ADP. Moreover, the absence of ATP within the main blood stream would not influence the distribution of cw−ATP+ADP. In conclusion, the concentration distribution of nucleotides along the aortic wall was quite uneven and determined by both the ATP release rate and the blood flow pattern in the aorta.

Structure and Dynamics of the ATP-Bound Open Conformation of Hsp70 Chaperones

Central to the chaperone function of Hsp70s is the transition between open and closed conformations of their polypeptide substrate binding domain (SBD), which is regulated through an allosteric mechanism via ATP binding and hydrolysis in their nucleotide binding domain (NBD). Although the structure of the closed conformation of Hsp70s is well studied, the open conformation has remained elusive. Here, we report on the 2.4Å crystal structure of the ATP-bound open conformation of the Escherichia coli Hsp70 homolog DnaK. In the open DnaK structure, the β sheet and α-helical lid subdomains of the SBD are detached from one another and docked to different faces of the NBD. The contacts between the β sheet subdomain and the NBD reveal the mechanism of allosteric regulation. In addition, we demonstrate that docking of the β sheet and α-helical lid subdomains to the NBD is a sequential process influenced by peptide and protein substrates.

Membrane Vesicle ABC Transporter Assays for Drug Safety Assessment

The use of plasma membrane vesicles that overexpress the bile salt export pump (BSEP) or multidrug resistance-associated protein 2, 3, or 4 (MRP2-4) with an in vitro vacuum filtration system offers a rapid and reliable means for screening drug candidates for their effects on transporter function in hepatocytes and thus their potential for causing drug-induced liver injury (DILI). Comparison of transporter activity in the presence and absence of ATP allows for determination of a specific assay window for each transporter. This window is used to determine the degree to which each test compound inhibits transporter activity. This assay battery is helpful for prioritizing and rank-ordering compounds within a chemical series with respect to each other and in the context of known inhibitors of transporter activity and/or liver injury. This model can be used to influence the drug development process at an early stage and provide rapid feedback regarding the selection of compounds for advancement to in vivo safety evaluations. A detailed protocol for the high-throughput assessment of ABC transporter function is provided, including specific recommendations for curve-fitting to help ensure consistent results.

Covalent Modification of Mutant Rat P2X2 Receptors with a Thiol-Reactive Fluorophore Allows Channel Activation by Zinc or Acidic pH without ATP

Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C5-maleimide (AM546). Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM) or acidic external solution (pH 6.5) elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.

A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

BackgroundDeep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available.ResultsWe demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum) using the T4 RNA ligase 1 adenylated adapter.ConclusionWe provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.

Studying the interaction between gyrase and DNA using magnetic tweezers

The DNA gyrase of Escherichia coli plays an essential role in the life of this microorganism. It is unique among all topoisomerases because of its ability to introduce negative supercoils into DNA. This study investigated the single molecular interaction of E. coli gyrase with DNA using magnetic tweezers. The results showed that, in the absence of ATP, gyrase weakly binds the G and T segments. The stretched force of 0.7 pN can gradually destroy the binding, whereas that of 5.9 pN directly destroys it. Addition of high concentrations of norfloxacin enhances gyrase binding to both segments, making them adapt to 5.9 pN. DNA gyrase reduces the plectonemic dimension, which was determined by the bacterial enzyme and not by the pull force. Moreover, it has different affinities for positive supercoils, which it prefers, and negative supercoils. The time distribution of the dissociation of gyrase from DNA has a double-exponential form. We herein propose a model to explain this distribution and compare the results with those of other models.

Inhibition of the intracellular Ca2+ transporter SERCA (Sarco-Endoplasmic Reticulum Ca2+-ATPase) by the natural polyphenol epigallocatechin-3-gallate

The use of a microsomal preparation from skeletal muscle revealed that both Ca(2+) transport and Ca(2+)-dependent ATP hydrolysis linked to Sarco-Endoplasmic Reticulum Ca(2+)-ATPase are inhibited by epigallocatechin-3-gallate (EGCG). A half-maximal effect was achieved at approx. 12μM. The presence of the galloyl group was essential for the inhibitory effect of the catechin. The relative inhibition of the Ca(2+)-ATPase activity decreased when the Ca(2+) concentration was raised but not when the ATP concentration was elevated. Data on the catalytic cycle indicated inhibition of maximal Ca(2+) binding and a decrease in Ca(2+) binding affinity when measured in the absence of ATP. Moreover, the addition of ATP to samples in the presence of EGCG and Ca(2+) led to an early increase in phosphoenzyme followed by a time-dependent decay that was faster when the drug concentration was raised. However, phosphorylation following the addition of ATP plus Ca(2+) led to a slow rate of phosphoenzyme accumulation that was also dependent on EGCG concentration. The results are consistent with retention of the transporter conformation in the Ca(2+)-free state, thus impeding Ca(2+) binding and therefore the subsequent steps when ATP is added to trigger the Ca(2+) transport process. Furthermore, phosphorylation by inorganic phosphate in the absence of Ca(2+) was partially inhibited by EGCG, suggesting alteration of the native Ca(2+)-free conformation at the catalytic site.

RAPADILINO RECQL4 mutant protein lacks helicase and ATPase activity

The RecQ family of helicases has been shown to play an important role in maintaining genomic stability. In humans, this family has five members and mutations in three of these helicases, BLM, WRN and RECQL4, are associated with disease. Alterations in RECQL4 are associated with three diseases, Rothmund–Thomson syndrome, Baller–Gerold syndrome, and RAPADILINO syndrome. One of the more common mutations found in RECQL4 is the RAPADILINO mutation, c.1390+2delT which is a splice-site mutation leading to an in-frame skipping of exon 7 resulting in 44 amino acids being deleted from the protein (p.Ala420–Ala463del). In order to characterize the RAPADILINO RECQL4 mutant protein, it was expressed in bacteria and purified using an established protocol. Strand annealing, helicase, and ATPase assays were conducted to characterize the protein's activities relative to WT RECQL4. Here we show that strand annealing activity in the absence of ATP is unchanged from that of WT RECQL4. However, the RAPADILINO protein variant lacks helicase and ssDNA-stimulated ATPase activity. These observations help explain the underlying molecular etiology of the disease and our findings provide insight into the genotype and phenotype association among RECQL4 syndromes.

Functional Characterization of the Role of the Chromosome I Partitioning System in Genome Segregation in Deinococcus radiodurans

Deinococcus radiodurans, a radiation-resistant bacterium, harbors a multipartite genome. Chromosome I contains three putative centromeres (segS1, segS2, and segS3), and ParA (ParA1) and ParB (ParB1) homologues. The ParB1 interaction with segS was sequence specific, and ParA1 was shown to be a DNA binding ATPase. The ATPase activity of ParA1 was stimulated when segS elements were coincubated with ParB1, but the greatest increase was observed with segS3. ParA1 incubated with the segS-ParB1 complex showed increased light scattering in the absence of ATP. In the presence of ATP, this increase was continued with segS1-ParA1B1 and segS2-ParA1B1 complexes, while it decreased rapidly after an initial increase for 30 min in the case of segS3. D. radiodurans cells expressing green fluorescent protein (GFP)-ParB1 produced foci on nucleoids, and the ΔparB1 mutant showed growth retardation and ∼13%-higher anucleation than the wild type. Unstable mini-F plasmids carrying segS1 and segS2 showed inheritance in Escherichia coli without ParA1B1, while segS3-mediated plasmid stability required the in trans expression of ParA1B1. Unlike untransformed E. coli cells, cells harboring pDAGS3, a plasmid carrying segS3 and also expressing ParB1-GFP, produced discrete GFP foci on nucleoids. These findings suggested that both segS elements and the ParA1B1 proteins of D. radiodurans are functionally active and have a role in genome segregation.

ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote

The heterohexameric minichromosome maintenance (MCM2-7) complex is an ATPase that serves as the central replicative helicase in eukaryotes. During initiation, the ring-shaped MCM2-7 particle is thought to open to facilitate loading onto DNA. The conformational state accessed during ring opening, the interplay between ATP binding and MCM2-7 architecture, and the use of these events in the regulation of DNA unwinding are poorly understood. To address these issues in isolation from the regulatory complexity of existing eukaryotic model systems, we investigated the structure/function relationships of a naturally minimized MCM2-7 complex from the microsporidian parasite Encephalitozoon cuniculi. Electron microscopy and small-angle X-ray scattering studies show that, in the absence of ATP, MCM2-7 spontaneously adopts a left-handed, open-ring structure. Nucleotide binding does not promote ring closure but does cause the particle to constrict in a two-step process that correlates with the filling of high- and low-affinity ATPase sites. Our findings support the idea that an open ring forms the default conformational state of the isolated MCM2-7 complex, and they provide a structural framework for understanding the multiphasic ATPase kinetics observed in different MCM2-7 systems.

A potentiator induces conformational changes on the recombinant CFTR nucleotide binding domains in solution.

Nucleotide binding domains (NBD1 and NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, are responsible for controlling the gating of the chloride channel and are the putative binding sites for several candidate drugs in the disease treatment. We studied the effects of the application of 2-pyrimidin-7,8-benzoflavone (PBF), a strong potentiator of the CFTR, on the properties of recombinant and equimolar NBD1/NBD2 mixture in solution. The results indicate that the potentiator induces significant conformational changes of the NBD1/NBD2 dimer in solution. The potentiator does not modify the ATP binding constant, but reduces the ATP hydrolysis activity of the NBD1/NBD2 mixture. The intrinsic fluorescence and the guanidinium denaturation measurements indicate that the potentiator induces different conformational changes on the NBD1/NBD2 mixture in the presence and absence of ATP. It was confirmed from small-angle X-ray scattering experiments that, in absence of ATP, the NBD1/NBD2 dimer was disrupted by the potentiator, but in the presence of 2 mM ATP, the two NBDs kept dimerised, and a major change in the size and the shape of the structure was observed. We propose that these conformational changes could modify the NBDs-intracellular loop interaction in a way that would facilitate the open state of the channel.

The Intra-S Phase Checkpoint Targets Dna2 to Prevent Stalled Replication Forks from Reversing

When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ∼10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.

Specific Domains in Yeast Translation Initiation Factor eIF4G Strongly Bias RNA Unwinding Activity of the eIF4F Complex toward Duplexes with 5′-Overhangs

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex is recruited to the 5'-end of an mRNA through its interaction with the 7-methylguanosine cap, and it subsequently scans along the mRNA to locate the start codon. Both mRNA recruitment and scanning require the removal of secondary structure within the mRNA. Eukaryotic translation initiation factor 4A is an essential component of the translational machinery thought to participate in the clearing of secondary structural elements in the 5'-untranslated regions of mRNAs. eIF4A is part of the 5'-7-methylguanosine cap-binding complex, eIF4F, along with eIF4E, the cap-binding protein, and the scaffolding protein eIF4G. Here, we show that Saccharomyces cerevisiae eIF4F has a strong preference for unwinding an RNA duplex with a single-stranded 5'-overhang versus the same duplex with a 3'-overhang or without an overhang. In contrast, eIF4A on its own has little RNA substrate specificity. Using a series of deletion constructs of eIF4G, we demonstrate that its three previously elucidated RNA binding domains work together to provide eIF4F with its 5'-end specificity, both by promoting unwinding of substrates with 5'-overhangs and inhibiting unwinding of substrates with 3'-overhangs. Our data suggest that the RNA binding domains of eIF4G provide the S. cerevisiae eIF4F complex with a second mechanism, in addition to the eIF4E-cap interaction, for directing the binding of pre-initiation complexes to the 5'-ends of mRNAs and for biasing scanning in the 5' to 3' direction.

Internucleosomal Interactions Mediated by Histone Tails Allow Distant Communication in Chromatin

Action across long distances on chromatin is a hallmark of eukaryotic transcriptional regulation. Although chromatin structure per se can support long-range interactions, the mechanisms of efficient communication between widely spaced DNA modules in chromatin remain a mystery. The molecular simulations described herein suggest that transient binary internucleosomal interactions can mediate distant communication in chromatin. Electrostatic interactions between the N-terminal tails of the core histones and DNA enhance the computed probability of juxtaposition of sites that lie far apart along the DNA sequence. Experimental analysis of the rates of communication in chromatin constructs confirms that long-distance communication occurs efficiently and independently of distance on tail-containing, but not on tailless, chromatin. Taken together, our data suggest that internucleosomal interactions involving the histone tails are essential for highly efficient, long-range communication between regulatory elements and their targets in eukaryotic genomes.

Inhibition of interdomain motion in g‐actin by the natural product latrunculin: A molecular dynamics study

As part of the cytoskeleton, actin is essential for the morphology, motility, and division of eukaryotic cells. Recent X-ray fiber diffraction studies have shown that the conformation of monomeric actin is flattened upon incorporation into the filament by a relative rotation of its two major domains. The antiproliferative activity of latrunculin, a macrolide toxin produced by sponges, seems to be related to its binding to monomeric actin and inhibition of polymerization. Yet, the mechanism of inhibition is not known in detail. Here, multiple explicit water molecular dynamics simulations show that latrunculin binding hinders the conformational transition related to actin polymerization. In particular, the presence of latrunculin at the interface of the two major domains of monomeric actin reduces the correlated displacement of Domain 2 with respect to Domain 1. Moreover, higher rotational flexibility between the two major domains is observed in the absence of ATP as compared to ATP-bound actin, offering a possible explanation as to why actin polymerizes more favorably in the absence of nucleotides.

E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2

This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb+ deocclusion stimulated by Na+ in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb+ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb+ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb+]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb+-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na+-stimulated Rb+ deocclusion would require the release of at least one Rb+ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb+ in equilibrium conditions. Results show that, while Mg2+ decreases the affinity for Rb+, addition of vanadate offsets this effect, increasing the affinity for Rb+. In transient experiments, we investigated the exchange of Rb+ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na+ without significantly affecting the rate of Rb+ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb+ occlusion in the enzyme–vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb+.

A new role for the P2X7 receptor: a scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP

The P2X7 receptor is widely recognized to mediate the proinflammatory effects of extracellular ATP. However this receptor in the absence of ATP may have a function unrelated to inflammation. Our data show that P2X7 expressed on the surface of monocyte/macrophages or on epithelial HEK-293 cells greatly augments the engulfment of latex beads and live and heat-killed bacteria by effector phagocyte in the absence of ATP and serum. The expression of P2X7 on the effector also confers the ability to phagocytose apoptotic target cells and an accumulation of P2X7 can be seen at the attachment point to the target. Activation of the P2X7 receptor by ATP causes a slow dissociation (over 10-15min) of nonmuscle myosin from the P2X7 membrane complex and abolishes further P2X7-mediated phagocytosis of these targets. The recent crystal structure of the homologous zebrafish P2X4 receptor shows an exposed "nose" of the ectodomain (residues 115-162) which contains three of the five disulfide bonds conserved in all P2X receptors. Three short biotin-labeled peptides mimicking sequence of this exposed region bound to apoptotic target cells but not to either viable cells or to other target particles. All three peptides contained one or two cysteine residues and their replacement by alanine abolished peptide binding. These data implicate thiol-disulfide exchange reactions in the initial tethering of apoptotic cells to macrophage and establish P2X7 as one of the scavenger receptors involved in the recognition and removal of apoptotic cells in the absence of extracellular ATP and serum.

ATP alters IKK‐2 inhibitor affinity for IKK2 enzyme and reveals non‐equivalent inhibitor binding sites

MethodInhibitor off‐rate was measured by two methods: (1) pre‐binding the enzyme with inhibitor followed by measuring the recovery of enzyme activity upon a 200‐fold dilution of the enzyme‐inhibitor complex in the presence of 1mM ATP and (2) measuring the shift in the compound potency (IC50) curves obtained by prebinding the enzyme and inhibitor in the absence of ATP followed by addition of either 1μM or 1mM ATP.ResultsThree literature inhibitors with potency (IC50) ranging from <1 to 35 nM were used for this study: PHA‐408 (Pfizer), GSK‐082 (GSK), and AVE‐002 (Aventis). The off‐rates (T1/2) measured by the dilution method are 124 and <15 min for PHA‐408 and GSK‐082, respectively. The off‐rate for AVE‐002 was biphasic with half of the enzyme‐inhibitor complex dissociating immediately (T1/2 < 2 min) and the other half bound tightly (T1/2 54 min). The IC50 curves at 1μM and 1mM ATP for PHA‐408 showed no shift using a short 2 min assay and is consistent with the slow off‐rate. GSK‐082 showed a > 10 fold loss of potency with 1mM ATP, as expected for a fast off‐rate inhibitor. AVE‐002 IC50 curve with 1μM ATP was monophasic but displayed a biphasic response with 1mM ATP.ConclusionATP can change the off‐rate of IKK‐2 inhibitor bound to the enzyme. Additionally, high ATP revealed that AVE‐002 bound in the two inhibitor binding sites of the IKK‐2 homodimer non‐equivalently.