ATP Analog Research Articles

Structural and Functional Characterization of Plasmodium falciparum Nicotinic Acid Mononucleotide Adenylyltransferase

Nicotinic acid mononucleotide adenylyltransferase (NaMNAT) is an indispensable enzyme for the synthesis of NAD and NAD phosphate. It catalyzes the adenylylation of nicotinic acid mononucleotide (NaMN) to yield nicotinic acid adenine dinucleotide (NaAD). Since NAD(H) and NAD phosphate(H) are essentially involved in metabolic and redox regulatory reactions, NaMNAT is an attractive drug target in the fight against bacterial and parasitic infections. Notably, NaMNAT of the malaria parasite Plasmodium falciparum possesses only 20% sequence identity with the homologous human enzyme. Here, we present for the first time the two X-ray structures of P. falciparum NaMNAT (PfNaMNAT)—in the product-bound state with NaAD and complexed with an α,β-non-hydrolizable ATP analog—the structures were determined to a resolution of 2.2Å and 2.5Å, respectively. The overall architecture of PfNaMNAT was found to be more similar to its bacterial homologs than its human counterparts although the PPHK motif conserved in bacteria is missing. Furthermore, PfNaMNAT possesses two cysteine residues within the active site that have not been described for any other NaMNATase so far and are likely to be involved in redox regulation of PfNaMNAT activity. Enzymatic studies and surface plasmon resonance data reveal that PfNaMNAT is capable of utilizing NaMN and nicotinamide mononucleotide with a slight preference for NaMN. Surprisingly, a comparison with the active site of Escherichia coli NaMNAT showed very similar architectures, despite different substrate preferences.

2',3'-O-Substituted ATP derivatives as potent antagonists of purinergic P2X3 receptors and potential analgesic agents.

Blocking membrane currents evoked by the activation of purinergic P2X3 receptors localized on nociceptive neurons represents a promising strategy for the development of agents useful for the treatment of chronic pain conditions. Among compounds endowed with such antagonistic action, 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) is an ATP analogue, whose inhibitory activity on P2X receptors has been previously reported. Based on the results of molecular modelling studies performed with homology models of the P2X3 receptor, novel adenosine nucleotide analogues bearing cycloalkyl or arylalkyl substituents replacing the trinitrophenyl moiety of TNP-ATP were designed and synthesized. These new compounds were functionally evaluated on native P2X3 receptors from mouse trigeminal ganglion (TG) sensory neurons using patch clamp recordings under voltage clamp configuration. Our data show that some of these molecules are potent (nanomolar range) and reversible inhibitors of P2X3 receptors, without any apparent effect on trigeminal GABAA and 5-HT3 receptors, whose membrane currents were unaffected by the tested compounds.

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.

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid-State NMR Spectroscopy.

DnaB helicases are bacterial, ATP-driven enzymes that unwind double-stranded DNA during DNA replication. Herein, we study the sequential binding of the "non-hydrolysable" ATP analogue AMP-PNP and of single-stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid-state NMR. Phosphorus cross-polarization experiments monitor the binding of AMP-PNP and DNA to the helicase. 13 C chemical-shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP-PNP addition into a conformation apt for ssDNA binding, and AMP-PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP-PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid-state NMR can provide for the characterization of protein-DNA interactions and the interplay with ATP or its analogues.

Nouveaux traitements : de l’autorisation de mise sur le marché aux bonnes pratiques

One major breakthrough in cystic fibrosis research in the past decade is the development of drugs that target the root cause of the disease — dysfunctional CFTR protein. One of the compounds, Ivacaftor or Kalydeco, which has been approved for clinical use since 2012, acts by promoting the gating function of CFTR. Our recent studies have led to a gating model that features energetic coupling between nucleotide-binding domain (NBD) dimerization and gate opening/closing in CFTR's transmembrane domains (TMDs). Based on this model, we showed that ATP analogs can enhance CFTR gating by facilitating NBD dimerization, whereas Ivacaftor works by stabilizing the open channel conformation of the TMDs. This latter idea also explains the near omnipotence of Ivacaftor. Furthermore, this model identifies multiple approaches to synergistically boost the open probability of CFTR by influencing distinct molecular events that control gating conformational changes.

Imatinib alters cell viability but not growth factors levels in TM4 Sertoli cells

The anticancer agent imatinib (IM) is a small molecular analog of ATP that inhibits tyrosine kinase activity of platelet derived growth factors (PDGFs) and stem cell factor (SCF) receptor in cancer cells. However these factors have a key role in regulating growth and development of normal Sertoli, Leydig and germ cells. The aim of this study was to determine cell viability, PDGF and SCF levels in mouse normal Sertoli cells exposed to IM. In this experimental study, the mouse TM4 Sertoli cells were treated with 0, 2.5, 5, 10 and 20 μM IM for 2, 4 or 6 days. The cell viability and growth factors levels were assessed by MTT and ELISA methods, respectively. For statistical analysis, One-Way ANOVA was performed. IM showed significant decrease in Sertoli cell viability compared to control group (p=0.001). However, IM increased PDGF and SCF level insignificantly (p>0.05). Results suggested that IM treatment induced a dose dependent reduction of cell viability in Sertoli cells. It seems that treatment with this anticancer drug is involved in the fertility process. Further studies are needed to evaluate the role of PDGF and SCF in this cell.

The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State

Single-molecule microscopy and stopped-flow kinetics assays were carried out to understand the microtubule polymerase activity of kinesin-5 (Eg5). Four lines of evidence argue that the motor primarily resides in a two-heads-bound (2HB) state. First, upon microtubule binding, dimeric Eg5 releases both bound ADPs. Second, microtubule dissociation in saturating ADP is 20-fold slower for the dimer than for the monomer. Third, ATP-triggered mant-ADP release is 5-fold faster than the stepping rate. Fourth, ATP binding is relatively fast when the motor is locked in a 2HB state. Shortening the neck-linker does not facilitate rear-head detachment, suggesting a minimal role for rear-head-gating. This 2HB state may enable Eg5 to stabilize incoming tubulin at the growing microtubule plus-end. The finding that slowly hydrolyzable ATP analogs trigger slower nucleotide release than ATP suggests that ATP hydrolysis in the bound head precedes stepping by the tethered head, leading to a mechanochemical cycle in which processivity is determined by the race between unbinding of the bound head and attachment of the tethered head.

ATP-sensitive muscle afferents activate spinal trigeminal neurons with meningeal afferent input in rat - pathophysiological implications for tension-type headache.

BackgroundTension-type headache and other primary headaches may be triggered or aggravated by disorders of pericranial muscles, which is possibly due to convergent or collateral afferent input from meningeal and muscular receptive areas. In rodent models high extracellular concentrations of ATP caused muscle nociception and central sensitization of second order neurons. In a rat model of meningeal nociception we asked if spinal trigeminal activity induced by ATP can be modulated by local anaesthesia of distinct muscles.MethodsOngoing activity was recorded from spinal trigeminal neurons with afferent input from the cranial dura mater, the temporal muscle and neck muscles. The stable ATP analogue α,β-methylene adenosine 5′-triphosphate (α,β-meATP, 10 mM) was injected into the ipsilateral temporal muscle, 30 min later followed by injection of local anaesthetics (lidocaine, 2 %) into the ipsilateral neck muscles and/or the temporal muscle.ResultsInjection of α,β-meATP into the temporal muscle caused progressive increase in ongoing activity of most of the spinal trigeminal neurons within 30 min. Injection of lidocaine into the neck muscles and/or the temporal muscle reduced this activation to previous levels within 10 min.ConclusionsDistinct spinal trigeminal neurons processing meningeal nociceptive information are under the control of convergent afferent input from several pericranial muscles. Blockade of at least one of these inputs can normalize central trigeminal activity. This may explain why therapeutic manipulations of head muscles can be beneficial in primary headaches.

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.

Adenine Nucleotides Control Proliferation In Vivo of Rat Retinal Progenitors by P2Y1 Receptor.

Previous studies demonstrated that exogenous ATP is able to regulate proliferation of retinal progenitor cells (RPCs) in vitro possibly via P2Y1 receptor, a G protein-coupled receptor. Here, we evaluated the function of adenine nucleotides in vivo during retinal development of newborn rats. Intravitreal injection of apyrase, an enzyme that hydrolyzes nucleotides, reduced cell proliferation in retinas at postnatal day 2 (P2). This decrease was reversed when retinas were treated together with ATPγ-S or ADPβ-S, two hydrolysis-resistant analogs of ATP and ADP, respectively. During early postnatal days (P0 to P5), an increase in ectonucleotidase (E-NTPDase) activity was observed in the retina, suggesting a decrease in the availability of adenine nucleotides, coinciding with the end of proliferation. Interestingly, intravitreal injection of the E-NTPDase inhibitor ARL67156 increased proliferation by around 60% at P5 rats. Furthermore, immunolabeling against P2Y1 receptor was observed overall in retina layers from P2 rats, including proliferating Ki-67-positive cells in the neuroblastic layer (NBL), suggesting that this receptor could be responsible for the action of adenine nucleotides upon proliferation of RPCs. Accordingly, intravitreal injection of MRS2179, a selective antagonist of P2Y1 receptors, reduced cell proliferation by approximately 20% in P2 rats. Moreover, treatment with MRS 2179 caused an increase in p57KIP2 and cyclin D1 expression, a reduction in cyclin E and Rb phosphorylated expression and in BrdU-positive cell number. These data suggest that the adenine nucleotides modulate the proliferation of rat RPCs via activation of P2Y1 receptors regulating transition from G1 to S phase of the cell cycle.

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.

ATPγS stalls splicing after B complex formation but prior to spliceosome activation.

The ATP analog ATPγS inhibits pre-mRNA splicing in vitro, but there have been conflicting reports as to which step of splicing is inhibited by this small molecule and its inhibitory mechanism remains unclear. Here we have dissected the effect of ATPγS on pre-mRNA splicing in vitro. Addition of ATPγS to splicing extracts depleted of ATP inhibited both catalytic steps of splicing. At ATPγS concentrations ≥0.5 mM, precatalytic B complexes accumulate, demonstrating a block prior to or during the spliceosome activation stage. Affinity purification of the ATPγS-stalled B complexes (BATPγS) and subsequent characterization of their abundant protein components by 2D gel electrophoresis revealed that BATPγS complexes are compositionally more homogeneous than B complexes previously isolated in the presence of ATP. In particular, they contain little or no Prp19/CDC5L complex proteins, indicating that these proteins are recruited after assembly of the precatalytic spliceosome. Under the electron microscope, BATPγS complexes exhibit a morphology highly similar to B complexes, indicating that the ATPγS-induced block in the transformation of the B to Bact complex is not due to a major structural defect. Likely mechanisms whereby ATPγS blocks spliceosome assembly at the activation stage, including inhibition of the RNA helicase Brr2, are discussed. Given their more homogeneous composition, B complexes stalled by ATPγS may prove highly useful for both functional and structural analyses of the precatalytic spliceosome and its conversion into an activated Bact spliceosomal complex.

DDK dependent regulation of TOP2A at centromeres revealed by a chemical genetics approach.

In eukaryotic cells the CDC7/DBF4 kinase, also known as DBF4-dependent kinase (DDK), is required for the firing of DNA replication origins. CDC7 is also involved in replication stress responses and its depletion sensitises cells to drugs that affect fork progression, including Topoisomerase 2 poisons. Although CDC7 is an important regulator of cell division, relatively few substrates and bona-fide CDC7 phosphorylation sites have been identified to date in human cells. In this study, we have generated an active recombinant CDC7/DBF4 kinase that can utilize bulky ATP analogues. By performing in vitro kinase assays using benzyl-thio-ATP, we have identified TOP2A as a primary CDC7 substrate in nuclear extracts, and serine 1213 and serine 1525 as in vitro phosphorylation sites. We show that CDC7/DBF4 and TOP2A interact in cells, that this interaction mainly occurs early in S-phase, and that it is compromised after treatment with CDC7 inhibitors. We further provide evidence that human DBF4 localises at centromeres, to which TOP2A is progressively recruited during S-phase. Importantly, we found that CDC7/DBF4 down-regulation, as well S1213A/S1525A TOP2A mutations can advance the timing of centromeric TOP2A recruitment in S-phase. Our results indicate that TOP2A is a novel DDK target and have important implications for centromere biology.

Resonance Raman spectroscopic study of the interaction between Co(II)rrinoids and the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri.

The human-type ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri (LrPduO) catalyzes the adenosylation of Co(II)rrinoids to generate adenosylcobalamin (AdoCbl) or adenosylcobinamide (AdoCbi(+)). This process requires the formation of "supernucleophilic" Co(I)rrinoid intermediates in the enzyme active site which are properly positioned to abstract the adeonsyl moiety from co-substrate ATP. Previous magnetic circular dichroism (MCD) spectroscopic and X-ray crystallographic analyses revealed that LrPduO achieves the thermodynamically challenging reduction of Co(II)rrinoids by displacing the axial ligand with a non-coordinating phenylalanine residue to produce a four-coordinate species. However, relatively little is currently known about the interaction between the tetradentate equatorial ligand of Co(II)rrinoids (the corrin ring) and the enzyme active site. To address this issue, we have collected resonance Raman (rR) data of Co(II)rrinoids free in solution and bound to the LrPduO active site. The relevant resonance-enhanced vibrational features of the free Co(II)rrinoids are assigned on the basis of rR intensity calculations using density functional theory to establish a suitable framework for interpreting rR spectral changes that occur upon Co(II)rrinoid binding to the LrPduO/ATP complex in terms of structural perturbations of the corrin ring. To complement our rR data, we have also obtained MCD spectra of Co(II)rrinoids bound to LrPduO complexed with the ATP analogue UTP. Collectively, our results provide compelling evidence that in the LrPduO active site, the corrin ring of Co(II)rrinoids is firmly locked in place by several amino acid side chains so as to facilitate the dissociation of the axial ligand.

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.

Molecular Mechanism of Mot1, a TATA-binding Protein (TBP)-DNA Dissociating Enzyme

The essential Saccharomyces cerevisiae ATPase Mot1 globally regulates transcription by impacting the genomic distribution and activity of the TATA-binding protein (TBP). In vitro, Mot1 forms a ternary complex with TBP and DNA and can use ATP hydrolysis to dissociate the TBP-DNA complex. Prior work suggested an interaction between the ATPase domain and a functionally important segment of DNA flanking the TATA sequence. However, how ATP hydrolysis facilitates removal of TBP from DNA is not well understood, and several models have been proposed. To gain insight into the Mot1 mechanism, we dissected the role of the flanking DNA segment by biochemical analysis of complexes formed using DNAs with short single-stranded gaps. In parallel, we used a DNA tethered cleavage approach to map regions of Mot1 in proximity to the DNA under different conditions. Our results define non-equivalent roles for bases within a broad segment of flanking DNA required for Mot1 action. Moreover, we present biochemical evidence for two distinct conformations of the Mot1 ATPase, the detection of which can be modulated by ATP analogs as well as DNA sequence flanking the TATA sequence. We also show using purified complexes that Mot1 dissociation of a stable, high affinity TBP-DNA interaction is surprisingly inefficient, suggesting how other transcription factors that bind to TBP may compete with Mot1. Taken together, these results suggest that TBP-DNA affinity as well as other aspects of promoter sequence influence Mot1 function in vivo.

The α-thio and/or β-γ-hypophosphate analogs of ATP as cofactors of T4 DNA ligase

T4 DNA ligase is one of the most commonly used enzymes for in vitro molecular research and a useful model for testing the ligation mechanism of ATP-dependent DNA ligation. To better understand the influence of phosphate group modifications in the ligation process, a series of ATP analogs were tested as cofactors. P-diastereomers of newly developed β,γ-hypo-ATPαS (thio) and β,γ-hypo-ATP (oxo) were synthesized and their activity was compared to ATPαS and their natural precursors. The evaluation of presented ATP analogs revealed the importance of the α-phosphate stereogenic center in ATPαS for the T4 DNA ligase activity and sheds new light on the interaction between ATP-dependent DNA ligases and cofactors.

Purification, crystallization, and preliminary X-ray crystallographic analysis of the Group III chaperonin from Carboxydothermus hydrogenoformans.

Chaperonins (CPNs) are megadalton sized ATP-dependent nanomachines that facilitate protein folding through complex cycles of complex allosteric articulation. They consist of two back-to-back stacked multisubunit rings. CPNs are usually classified into Group I and Group II. Here, we report the crystallization of both the AMPPNP (an ATP analogue) and ADP bound forms of a novel CPN, classified as belonging to a third Group, recently discovered in the extreme thermophile Carboxydothermus hydrogenoformans. Crystals of the two forms were grown by the vapor batch crystallization method at 295 K. Crystals of the Ch-CPN/AMPPNP complex diffracted to 3.0 Å resolution and belonged to the space group P422, with unit-cell parameters a = b = 186.166, c = 160.742 Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 60.02%. Crystals of the Ch-CPN/ADP complex diffracted to 4.0 Å resolution and belonged to the space group P4212, with unit-cell parameters a = b = 209.780, c = 169.813Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 70.19%.