Tracking the ATP-binding response in adenylate kinase in real time.
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ABSTRACT:
SUBMITTER: Oradd F
PROVIDER: S-EPMC8597995 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure-function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.
| S-EPMC5866598 | biostudies-literature
Project description:Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P(1),P(5)-di(adenosine-5') pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.
| S-EPMC3779764 | biostudies-literature
Project description:Adenylate kinase 4 (AK4) is a unique member with no enzymatic activity in vitro in the adenylate kinase (AK) family although it shares high sequence homology with other AKs. It remains unclear what physiological function AK4 might play or why it is enzymatically inactive. In this study, we showed increased AK4 protein levels in cultured cells exposed to hypoxia and in an animal model of the neurodegenerative disease amyotrophic lateral sclerosis. We also showed that short hairpin RNA (shRNA)-mediated knockdown of AK4 in HEK293 cells with high levels of endogenous AK4 resulted in reduced cell proliferation and increased cell death. Furthermore, we found that AK4 over-expression in the neuronal cell line SH-SY5Y with low endogenous levels of AK4 protected cells from H(2)O(2) induced cell death. Proteomic studies revealed that the mitochondrial ADP/ATP translocases (ANTs) interacted with AK4 and higher amount of ANT was co-precipitated with AK4 when cells were exposed to H(2)O(2) treatment. In addition, structural analysis revealed that, while AK4 retains the capability of binding nucleotides, AK4 has a glutamine residue instead of a key arginine residue in the active site well conserved in other AKs. Mutation of the glutamine residue to arginine (Q159R) restored the adenylate kinase activity with GTP as substrate. Collectively, these results indicate that the enzymatically inactive AK4 is a stress responsive protein critical to cell survival and proliferation. It is likely that the interaction with the mitochondrial inner membrane protein ANT is important for AK4 to exert the protective benefits to cells under stress.
| S-EPMC2676352 | biostudies-literature
Project description:The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg2+ was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount.
| S-EPMC6400584 | biostudies-literature
Project description:The complex interplay of dynamic protein plasticity and specific side-chain interactions with substrate molecules that allows enzymes to catalyze reactions has yet to be fully unraveled. Top-down ultraviolet photodissociation (UVPD) mass spectrometry is used to track snapshots of conformational fluctuations in the phosphotransferase adenylate kinase (AK) throughout its active reaction cycle by characterization of complexes containing AK and each of four different adenosine phosphate ligands. Variations in efficiencies of UVPD backbone cleavages were consistently observed for three ?-helices and the adenosine binding regions for AK complexes representing different steps of the catalytic cycle, implying that these stretches of the protein sample various structural microstates as the enzyme undergoes global open-to-closed transitions. Focusing on the conformational impact of recruiting or releasing the Mg2+ cofactor highlights two loop regions for which fragmentation increases upon UVPD, signaling an increase in loop flexibility as the metal cation disrupts the loop interactions with the substrate ligands. Additionally, the observation of holo ions and variations in UVPD backbone cleavage efficiency at R138 implicate this conserved active site residue in stabilizing the donor phosphoryl group during catalysis. This study showcases the utility of UVPD-MS to provide insight into conformational fluctuations of single residues for active enzymes.
| S-EPMC5750083 | biostudies-literature
Project description:Fiber tracking is a technique that, based on a diffusion tensor magnetic resonance imaging dataset, locates the fiber bundles in the human brain. Because it is a computationally expensive process, the interactivity of current fiber tracking tools is limited. We propose a new approach, which we termed real-time interactive fiber tracking, which aims at providing a rich and intuitive environment for the neuroradiologist. In this approach, fiber tracking is executed automatically every time the user acts upon the application. Particularly, when the volume of interest from which fiber trajectories are calculated is moved on the screen, fiber tracking is executed, even while it is being moved. We present our fiber tracking tool, which implements the real-time fiber tracking concept by using the video card's graphics processing units to execute the fiber tracking algorithm. Results show that real-time interactive fiber tracking is feasible on computers equipped with common, low-cost video cards.
| S-EPMC3056974 | biostudies-literature
Project description:During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.
| S-EPMC7289862 | biostudies-literature
Project description:The enzyme adenylate kinase (ADK) features two substrate binding domains that undergo large-scale motions during catalysis. In the apo state, the enzyme preferentially adopts a globally open state with accessible binding sites. Binding of two substrate molecules (AMP + ATP or ADP + ADP) results in a closed domain conformation, allowing efficient phosphoryl-transfer catalysis. We employed molecular dynamics simulations to systematically investigate how the individual domain motions are modulated by the binding of substrates. Two-dimensional free-energy landscapes were calculated along the opening of the two flexible lid domains for apo and holo ADK as well as for all single natural substrates bound to one of the two binding sites of ADK. The simulations reveal a strong dependence of the conformational ensembles on type and binding position of the bound substrates and a nonsymmetric behavior of the lid domains. Altogether, the ensembles suggest that, upon initial substrate binding to the corresponding lid site, the opposing lid is maintained open and accessible for subsequent substrate binding. In contrast, ATP binding to the AMP-lid induces global domain closing, preventing further substrate binding to the ATP-lid site. This might constitute a mechanism by which the enzyme avoids the formation of a stable but enzymatically unproductive state.
| S-EPMC4643206 | biostudies-literature
Project description:BackgroundIt has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 μM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP) as a molecular probe with site directed mutagenesis (SDM) of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings.ResultsWe have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK) and adenylate kinase 1 (AK1), are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP.ConclusionsThe data outlined serves to demonstrate the "push" mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It is therefore conceivable that kinase enzymes achieve the observed 2,500-fold variation in KM through a combination of the various conserved "push" and "pull" mechanisms associated with the release of C8-H, the proton transfer cascades unique to the class of kinase in question and the resultant/concomitant creation of a pentavalent species from the γ-phosphate group of ATP. Also demonstrated is the interplay between the role of the C8-H of ATP and the ATP concentration in the observed enzyme activity. The lability of the C8-H mediated by active site residues co-ordinated to the purine ring of ATP therefore plays a significant role in explaining the broad KM range associated with kinase steady state enzyme activities.
| S-EPMC3537612 | biostudies-literature
Project description:The drug Rimantadine binds to two different sites in the M2 protein from influenza A, a peripheral site and a pore site that is the primary site of efficacy. It remained enigmatic that pore binding did not occur in certain detergent micelles, and in particular incomplete binding was observed in a mixture of lipids selected to match the viral membrane. Here we show that two effects are responsible, namely changes in the protein upon pore binding that prevented detergent solubilization, and slow binding kinetics in the lipid samples. Using 55-100 kHz magic-angle spinning NMR, we characterize kinetics of drug binding in three different lipid environments: DPhPC, DPhPC with cholesterol and viral mimetic membrane lipid bilayers. Slow pharmacological binding kinetics allowed the characterization of spectral changes associated with non-specific binding to the protein periphery in the kinetically trapped pore-apo state. Resonance assignments were determined from a set of proton-detected 3D spectra. Chemical shift changes associated with functional binding in the pore of M2 were tracked in real time in order to estimate the activation energy. The binding kinetics are affected by pH and the lipid environment and in particular cholesterol. We found that the imidazole-imidazole hydrogen bond at residue histidine 37 is a stable feature of the protein across several lipid compositions. Pore binding breaks the imidazole-imidazole hydrogen bond and limits solubilization in DHPC detergent.
| S-EPMC10285276 | biostudies-literature