Mode Of ATP Binding Research Articles

Conformation selection by ATP-competitive inhibitors and allosteric communication in ERK2.

Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named 'L' and 'R,' where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

Conformation selection by ATP-competitive inhibitors and allosteric communication in ERK2

Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named ‘L’ and ‘R,’ where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

Structure-based design and modular synthesis of novel PI4K class II inhibitors bearing a 4-aminoquinazoline scaffold

Novel 4-aminoquinazoline-6-carboxamide derivatives bearing differently substituted aryl or heteroaryl groups at position 7 in the core were rationally designed, synthesized and evaluated for biological activity in vitro as phosphatidylinositol 4-kinase IIα (PI4K2A) inhibitors. The straightforward approach described here enabled the sequential, modular synthesis and broad functionalization of the scaffold in a mere six steps. The SAR investigation reported here is based on detailed structural analysis of the conserved binding mode of ATP and other adenine derivatives to the catalytic site of type II PI4Ks, combined with extensive docking studies. Several compounds exhibited significant activity against PI4K2A. Moreover, we solved a crystal structure of PI4K2B in complex with one of our lead ligand candidates, which validated the ligand binding site and pose predicted by our docking-based ligand model. These discoveries suggest that our structure-based approach may be further developed and employed to synthesize new inhibitors with optimized potency and selectivity for this class of PI4Ks.

Nucleotide Binding, Evolutionary Insights, and Interaction Partners of the Pseudokinase Unc-51-like Kinase 4

Unc-51-like kinase 4 (ULK4) is a pseudokinase that has been linked to the development of several diseases. Even though sequence motifs required for ATP binding in kinases are lacking, ULK4 still tightly binds ATP and the presence of the co-factor is required for structural stability of ULK4. Here, we present a high-resolution structure of a ULK4-ATPγS complex revealing a highly unusual ATP binding mode in which the lack of the canonical VAIK motif lysine is compensated by K39, located N-terminal to αC. Evolutionary analysis suggests that degradation of active site motifs in metazoan ULK4 has co-occurred with an ULK4-specific activation loop, which stabilizes the C helix. In addition, cellular interaction studies using BioID and biochemical validation data revealed high confidence interactors of the pseudokinase and armadillo repeat domains. Many of the identified ULK4 interaction partners were centrosomal and tubulin-associated proteins and several active kinases suggesting interesting regulatory roles for ULK4.

Structural bioinformatics insights into ATP binding mechanism in zebrafish (Danio rerio) cyclin-dependent kinase-like 5 (zCDKL5) protein.

In mammalian systems, the conserved cyclin-dependent protein kinases (CDKs) control the process of cell division and curb the transcription mechanism in response to diverse signaling events that are essential for the catalytic activity. In zebrafish, zCDKL5 portrays differential expression profiling in several tissues and presumed to play a vital role in the neuronal development. In this present study, the sequence-structure relationship and mode of ATP binding in zCDKL5 was unveiled through theoretical modeling, molecular docking, and MD simulations. Like human CDKs, the modeled zCDKL5 was found to be bipartite in nature, where, ATP binds to the central cavity of the catalytic domain through a strong network of H-bonding, electrostatic, and hydrophobic interactions. MD simulation portrayed that conserved residues, viz, Ile10, Gly11, Glu12, Val18, Val64, Glu81, Cys143, and Asp144 were indispensable for tight anchoring of ATP and contribute to the stability of the zCDKL5-ATP complex. MM/PBSA binding free energy analysis displayed that van der Waal energy (ΔG vwd ) and Electrostatic energy (ΔG ele ) were the major contributors towards the overall binding free energy. Thus, the comparative structural bioinformatics approach has shed new insights into the dynamics and ATP binding mechanism of zCDKL5. The results from the study will help to undertake further research on the role of phosphorylated CDKL5 in the onset of neurodevelopmental disorders caused by mutations in higher eukaryotic systems.

Cryo-EM structure of the bacterial actin AlfA reveals unique assembly and ATP-binding interactions and the absence of a conserved subdomain

Bacterial actins are an evolutionarily diverse family of ATP-dependent filaments built from protomers with a conserved structural fold. Actin-based segregation systems are encoded on many bacterial plasmids and function to partition plasmids into daughter cells. The bacterial actin AlfA segregates plasmids by a mechanism distinct from other partition systems, dependent on its unique dynamic properties. Here, we report the near-atomic resolution electron cryo-microscopy structure of the AlfA filament, which reveals a strikingly divergent filament architecture resulting from the loss of a subdomain conserved in all other actins and a mode of ATP binding. Its unusual assembly interfaces and nucleotide interactions provide insight into AlfA dynamics, and expand the range of evolutionary variation accessible to actin quaternary structure.

Deep insights into the mode of ATP-binding mechanism in Zebrafish cyclin-dependent protein kinase-like 1 (zCDKL1): A molecular dynamics approach

In eukaryotes, the serine/threonine kinases (STKs) belonging to cyclin-dependent protein kinases (CDKs) play significant role in control of cell division and curb transcription in response to several extra and intra-cellular signals indispensable for enzymatic activity. The zebrafish cyclin-dependent protein kinase-like 1 protein (zCDKL1) shares a high degree of sequence and structural similarity with mammalian orthologs and express in brain, ovary, testis, and low levels in other tissues. Regardless of its importance in the developmental process, the structure, function and mode of ATP recognition have not been investigated yet due to lack of experimental data. Henceforth, to gain atomistic insights in to the structural dynamics and mode of ATP binding, a series of computational techniques involving theoretical modeling, docking, molecular dynamics (MD) simulations and MM/PBSA binding free energies were employed. The modeled bi-lobed zCDKL1 shares a high degree of secondary structure topology with human orthologs where ATP prefers to lie in the central cavity of the bi-lobed catalytic domain enclosed by strong hydrogen bonding, electrostatic and hydrophobic contacts. Long range MD simulation portrayed that catalytic domain of zCDKL1 to be highly rigid in nature as compared to the complex (zCDKL1-ATP) form. Comparative analysis with its orthologs revealed that conserved amino acids i.e., Ile10, Gly11, Glu12, Val18, Arg31, Phe80, Glu 130, Cys143 and Asp144 were crucial for ATP binding mechanism, which needs further investigation for legitimacy. MM/PBSA method revealed that van der Waals, electrostatic and polar solvation energy mostly contributes towards negative free energy. The implications of ATP binding mechanism inferred through these structural bioinformatics approaches will help in understanding the catalytic mechanisms of important STKs in eukaryotic system.

Molecular Modeling and Dynamics Simulation Analysis of KATNAL1 for Identification of Novel Inhibitor of Sperm Maturation.

Hormone based birth control often causes various side effects. A recent study revealed that temporary infertility without changing hormone levels can be attained by inhibiting Katanin p60 ATPase-containing subunit A-like 1 protein (KATNAL1) which is critical for sperm maturation in the testes. This study aimed at attaining the most energetically stable three dimensional (3D) structure of KATNAL1 protein using comparative modeling followed by screening of a ligand library of known natural spermicidal compounds for their binding affinity with KATNAL1. This in turn may inhibit the development of mature sperm in the seminiferous epithelium. A series of computational techniques were used for building the 3D structure of KATNAL1 which was further optimized by molecular dynamics (MD) simulation. For revealing the ATP binding mode of KATNAL1, docking study was carried out using the optimized model obtained from the MD simulation. The docking study was also employed to test the binding efficiency of the ligand library. Molecular docking study confirmed the ATP binding of KATNAL1 with various hydrophobic and hydrogen bond interactions. Binding efficiency of the ligand library suggested that calotropin, a cardenolide of Calotropis procera showed the highest binding efficiency against the target protein without toxicity. MD simulation of the docked complex validated the results of the docking study. This study revealed the ATP binding mode of KATNAL1 and identified calotropin as a potential lead molecule against it showing high binding efficiency with good bioavailability and no mutagenicity. Further in vitro and in vivo bioassay of calotropin could facilitate the development of novel non-hormonal male-specific contraceptive in near future.

How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

Crystal structure of heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) and the inhibitory influence of citrate on substrate binding.

The heart-specific isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8 Å, respectively. Citrate, a TCA cycle intermediate and well-known inhibitor of PFKFB2, co-crystallized in the 2-kinase domains of both orthologues, occupying the fructose-6-phosphate binding-site and extending into the γ-phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ-phosphate site by citrate proved highly consequential to the binding of co-complexed ATP analogues. The bovine structure, which co-crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co-complexed with AMPPNP, which, unlike ADP, contains a γ-phosphate. The presence of this γ-phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding-pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate's inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate's negative feed-back loop of the glycolytic pathway via PFKFB2. Proteins 2016; 85:117-124. © 2016 Wiley Periodicals, Inc.

Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor.

V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A3B3DF (V1) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V1 crystals in ADP. In the presence of 20 μM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V1-ATPase rotational mechanism model.

Phosphorylation of spore coat proteins by a family of atypical protein kinases

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

Species-dependent variation in the structure of the CK2α-apigenin complex

Ser/Thr kinase CK2, consisting of two catalytic α subunits and two regulatory β subunits, represents various cell fates such as proliferation and is a crucial target for cancer and glomerulonephritis therapies. To date, several planar compounds have been identified as ATP-competitive CK2-inhibitors including apigenin, a flavonoid inhibitor. We here determined the crystal structures of human CK2α complexed with apigenin and an ATP analogue (AMPPNP) at 2.3 and 2.5 Å resolution, respectively and investigated the species specificity in the molecular recognition by the structural comparisons of these complexes with each maize equivalent. The pairs of the superimposed structures reveal that apigenin binds to human and maize CK2α in the quite distinct manners. while AMPPNP binds to the both species in a similar manner. Together with the chemical calculations, we deduce that the alternation at the Leu45/Val45 position of CK2α largely contributes to the species-dependent variation in the apigenin binding mode although the discrepancy is unrelated to the structural conservation in the ATP binding mode.

ATP-binding mode including a carbamoylated lysine and two Mg2+ ions, and substrate-binding mode in Acinetobacter baumannii MurF

MurF adds d-Ala-d-Ala dipeptide to UDP-N-acetylmuramyl-l-Ala-γ-d-Glu-m-DAP (or l-Lys) in an ATP-dependent manner, which is the last step in the biosynthesis of monomeric precursor of peptidoglycan. Here we report crystal structures of two MurF–ATP complexes: the MurF–ATP complex and the MurF–ATP–UDP complex. The ATP-binding mode revealed by the crystal structure of the MurF–ATP complex confirms the previous biochemical demonstration that a carbamoylated lysine and two Mg2+ ions are required for enzyme activity of MurF. The UDP–MurF interactions observed in the crystal structure of the MurF–ATP–UDP complex depict the characteristic substrate-binding mode of MurF. The emergence and dissemination of multidrug-resistant Acinetobacter baumannii strains are great threats to public health. Therefore, the structural information on A. baumannii MurF as a validated target for drug discovery will provide a framework to develop antibacterial agents against multidrug-resistant A. baumannii infections as well as to understand the reaction mechanism of MurF.

Conserved Inhibitory Mechanism and Competent ATP Binding Mode for Adenylyltransferases with Fic Fold

The ubiquitous FIC domain is evolutionarily conserved from bacteria to human and has been shown to catalyze AMP transfer onto protein side-chain hydroxyl groups. Recently, it was predicted that most catalytically competent Fic proteins are inhibited by the presence of an inhibitory helix αinh that is provided by a cognate anti-toxin (class I), or is part of the N- or C-terminal part of the Fic protein itself (classes II and III). In vitro, inhibition is relieved by mutation of a conserved glutamate of αinh to glycine. For the class III bacterial Fic protein NmFic from Neisseria meningitidis, the inhibitory mechanism has been elucidated. Here, we extend above study by including bacterial class I and II Fic proteins VbhT from Bartonella schoenbuchensis and SoFic from Shewanella oneidensis, respectively, and the respective E->G mutants. Comparative enzymatic and crystallographic analyses show that, in all three classes, the ATP substrate binds to the wild-type FIC domains, but with the α-phosphate in disparate and non-competent orientations. In the E->G mutants, however, the tri-phosphate moiety is found reorganized to the same tightly bound structure through a unique set of hydrogen bonds with Fic signature motif residues. The γ-phosphate adopts the location that is taken by the inhibitory glutamate in wild-type resulting in an α-phosphate orientation that can be attacked in-line by a target side-chain hydroxyl group. The latter is properly registered to the Fic active center by main-chain β-interactions with the β-hairpin flap. These data indicate that the active site motif and the exposed edge of the flap are both required to form an adenylylation-competent Fic protein.

Structural Basis for dsRNA Recognition, Filament Formation, and Antiviral Signal Activation by MDA5

MDA5, a viral double-stranded RNA (dsRNA) receptor, shares sequence similarity and signaling pathways with RIG-I yet plays essential functions in antiviral immunity through distinct specificity for viral RNA. Revealing the molecular basis for the functional divergence, we report here the crystal structure ofMDA5 bound to dsRNA, which shows how, using the same domain architecture, MDA5 recognizes the internal duplex structure, whereas RIG-I recognizes the terminus of dsRNA. We further show that MDA5 uses direct protein-protein contacts to stack along dsRNA in a head-to-tail arrangement, and that the signaling domain (tandem CARD), which decorates the outside of the core MDA5 filament, also has an intrinsic propensity to oligomerize into an elongated structure that activates the signaling adaptor, MAVS. These data support a model in which MDA5 uses long dsRNA as a signaling platform to cooperatively assemble the core filament, which in turn promotes stochastic assembly of the tandem CARD oligomers for signaling.

Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family.

Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family.

Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor

P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120-I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP-induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.

The Binding Mode of ATP Revealed by the Solution Structure of the N-domain of Human ATP7A

We report the solution NMR structures of the N-domain of the Menkes protein (ATP7A) in the ATP-free and ATP-bound forms. The structures consist of a twisted antiparallel six-stranded beta-sheet flanked by two pairs of alpha-helices. A protein loop of 50 amino acids located between beta 3 and beta 4 is disordered and mobile on the subnanosecond time scale. ATP binds with an affinity constant of (1.2 +/- 0.1) x 10(4) m(-1) and exchanges with a rate of the order of 1 x 10(3) s(-1). The ATP-binding cavity is considerably affected by the presence of the ligand, resulting in a more compact conformation in the ATP-bound than in the ATP-free form. This structural variation is due to the movement of the alpha1-alpha2 and beta2-beta 3 loops, both of which are highly conserved in copper(I)-transporting P(IB)-type ATPases. The present structure reveals a characteristic binding mode of ATP within the protein scaffold of the copper(I)-transporting P(IB)-type ATPases with respect to the other P-type ATPases. In particular, the binding cavity contains mainly hydrophobic aliphatic residues, which are involved in van der Waal's interactions with the adenine ring of ATP, and a Glu side chain, which forms a crucial hydrogen bond to the amino group of ATP.

Analysis of Structural Rearrangements during P2X1 Receptor Activation by Voltage Clamp Fluorometry

P2X receptors (P2XRs) are non-selective cation channels which are activated upon binding of extracellular ATP. They are assembled as homo- or heteromers from three subunits with two transmembrane domains each and show no sequence homology to any other known ion channel or ATP-binding protein. Most recently, the first crystal structure of a P2X receptor has been resolved confirming many predictions that were based on the interpretation of mutagenesis studies. However, the conformational changes governing channel opening, desensitization and recovery remain unknown, as does the exact mode of ATP binding. The P2X1 receptor subtype is characterized by nanomolar affinity for ATP and a rapid desensitization, followed by a prolonged recovery period before reactivation is possible. Here we used voltage clamp fluorometry to identify domains undergoing conformational changes during ligand binding, activation, desensitization and recovery from desensitization of the P2X1 receptor. We have identified six residues in the extracellular domain of the P2X1 receptor that, upon substitution with cysteine, are accessible for TMRM labeling. Upon activation by ATP, five of these mutants showed significant changes in fluorescence. The kinetics of the fluorescence changes could be correlated with receptor activation or desensitization indicating that the changes were reporting discrete conformational changes. In addition, binding of the competitive antagonist NF449 produced a fluorescence change in three of these mutants. In conclusion, our observations provide insight into the conformational changes occurring during the P2X opening and desensitization and suggest that movements in a cysteine rich domain that projects over the supposed ATP binding site are involved in these processes.