Phosphoryl Group Transfer Research Articles

Crystal Structures of the Acinetobacter baumannii Macrolide Phosphotransferase E.

Acinetobacter baumannii (A. baumannii) challenges clinical infection treatment due to its resistance to various antibiotics. Multiple resistance genes in the core genome or mobile elements contribute to multidrug resistance in A. baumannii. Macrolide phosphotransferase gene mphE has been identified in A. baumannii, which is particularly relevant to macrolide antibiotics. Here, we determined the structure of MphE protein in three states: the apo state, the complex state with erythromycin and guanosine triphosphate (GTP), and the complex state with azithromycin and guanosine. Interestingly, GTP and two magnesium ions were observed in the erythromycin-bound MphE complex. This structure captured the active state of MphE, in which the magnesium ions stabilized the active site and assisted the transfer of phosphoryl groups. Based on these structures, we verified that the conserved residues Asp29, Asp194, His199, and Asp213 play an important role in the catalytic phosphorylation of MphE leading to drug resistance. Our work helps to understand the molecular basis of drug resistance and provides reference targets for optimizing macrolide antibiotics.

Metal fluorides—multi-functional tools for the study of phosphoryl transfer enzymes, a practical guide

Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein families across all kingdoms of life. They make up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyze these reactions is essential in characterizing the processes they regulate. Metal fluorides can be used as multifunctional tools to study these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogs. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryo-EM, and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal using soft X-rays.

An allosteric switch ensures efficient unidirectional information transmission by the histidine kinase DesK from Bacillus subtilis.

Phosphorylation carries chemical information in biological systems. In two-component systems (TCSs), the sensor histidine kinase and the response regulator are connected through phosphoryl transfer reactions that may be uni- or bidirectional. Directionality enables the construction of complex regulatory networks that optimize signal propagation and ensure the forward flow of information. We combined x-ray crystallography, hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, and systems-integrative kinetic modeling approaches to study phosphoryl flow through the Bacillus subtilis thermosensing TCS DesK-DesR. The allosteric regulation of the histidine kinase DesK was critical to avoid back transfer of phosphoryl groups and futile phosphorylation-dephosphorylation cycles by isolating phosphatase, autokinase, and phosphotransferase activities. Interactions between the kinase's ATP-binding domain and the regulator's receiver domain placed the regulator in two distinct positions in the phosphotransferase and phosphatase complexes, thereby determining whether a key glutamine residue in DesK was properly situated to assist in the dephosphorylation reaction. Moreover, an energetically unfavorable phosphotransferase conformation when DesK was not phosphorylated minimized reverse phosphoryl transfer. DesR dimerization and a dissociative phosphoryl transfer reaction also enforced the direction of phosphoryl flow. Shorter or longer distances between the phosphoryl acceptor and donor residues shifted the phosphoryl transfer equilibrium by modulating the stabilizing effect of the Mg2+ cofactor. These mechanisms control the directionality of signal transmission and show how structure-encoded allostery stores and transmits information in signaling systems.

An Enzyme with High Catalytic Proficiency Utilizes Distal Site Substrate Binding Energy to Stabilize the Closed State but at the Expense of Substrate Inhibition.

Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to achieve high catalytic proficiencies by utilizing the binding energy derived from nonreacting portions of the substrate. In particular, enzymes with substrates containing a nonreacting phosphodianion group coordinated in a distal site have been suggested to exploit this binding energy primarily to facilitate a conformational change from an open inactive form to a closed active form, rather than to either induce ground state destabilization or stabilize the transition state. However, detailed structural evidence for the model is limited. Here, we use β-phosphoglucomutase (βPGM) to investigate the relationship between binding a phosphodianion group in a distal site, the adoption of a closed enzyme form, and catalytic proficiency. βPGM catalyzes the isomerization of β-glucose 1-phosphate to glucose 6-phosphate via phosphoryl transfer reactions in the proximal site, while coordinating a phosphodianion group of the substrate(s) in a distal site. βPGM has one of the largest catalytic proficiencies measured and undergoes significant domain closure during its catalytic cycle. We find that side chain substitution at the distal site results in decreased substrate binding that destabilizes the closed active form but is not sufficient to preclude the adoption of a fully closed, near-transition state conformation. Furthermore, we reveal that binding of a phosphodianion group in the distal site stimulates domain closure even in the absence of a transferring phosphoryl group in the proximal site, explaining the previously reported β-glucose 1-phosphate inhibition. Finally, our results support a trend whereby enzymes with high catalytic proficiencies involving phosphorylated substrates exhibit a greater requirement to stabilize the closed active form.

The Relationship between Enzyme Conformational Change, Proton Transfer, and Phosphoryl Transfer in β-Phosphoglucomutase

Molecular details for the timing and role of proton transfer in phosphoryl transfer reactions are poorly understood. Here, we have combined QM models, experimental NMR measurements, and X-ray structures to establish that the transition of an archetypal phosphoryl transfer enzyme, βPGM, from a very closed near-attack conformation to a fully closed transition state analogue (TSA) conformation triggers both partial proton transfer from the general acid–base residue to the leaving group oxygen and partial dissociation of the transferring phosphoryl group from the leaving group oxygen. Proton transfer continues but is not completed throughout the reaction path of the phosphoryl transfer with the enzyme in the TSA conformation. Moreover, using interacting quantum atoms (IQA) and relative energy gradient (REG) analysis approaches, we observed that the change in the position of the proton and the corresponding increased electrostatic repulsion between the proton and the phosphorus atom provide a stimulus for phosphoryl transfer in tandem with a reduction in the negative charge density on the leaving group oxygen atom. The agreement between solution-phase 19F NMR measurements and equivalent QM models of βPGMWT and βPGMD10N TSA complexes confirms the protonation state of G6P in the two variants, validating the employed QM models. Furthermore, QM model predictions of an AlF4 distortion in response to the proton position are confirmed using high resolution X-ray crystal structures, not only providing additional validation to the QM models but also further establishing metal fluorides as highly sensitive experimental predictors of active-site charge density distributions.

Adenylate Kinase-Catalyzed Reaction of AMP in Pieces: Enzyme Activation for Phosphoryl Transfer to Phosphite Dianion.

The binding of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP) to adenylate kinase (AdK) drives closure of lids over the substrate adenosyl groups. We test the hypothesis that this conformational change activates AdK for catalysis. The rate constants for Homo sapiens adenylate kinase 1 (HsAdK1)-catalyzed phosphoryl group transfer to AMP, kcat/Km = 7.0 × 106 M-1 s-1, and phosphite dianion, (kHPi)obs ≤1 × 10-4 M-1 s-1, show that the binding energy of the adenosyl group effects a ≥7.0 × 1010-fold rate acceleration of phosphoryl transfer from ATP. The third-order rate constant of kcat/KHPiKEA = 260 M-2 s-1 for 1-(β-d-erythrofuranosyl)adenine (EA)-activated phosphoryl transfer to phosphite dianion was determined, and the isohypophosphate reaction product characterized by 31P NMR. The results demonstrate the following: (i) a ≥14.7 kcal/mol stabilization of the transition state for phosphoryl transfer by the adenosyl group of AMP and a ≥2.6 × 106-fold rate acceleration from the EA-driven conformational change and (ii) the recovery of ≥8.7 kcal/mol of this transition state stabilization for EA-activated phosphoryl transfer from ATP to phosphite.

The Burden Borne by Protein Methyltransferases: Rates and Equilibria of Non-enzymatic Methylation of Amino Acid Side Chains by SAM in Water.

SAM is a powerful methylating agent, with a methyl group transfer potential matching the phosphoryl group transfer potential of ATP. SAM-dependent N-methyltransferases have evolved to catalyze the modification of specific lysine residues in histones and transcription factors, in addition to generating epinephrine, N-methylnicotinamide, and a quaternary amine (betaine) that is used to maintain osmotic pressure in plants and halophilic bacteria. To assess the catalytic power of these enzymes and their potential susceptibility to transition state and multisubstrate analogue inhibitors, we determined the rates and positions of the equilibrium of methyl transfer from the trimethylsulfonium ion to model amines in the absence of a catalyst. Unlike the methyl group transfer potential of SAM, which becomes more negative with an increase in pH throughout the normal pH range, equilibrium constants for the hydrolytic demethylation of secondary, tertiary, and quaternary amines are found to be insensitive to a change in pH and resemble each other in magnitude, with an average ΔG value of approximately -0.7 kcal/mol at pH 7. Thus, each of the three steps in the mono-, di-, and trimethylation of lysine by SAM is accompanied by a change in free energy of -7.5 kcal/mol in a neutral solution. Arrhenius analysis of the uncatalyzed reactions shows that the unprotonated form of glycine attacks the trimethylsulfonium ion (TMS+) with second-order rates constant of 1.8 × 10-7 M-1 s-1 at 25 °C (ΔH⧧ = 22 kcal/mol, and TΔS⧧ = -6 kcal/mol). Comparable values are observed for the methylation of secondary and tertiary amines, with k25 values of 1.1 × 10-7 M-1 s-1 for sarcosine and 4.3 × 10-8 M-1 s-1 for dimethylglycine. The non-enzymatic methylations of imidazole and methionine by TMS+, benchmarks for the methylation of histidine and methionine residues by SETD3, exhibit k25 values of 3.3 × 10-9 and 1.2 × 10-9 M-1 s-1, respectively. Lysine methylation by SAM, although slow under physiological conditions (t1/2 = 7 weeks at 25 °C), is accelerated 1.1 × 1012 -fold at the active site of a SET domain methyltransferase.

Identification of Z nucleotides as an ancient signal for two-component system activation in bacteria

Two-component systems (TCSs) in bacteria are molecular circuits that allow the perception of and response to diverse stimuli. These signaling circuits rely on phosphoryl-group transfers between transmitter and receiver domains of sensor kinase and response regulator proteins, and regulate several cellular processes in response to internal or external cues. Phosphorylation, and thereby activation, of response regulators has been demonstrated to occur by their cognate histidine kinases but also by low molecular weight phosphodonors such as acetyl phosphate and carbamoyl phosphate. Here, we present data indicating that the intermediates of the de novo syntheses of purines and histidine, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP) and/or 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-triphosphate (ZTP), activate the response regulator UvrY, by promoting its autophosphorylation at the conserved aspartate at position 54. Moreover, these Z nucleotides are shown to also activate the nonrelated response regulators ArcA, CpxR, RcsB, and PhoQ. We propose that ZMP and/or ZTP act as alarmones for a wide range of response regulators in vivo, providing a novel mechanism by which they could impact gene expression in response to metabolic cues.

Deciphering the Che2 chemosensory pathway and the roles of individual Che2 proteins from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that senses and responds to its environment via four chemosensory systems. Oxygen activates the Che2 chemosensory system by binding to the PAS-heme domain of the Aer2 receptor. Ostensibly, the output of Che2 occurs via its response regulator CheY2, but controversy persists over CheY2's exact role. In this study, we show that CheY2 does not interact with the flagellar motor and that the Che2 system does not transfer phosphoryl groups to the chemotaxis (Che) system. We show that CheY2 instead provides feedback control of Aer2 adaptation. In the presence of O2 , Aer2 signaling increases the autophosphorylation of the histidine kinase CheA2, followed by CheY2-mediated dephosphorylation. CheY2 does not stably retain phosphate and may not signal the output of the Che2 system. Rather, CheY2 activity enhances the direct interaction of CheY2 with the adaptation protein CheD (a role often facilitated by CheC, which P. aeruginosa lacks). In the absence of O2 , Aer2 does not signal, and CheY2/CheD interactions attenuate. This frees CheD to augment CheR2-mediated methylation of Aer2, which enhances Aer2 signaling. CheD does not interact with CheR2, but most likely interacts with Aer2 via conserved CheD-binding motifs to make Aer2 a better methylation substrate.

Conformational Transitions of Histidine Kinases using Molecular Dynamics

One of the main signal transduction mechanisms in bacteria are two-component systems (TCS), which are comprised of homodimeric sensor histidine kinase (HK) and response regulator (RR) proteins (1). Histidine kinases are multidomain, transmembrane proteins which function by detecting a stimulus and then propagate this signal across the domains via a series of conformational transitions ruled by unstable transient interactions (2), subsequently resulting in an appropriate cellular response. The dimerization histidine phosphotransfer (DHp) domain, contains a phosphorylatable conserved histidine residue. Secondly, the catalytic ATP-binding (CA) domain, binds ATP and hence provides the phosphoryl group necessary for phosphorylating the histidine residue in the DHp. Finally, the phosphoryl group from the histidine residue of the DHp of HK is transferred to an aspartate residue of the RR protein. We investigate this signal transduction using coarse-grained and all-atom molecular dynamics simulations. A specific focus are the conformational transitions between the active and inactive states of the conserved kinase core. In the active conformation, the ATP of CA and phosphorylatable histidine of DHp are in an ideal orientation for phosphoryl group transfer whereas in the inactive conformation, the two domains are too distant.

Asymmetric Electrophilic Reactions in Phosphorus Chemistry

This review is devoted to the theoretic and synthetic aspects of asymmetric electrophilic substitution reactions at the stereogenic phosphorus center. The stereochemistry and mechanisms of electrophilic reactions are discussed—the substitution, addition and addition-elimination of many important reactions. The reactions of bimolecular electrophilic substitution SE2(P) proceed stereospecifically with the retention of absolute configuration at the phosphorus center, in contrast to the reactions of bimolecular nucleophilic substitution SN2(P), proceeding with inversion of absolute configuration. This conclusion was made based on stereochemical analysis of a wide range of trivalent phosphorus reactions with typical electrophiles and investigation of examples of a sizeable number of diverse compounds. The combination of stereospecific electrophilic reactions and stereoselective nucleophilic reactions is useful and promising for the further development of organophosphorus chemistry. The study of phosphoryl group transfer reactions is important for biological and molecular chemistry, as well as in studying mechanisms of chemical processes involving organophosphorus compounds. New versions of asymmetric electrophilic reactions applicable for the synthesis of enantiopure P-chiral secondary and tertiary phosphines are discussed.

Zooming in on protons: Neutron structure of protein kinase A trapped in a product complex.

The question vis-à-vis the chemistry of phosphoryl group transfer catalyzed by protein kinases remains a major challenge. The neutron diffraction structure of the catalytic subunit of cAMP-dependent protein kinase (PKA-C) provides a more complete chemical portrait of key proton interactions at the active site. By using a high-affinity protein kinase substrate (PKS) peptide, we captured the reaction products, dephosphorylated nucleotide [adenosine diphosphate (ADP)] and phosphorylated PKS (pPKS), bound at the active site. In the complex, the phosphoryl group of the peptide is protonated, whereas the carboxyl group of the catalytic Asp166 is not. Our structure, including conserved waters, shows how the peptide links the distal parts of the cleft together, creating a network that engages the entire molecule. By comparing slow-exchanging backbone amides to those determined by the NMR analysis of PKA-C with ADP and inhibitor peptide (PKI), we identified exchangeable amides that likely distinguish catalytic and inhibited states.

Abstract P2-02-01: Inhibition of creatine kinase metabolism represses invasion and sensitizes estrogen-receptor negative breast cancer cells to standard chemotherapies

Abstract Dysregulated tumor cell metabolism is a hallmark of cancer progression and therapeutic resistance. In a screen for Hypoxia-Inducible Factor (HIF)-dependent genes regulating metabolism, we identified creatine kinase, brain isoform (CKB) as down-regulated in HIF-1 knockout mammary tumor cells. CKB is a member of a family of cytosolic and mitochondrial creatine kinases (CKs) that reversibly catalyze the transfer of a high-energy phosphoryl group from ATP to creatine, generating local stores of phosphocreatine in the forward reaction, and re-generating ATP in the reverse reaction. Creatine kinases are up-regulated in a variety of solid tumors, including breast cancers. CK activity is inhibited by cyclocreatine (cCr), a creatine kinase substrate that represses the CK-dependent generation of ATP from phosphocreatine >170-fold. Knockdown of CKB in tumor cells derived from the polyoma middle T (PyMT) transgenic model of metastatic breast cancer did not impair cell proliferation or wound healing, but potently suppressed invasion in vitro, suggesting utility for treating stage IV disease. When female FVB/Nj mice were injected with wild type PyMT cells in a tail vein assay and then treated with cCr, lung metastasis was repressed to the same extent as CKB gene knockdown. These data were validated using a variety of human estrogen receptor (ER)-negative breast cancer cell lines. CKB loss- and gain-of-function models were created and effects on cell proliferation, survival, wound healing and ATP production were compared to cCr treatment. Overall, whereas deletion of CKB had no effect on cell proliferation or survival in either adherent or suspension conditions, either deletion of CKB or cCr therapy potently reduced intracellular ATP levels, which was associated with reduced invasive potential. In contrast, over-expression of CKB enhanced chemotaxis to EGF and invasion towards serum. Whereas cCr monotherapy is generally cytostatic, combination with either doxorubicin or paclitaxel is synergistic to kill tumor cells. Together, these data suggest that inhibition of CK activity may be effective in treating stage IV breast cancer patients with established metastatic disease. We are currently exploring the cell signaling and cell survival pathways that are altered by CKB gene deletion or cCr treatment to understand how the creatine kinase arm of metabolism promotes tumor progression, metastasis and response to conventional chemotherapies. Citation Format: Barch HP, Krutilina R, Brooks DL, Parke D, Seagroves TN. Inhibition of creatine kinase metabolism represses invasion and sensitizes estrogen-receptor negative breast cancer cells to standard chemotherapies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-01.

Mecanismos de autofosforilación y transfosforilación en sistemas de dos componentes bacterianos

Los Sistemas de señalización de Dos Componentes (SDCs) permiten a las bacterias detectar estímulos ambientales y responder a estos de manera adaptativa. Estos sistemas de transducción de señales se basan en la autofosforilación y transferencia de grupos fosforilo entre residuos de histidina y aspartato de una proteína cinasa sensora y un regulador de respuesta. Cuando la cinasa sensora percibe un estímulo específico se autofosforila, en una reacción que puede ser inter o intramolecular, y transfosforila a su regulador de respuesta cognado, que actúa generalmente como regulador transcripcional para ejercer una respuesta fisiológica. Con frecuencia, en ausencia de estímulo, la misma cinasa sensora se encarga de desfosforilar a su regulador de respuesta cognado. Además, algunas cinasas sensoras tienen módulos adicionales que participan en un fosforelevo que termina en la fosforilación del regulador de respuesta, y en un fosforelevo reverso que permite su desfosforilación. Al igual que en la autofosforilación, las trasferencias de grupos fosforilo implicadas en el fosforelevo y el fosforelevo reverso pueden ser inter o intramoleculares. En esta revisión exponemos algunas de las más importantes características de los SDCs bacterianos, poniendo especial énfasis en los procesos de autofosforilación y fosfotransferencia.

Mecanismos de autofosforilación y transfosforilación en sistemas de dos componentes bacterianos

Two-component signaling circuits (TCS) allow bacteria to detect environmental cues and to produce adaptive responses. These signaling systems are based on autophosphorylation and phosphoryl-group transfers between histidine and aspartate containing sensor kinase and response regulator proteins. Upon reception of a specific stimulus the sensor kinase protein autophosphorylates, by either an inter- or intra-molecular reaction, and transphosphorylates its cognate response regulator, which, typically, acts as a transcriptional regulator, thereby triggering physiological responses. Frequently, in the absence of the stimulus, the sensor kinase proteins are responsible for the dephosphorylation of their cognate response regulators. Furthermore, a group of sensor kinases have additional functional domains that are involved in a forward phosphorelay for signal transmission and in a reverse phosphorelay for signal decay. As is the case of the autophosphorylation reaction, the phosphoryl-group transfers involved in the forward and reverse phosphorelay can occur either intra- or inter-molecularly. In this review, we highlight some important features of bacterial TCS, with special emphasis on the autophosphorylation and phosphoryl-group transfer events.

Carbohydrate Transport by Group Translocation: The Bacterial Phosphoenolpyruvate: Sugar Phosphotransferase System.

The Bacterial Phosphoenolpyruvate (PEP) : Sugar Phosphotransferase System (PTS) mediates the uptake and phosphorylation of carbohydrates, and controls the carbon- and nitrogen metabolism in response to the availability of sugars. PTS occur in eubacteria and in a few archaebacteria but not in animals and plants. All PTS comprise two cytoplasmic phosphotransferase proteins (EI and HPr) and a species-dependent, variable number of sugar-specific enzyme II complexes (IIA, IIB, IIC, IID). EI and HPr transfer phosphorylgroups from PEP to the IIA units. Cytoplasmic IIA and IIB units sequentially transfer phosphates to the sugar, which is transported by the IIC and IICIID integral membrane protein complexes. Phosphorylation by IIB and translocation by IIC(IID) are tightly coupled. The IIC(IID) sugar transporters of the PTS are in the focus of this review. There are four structurally different PTS transporter superfamilies (glucose, glucitol, ascorbate, mannose) . Crystal structures are available for transporters of two superfamilies: bcIICmal (MalT, 5IWS, 6BVG) and bcIICchb (ChbC, 3QNQ) of B. subtilis from the glucose family, and IICasc (UlaA, 4RP9, 5ZOV) of E. coli from the ascorbate superfamily . They are homodimers and each protomer has an independent transport pathway which functions by an elevator-type alternating-access mechanism. bcIICmal and bcIICchb have the same fold, IICasc has a completely different fold. Biochemical and biophysical data accumulated in the past with the transporters for mannitol (IICBAmtl) and glucose (IICBglc) are reviewed and discussed in the context of the bcIICmal crystal structures. The transporters of the mannose superfamily are dimers of protomers consisting of a IIC and a IID protein chain. The crystal structure is not known and the topology difficult to predict. Biochemical data indicate that the IICIID complex employs a different transport mechanism . Species specific IICIID serve as a gateway for the penetration of bacteriophage lambda DNA across, and insertion of class IIa bacteriocins into the inner membrane. PTS transporters are inserted into the membrane by SecYEG translocon and have specific lipid requirements. Immunoelectron- and fluorescence microscopy indicate a non-random distribution and supramolecular complexes of PTS proteins.

Long-range molecular dynamics show that inactive forms of Protein Kinase A are more dynamic than active forms.

Many protein kinases are characterized by at least two structural forms corresponding to the highest level of activity (active) and low or no activity, (inactive). Further, protein dynamics is an important consideration in understanding the molecular and mechanistic basis of enzyme function. In this work, we use protein kinase A (PKA) as the model system and perform microsecond range molecular dynamics (MD) simulations on six variants which differ from one another in terms of active and inactive form, with or without bound ligands, C-terminal tail and phosphorylation at the activation loop. We find that the root mean square fluctuations in the MD simulations are generally higher for the inactive forms than the active forms. This difference is statistically significant. The higher dynamics of inactive states has significant contributions from ATP binding loop, catalytic loop, and αG helix. Simulations with and without C-terminal tail show this differential dynamics as well, with lower dynamics both in the active and inactive forms if C-terminal tail is present. Similarly, the dynamics associated with the inactive form is higher irrespective of the phosphorylation status of Thr 197. A relatively stable stature of active kinases may be better suited for binding of substrates and detachment of the product. Also, phosphoryl group transfer from ATP to the phosphosite on the substrate requires precise transient coordination of chemical entities from three different molecules, which may be facilitated by the higher stability of the active state.

Allosteric coupling between autophosphorylation and phosphoryl-group transfer in a prototypical two-component signal transduction system

Allosteric coupling between autophosphorylation and phosphoryl-group transfer in a prototypical two-component signal transduction system

Routes of phosphoryl group transfer during signal transmission and signal decay in the dimeric sensor histidine kinase ArcB

The Arc (anoxic redox control) two-component system of Escherichia coli, comprising ArcA as the response regulator and ArcB as the sensor histidine kinase, modulates the expression of numerous genes in response to respiratory growth conditions. Under reducing growth conditions, ArcB autophosphorylates at the expense of ATP, and transphosphorylates ArcA via a His292 → Asp576 → His717 → Asp54 phosphorelay, whereas under oxidizing growth conditions, ArcB catalyzes the dephosphorylation of ArcA-P by a reverse Asp54 → His717 → Asp576 → Pi phosphorelay. However, the exact phosphoryl group transfer routes and the molecular mechanisms determining their directions are unclear. Here, we show that, during signal propagation, the His292 → Asp576 and Asp576 → His717 phosphoryl group transfers within ArcB dimers occur intra- and intermolecularly, respectively. Moreover, we report that, during signal decay, the phosphoryl group transfer from His717 to Asp576 takes place intramolecularly. In conclusion, we present a mechanism that dictates the direction of the phosphoryl group transfer within ArcB dimers and that enables the discrimination of the kinase and phosphatase activities of ArcB.

1H, 15N, 13C backbone resonance assignments of human phosphoglycerate kinase in a transition state analogue complex with ADP, 3-phosphoglycerate and magnesium trifluoride

Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (3PG) and ATP. PGK is composed of two α/β Rossmann-fold domains linked by a central α-helix and the active site is located in the cleft formed between the N-domain which binds BPG or 3PG, and the C-domain which binds the nucleotides ADP or ATP. Domain closure is required to bring the two substrates into close proximity for phosphoryl transfer to occur, however previous structural studies involving a range of native substrates and substrate analogues only yielded open or partly closed PGK complexes. X-ray crystallography using magnesium trifluoride (MgF3−) as a isoelectronic and near-isosteric mimic of the transferring phosphoryl group (PO3−), together with 3PG and ADP has been successful in trapping human PGK in a fully closed transition state analogue (TSA) complex. In this work we report the 1H, 15N and 13C backbone resonance assignments of human PGK in the solution conformation of the fully closed PGK:3PG:MgF3:ADP TSA complex. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97% of all backbone resonances were assigned in the complex, with 385 out of a possible 399 residues assigned in the 1H–15N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS-N webserver is in good agreement with the published X-ray crystal structure of this complex.