Elucidation of the Mechanisms of Inter-domain Coupling in the Monomeric State of Enzyme I by High-pressure NMR.

The catalytic cycle of Enzyme I (EI), a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate, is characterized by a series of local and global conformational rearrangements. This multistep process includes a monomer-to-dimer transition, followed by an open-to-closed rearrangement of the dimeric complex upon PEP binding. In the present study, we investigate the thermodynamics of EI dimerization using a range of high-pressure solution NMR techniques complemented by SAXS experiments. 1H-15N TROSY and 1H-13C methyl TROSY NMR spectra combined with 15N relaxation measurements revealed that a native-like engineered variant of full-length EI fully dissociates into stable monomeric state above 1.5 kbar. Conformational ensembles of EI monomeric state were generated via a recently developed protocol combining coarse-grained molecular simulations with experimental backbone residual dipolar coupling measurements. Analysis of the structural ensembles provided detailed insights into the molecular mechanisms driving formation of the catalytically competent dimeric state, and reveals that each step of EI catalytical cycle is associated with a significant reduction in either inter- or intra-domain conformational entropy. Altogether, this study completes a large body work conducted by our group on EI and establishes a comprehensive structural and dynamical description of the catalytic cycle of this prototypical multidomain, oligomeric enzyme.

Structure elucidation of the elusive Enzyme I monomer reveals the molecular mechanisms linking oligomerization and enzymatic activity.

Enzyme I (EI) is a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate. This reaction initiates a five-step phosphorylation cascade in the bacterial phosphotransferase (PTS) transduction pathway. Under physiological conditions, EI exists in an equilibrium between a functional dimer and an inactive monomer. The monomer-dimer equilibrium is a crucial factor regulating EI activity and the phosphorylation state of the overall PTS. Experimental studies of EI's monomeric state have yet been hampered by the dimer's high thermodynamic stability, which prevents its characterization by standard structural techniques. In this study, we modified the dimerization domain of EI (EIC) by mutating three amino acids involved in the formation of intersubunit salt bridges. The engineered variant forms an active dimer in solution that can bind and hydrolyze PEP. Using hydrostatic pressure as an additional perturbation, we were then able to study the complete dissociation of the variant from 1 bar to 2.5 kbar in the absence and the presence of EI natural ligands. Backbone residual dipolar couplings collected under high-pressure conditions allowed us to determine the conformational ensemble of the isolated EIC monomeric state in solution. Our calculations reveal that three catalytic loops near the dimerization interface become unstructured upon monomerization, preventing the monomeric enzyme from binding its natural substrate. This study provides an atomic-level characterization of EI's monomeric state and highlights the role of the catalytic loops as allosteric connectors controlling both the activity and oligomerization of the enzyme.

Hybrid Thermophilic/Mesophilic Enzymes Reveal a Role for Conformational Disorder in Regulation of Bacterial Enzyme I.

Conformational disorder is emerging as an important feature of biopolymers, regulating a vast array of cellular functions, including signaling, phase separation, and enzyme catalysis. Here we combine NMR, crystallography, computer simulations, protein engineering, and functional assays to investigate the role played by conformational heterogeneity in determining the activity of the C-terminal domain of bacterial Enzyme I (EIC). In particular, we design chimeric proteins by hybridizing EIC from thermophilic and mesophilic organisms, and we characterize the resulting constructs for structure, dynamics, and biological function. We show that EIC exists as a mixture of active and inactive conformations and that functional regulation is achieved by tuning the thermodynamic balance between active and inactive states. Interestingly, we also present a hybrid thermophilic/mesophilic enzyme that is thermostable and more active than the wild-type thermophilic enzyme, suggesting that hybridizing thermophilic and mesophilic proteins is a valid strategy to engineer thermostable enzymes with significant low-temperature activity.

Resonance assignment of the 128 kDa enzyme I dimer from Thermoanaerobacter tengcongensis.

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) utilizes phosphoenolpyruvate (PEP) as a source of energy in order to transport sugars across the cellular membrane. PEP binding to EI initiates a phosphorylation cascade that regulates a variety of essential pathways in the metabolism of bacterial cells. Given its central role in controlling bacterial metabolism, EI has been often suggested as a good target for antimicrobial research. Here, we report the 1HN, 15N, 13C', 1Hmethyl, and 13Cmethyl chemical shifts of the 128 kDa homodimer EI from the thermophile Thermoanaerobacter tengcongensis. In total 79% of the expected backbone amide correlations and 80% of the expected methyl TROSY peaks from U-[2H, 13C, 15N], Ileδ1-[13CH3], Val-Leu-[13CH3/12CD3] labeled EI were assigned. The reported assignments will enable future structural studies aimed at illuminating the fundamental mechanisms governing long-range interdomain communication in EI and at indicating new therapeutic strategies to combat bacterial infections.

The oligomerization state of bacterial enzyme I (EI) determines EI's allosteric stimulation or competitive inhibition by α-ketoglutarate.

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by phosphorylation of enzyme I (EI) by phosphoenolpyruvate (PEP). The EI phosphorylation state determines the phosphorylation states of all other PTS components and is thought to play a central role in the regulation of several metabolic pathways and to control the biology of bacterial cells at multiple levels, for example, affecting virulence and biofilm formation. Given the pivotal role of EI in bacterial metabolism, an improved understanding of the mechanisms controlling its activity could inform future strategies for bioengineering and antimicrobial design. Here, we report an enzymatic assay, based on Selective Optimized Flip Angle Short Transient (SOFAST) NMR experiments, to investigate the effect of the small-molecule metabolite α-ketoglutarate (αKG) on the kinetics of the EI-catalyzed phosphoryl transfer reaction. We show that at experimental conditions favoring the monomeric form of EI, αKG promotes dimerization and acts as an allosteric stimulator of the enzyme. However, when the oligomerization state of EI is shifted toward the dimeric species, αKG functions as a competitive inhibitor of EI. We developed a kinetic model that fully accounted for the experimental data and indicated that bacterial cells might use the observed interplay between allosteric stimulation and competitive inhibition of EI by αKG to respond to physiological fluctuations in the intracellular environment. We expect that the mechanism for regulating EI activity revealed here is common to several other oligomeric enzymes.

1H, 15N, 13C backbone resonance assignment of the C-terminal domain of enzyme I from Thermoanaerobacter tengcongensis.

Phosphoenolpyruvate binding to the C-terminal domain (EIC) of enzyme I of the bacterial phosphotransferase system (PTS) initiates a phosphorylation cascade that results in sugar translocation across the cell membrane and controls a large number of essential pathways in bacterial metabolism. EIC undergoes an expanded to compact conformational equilibrium that is regulated by ligand binding and determines the phosphorylation state of the overall PTS. Here, we report the backbone 1H, 15N and 13C chemical shift assignments of the 70 kDa EIC dimer from the thermophilic bacterium Thermoanaerobacter tengcongensis. Assignments were obtained at 70 °C by heteronuclear multidimensional NMR spectroscopy. In total, 90% of all backbone resonances were assigned, with 264 out of a possible 299 residues assigned in the 1H-15N TROSY spectrum. The secondary structure predicted from the assigned backbone resonance using the program TALOS+ is in good agreement with the X-ray crystal structure of T. tengcongensis EIC. The reported assignments will allow detailed structural and thermodynamic investigations on the coupling between ligand binding and conformational dynamics in EIC.

Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering.

Enzyme I (EI) is the first component in the bacterial phosphotransferase system, a signal transduction pathway in which phosphoryl transfer through a series of bimolecular protein-protein interactions is coupled to sugar transport across the membrane. EI is a multidomain, 128-kDa homodimer that has been shown to exist in two conformational states related to one another by two large (50-90°) rigid body domain reorientations. The open conformation of apo EI allows phosphoryl transfer from His189 located in the N-terminal domain α/ß (EIN(α/ß)) subdomain to the downstream protein partner bound to the EIN(α) subdomain. The closed conformation, observed in a trapped phosphoryl transfer intermediate, brings the EIN(α/ß) subdomain into close proximity to the C-terminal dimerization domain (EIC), thereby permitting in-line phosphoryl transfer from phosphoenolpyruvate (PEP) bound to EIC to His189. Here, we investigate the solution conformation of a complex of an active site mutant of EI (H189A) with PEP. Simulated annealing refinement driven simultaneously by solution small angle X-ray scattering and NMR residual dipolar coupling data demonstrates unambiguously that the EI(H189A)-PEP complex exists in a dynamic equilibrium between two approximately equally populated conformational states, one corresponding to the closed structure and the other to a partially closed species. The latter likely represents an intermediate in the open-to-closed transition.

Coupling between overall rotational diffusion and domain motions in proteins and its effect on dielectric spectra.

In this work, we formulate a closed-form solution of the model of a semirigid molecule for the case of fluctuating and reorienting molecular electric dipole moment. We illustrate with numeric calculations the impact of protein domain motions on dielectric spectra using the example of the 128 kDa protein dimer of Enzyme I. We demonstrate that the most drastic effect occurs for situations when the characteristic time of protein domain dynamics is comparable to the time of overall molecular rotational diffusion. We suggest that protein domain motions could be a possible explanation for the high-frequency contribution that accompanies the major relaxation dispersion peak in the dielectric spectra of protein aqueous solutions. We propose that the presented computational methodology could be used for the simultaneous analysis of dielectric spectroscopy and nuclear magnetic resonance data. Proteins 2015; 83:1571-1581. © 2015 Wiley Periodicals, Inc.

The substrate-free and -bound crystal structures of the duplicated taurocyamine kinase from the human parasite Schistosoma mansoni.

The taurocyamine kinase from the blood fluke Schistosoma mansoni (SmTK) belongs to the phosphagen kinase (PK) family and catalyzes the reversible Mg(2+)-dependent transfer of a phosphoryl group between ATP and taurocyamine. SmTK is derived from gene duplication, as are all known trematode TKs. Our crystallographic study of SmTK reveals the first atomic structure of both a TK and a PK with a bilobal structure. The two unliganded lobes present a canonical open conformation and interact via their respective C- and N-terminal domains at a helix-mediated interface. This spatial arrangement differs from that observed in true dimeric PKs, in which both N-terminal domains make contact. Our structures of SmTK complexed with taurocyamine or l-arginine compounds explain the mechanism by which an arginine residue of the phosphagen specificity loop is crucial for substrate specificity. An SmTK crystal was soaked with the dead end transition state analog (TSA) components taurocyamine-NO3 (2-)-MgADP. One SmTK monomer was observed with two bound TSAs and an asymmetric conformation, with the first lobe semiclosed and the second closed. However, isothermal titration calorimetry and enzyme kinetics experiments showed that the two lobes function independently. A small angle x-ray scattering model of SmTK-TSA in solution with two closed active sites was generated.

Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I.

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. Here we characterize the perturbations generated by two small molecules, the natural substrate phosphoenolpyruvate and the inhibitor α-ketoglutarate, on the structure and dynamics of EI using NMR, small-angle X-ray scattering and biochemical techniques. The results indicate unambiguously that the open-to-closed conformational switch of EI is triggered by complete suppression of micro- to millisecond dynamics within the C-terminal domain of EI. Indeed, we show that a ligand-induced transition from a dynamic to a more rigid conformational state of the C-terminal domain stabilizes the interface between the N- and C-terminal domains observed in the structure of the closed state, thereby promoting the resulting conformational switch and autophosphorylation of EI. The mechanisms described here may be common to several other multidomain proteins and allosteric systems.

The role of Y84 on domain 1 and Y87 on domain 2 of Paragonimus westermani taurocyamine kinase: Insights on the substrate binding mechanism of a trematode phosphagen kinase.

The two-domain taurocyamine kinase (TK) from Paragonimus westermani was suggested to have a unique substrate binding mechanism. We performed site-directed mutagenesis on each domain of this TK and compared the kinetic parameters Km(Tc) and Vmax with that of the wild-type to determine putative amino acids involved in substrate recognition and binding. Replacement of Y84 on domain 1 and Y87 on domain 2 with R resulted in the loss of activity for the substrate taurocyamine. Y84E mutant has a dramatic decrease in affinity and activity for taurocyamine while Y87E has completely lost catalytic activity. Substituting H and I on the said positions also resulted in significant changes in activity. Mutation of the residues A59 on the GS region of domain 1 also caused significant decrease in affinity and activity while mutation on the equivalent position on domain 2 resulted in complete loss of activity.

Characterization of a putative oomycete taurocyamine kinase: Implications for the evolution of the phosphagen kinase family.

Phosphagen kinases (PKs) are known to be distributed throughout the animal kingdom, but have recently been discovered in some protozoan and bacterial species. Within animal species, these enzymes play a critical role in energy homeostasis by catalyzing the reversible transfer of a high-energy phosphoryl group from Mg⋅ATP to an acceptor molecule containing a guanidinium group. In this work, a putative PK gene was identified in the oomycete Phytophthora sojae that was predicted, based on sequence homology, to encode a multimeric hypotaurocyamine kinase. The recombinant P. sojae enzyme was purified and shown to catalyze taurocyamine phosphorylation efficiently (kcat/KM (taurocyamine) = 2 × 10(5) M(-1) s(-1)) and glycocyamine phosphorylation only weakly (kcat/KM (glycocyamine) = 2 × 10(2) M(-1) s(-1)), but lacked any observable kinase activity with the more ubiquitous guanidinium substrates, creatine or arginine. Additionally, the enzyme was observed to be dimeric but lacked cooperativity between the subunits in forming a transition state analog complex. These results suggest that protozoan PKs may exhibit more diversity in substrate specificity than was previously thought.

Medicago truncatula histidine-containing phosphotransfer protein: structural and biochemical insights into the cytokinin transduction pathway in plants.

Histidine-containing phosphotransfer proteins (HPts) take part in hormone signal transduction in higher plants. The overall pathway of this process is reminiscent of the two-component system initially identified in prokaryotes. HPts function in histidine-aspartate phosphorelays in which they mediate the signal from sensory kinases (usually membrane proteins) to RRs in the nucleus. Here, we report the crystal structure of an HPt protein from Medicago truncatula (MtHPt1) determined at 1.45 Å resolution and refined to an R-factor of 16.7% using low-temperature synchrotron-radiation X-ray diffraction data. There is one MtHPt1 molecule in the asymmetric unit of the crystal lattice with P2(1)2(1)2(1) symmetry. The protein fold consists of six α helices, four of which form a C-terminal helix bundle. The coiled-coil structure of the bundle is stabilized by a network of S-aromatic interactions involving highly conserved sulfur-containing residues. The structure reveals a solvent-exposed side chain of His79, which is the phosphorylation site, as demonstrated by autoradiography combined with site-directed mutation. It is surrounded by highly conserved residues present in all plant HPts. These residues form a putative docking interface for either the receiver domain of the sensory kinase, or for the RR. The biological activity of MtHPt1 was tested by autoradiography. It demonstrated phosphorylation by the intracellular kinase domain of the cytokinin receptor MtCRE1. Complex formation between MtHPt1 and the intracellular fragment of MtCRE1 was confirmed by thermophoresis, with a dissociation constant K(d) of 14 µM.

Gene structure of the two-domain taurocyamine kinase from Paragonimus westermani: evidence for a distinct lineage of trematode phosphagen kinases.

Taurocyamine kinase (TK) is an enzyme that catalyzes the reversible transfer of a phosphate between ATP and taurocyamine. Annelid TKs were suggested to have evolved from a CK ancestor. However, TKs from the lung fluke Paragonimus westermani comprised another lineage. Construction of phylogenetic tree and comparison of exon/intron organization showed that P. westermani TK and other trematode TKs evolved from a molluscan arginine kinase (AK) gene. Exon shuffling probably caused the changes in amino acid sequence thereby changing the affinity from AK to TK. The present study provides new insights on the evolution of phosphagen kinases found in trematodes.

Phosphagen kinase in Schistosoma japonicum: characterization of its enzymatic properties and determination of its gene structure.

Phosphagen kinases (PKs) play a major role in the regulation of energy metabolism in animals. Creatine kinase (CK) is the sole PK in vertebrates, whereas several PKs are present in invertebrates. Here, we report the enzymatic properties and gene structure of PK in the trematode Schistosoma japonicum (Sj). SjPK has a unique contiguous dimeric structure comprising domain 1 (D1) and domain 2 (D2). The three states of the recombinant SjPK (D1, D2, and D1D2) show a specific activity for the substrate taurocyamine. The comparison of the two domains of SjPK revealed that D1 had a high turnover rate (kcat=52.91) and D2 exhibited a high affinity for taurocyamine (Km(Tauro) =0.53±0.06). The full-length protein exhibited higher affinity for taurocyamine (Km(Tauro) =0.47±0.03) than the truncated domains (D1=1.30±0.10, D2=0.53±0.06). D1D2 also exhibited higher catalytic efficiency (kcat/Km(Tauro) =82.98) than D1 (40.70) and D2 (29.04). These results demonstrated that both domains of SjTKD1D2 interacted efficiently and remained functional. The three-dimensional structure of SjPKD1 was constructed by the homology modeling based on the transition state analog complex state of Limulus AK. This protein model of SjPKD1 suggests that the overall structure is almost conserve between SjPKD1 and Limulus AK except for the flexible loops, that is, particularly guanidino-specificity (GS) region, which is associated with the recognition of the corresponding guanidino substrate. The constructed NJ tree and the comparison of exon/intron organization suggest that SjTK has evolved from an arginine kinase (AK) gene. SjTK has potential as a novel antihelminthic drug target as it is absent in mammals and its strong activity may imply a significant role for this protein in the energy metabolism of the parasite.

A novel taurocyamine kinase found in the protist Phytophthora infestans.

Phosphagen kinase (PK), which is typically in the form of creatine kinase (CK; EC 2.7.3.2) in vertebrates or arginine kinase (AK; EC 2.7.3.3) in invertebrates, plays a key role in ATP buffering systems of tissues and nerves that display high and variable rates of ATP turnover. The enzyme is also found with intermittent occurrence as AK in unicellular organisms, protist and bacteria species, suggesting an ancient origin of AK. Through a database search, we identified two novel PK genes, coding 40- and 80-kDa (contiguous dimer) enzymes in the protist Phytophthora infestans. Both enzymes showed strong activity for taurocyamine and, in addition, we detected taurocyamine in cell extracts of P. infestans. Thus, the enzyme was identified to be taurocyamine kinase (TK; EC 2.7.3.4). This was the first phosphagen kinase, other than AK, to be found in unicellular organisms. Their position on the phylogenetic tree indicates that P. infestans TKs evolved uniquely at an early stage of evolution. Occurrence of TK in protists suggests that PK enzymes show flexible substrate specificity.

Peptides as inhibitors of the first phosphorylation step of the Streptomyces coelicolor phosphoenolpyruvate: sugar phosphotransferase system.

The phosphotransferase system (PTS) controls the use of sugars in bacteria. The PTS is ubiquitous in bacteria, but it does not occur in plants and animals; it modulates catabolite repression, intermediate metabolism, gene expression, and chemotaxis. Its uniqueness and pleiotropic function make the PTS an attractive target for new antibacterial drugs. The PTS is constituted of two general proteins, namely, enzyme I (EI) and the histidine phosphocarrier (HPr), and various sugar-specific permeases. EI has two domains: the N-terminal domain (EIN), which binds to HPr, and the C-terminal domain (EIC), which contains the dimerization interface. In this work, we determined the binding affinities of peptides derived from EIN of Streptomyces coelicolor (EIN(sc)) against HPr of the same organism (HPr(sc)), by using nuclear magnetic resonance and isothermal titration calorimetry techniques. Furthermore, we measured the affinity of EIN(sc) for (i) a peptide derived from HPr(sc), containing the active-site histidine, and (ii) other peptides identified previously by phage display and combinatorial chemistry in Escherichia coli [Mukhija, S. L., et al (1998) Eur. J. Biochem. 254, 433-438; Mukhija, S., and Erni, B. (1997) Mol. Microbiol. 25, 1159-1166]. The affinities were in the range of ~10 µM, being slightly higher for the binding of EIN(sc) with peptides derived from HPr(sc), phage display, or combinatorial chemistry (K(D) ~ 5 µM). Because the affinity of intact EIN(sc) for the whole HPr(sc) is 12 µM, we suggest that the assayed peptides might be considered as good hit compounds for inhibiting the interaction between HPr(sc) and EIN(sc).

Identification of amino acid residues responsible for taurocyamine binding in mitochondrial taurocyamine kinase from Arenicola brasiliensis.

In order to investigate the residues associated with binding of the substrate taurocyamine in Arenicola mitochondrial taurocyamine kinase (TK), we performed Ala-scanning of the amino acid sequence HTKTV at positions 67-71 on the GS loop, and determined apparent K(m) and V(max) (appK(m) and appV(max), respectively) of the mutant forms for the substrates taurocyamine and glycocyamine. The appK(m) values for taurocyamine of the K69A, T70A and V71A mutants were significantly increased as compared with wild-type, suggesting that these residues are associated with taurocyamine binding. Of special interest is a property of V71A mutant: its catalytic efficiency for glycocyamine was twice that for taurocyamine, indicating that the V71A mutant acts like a glycocyamine kinase, rather than a TK. The role of the amino acid residue K95 of Arenicola MiTK was also examined. K95 was replaced with R, H, Y, I, A and E. K95R, K95H and K95I have a 3-fold higher affinity for taurocyamine, and activity was largely lost in K95E. On the other hand, the K95Y mutant showed a rather unique feature; namely, an increase in substrate concentration caused a decrease in initial velocity of the reaction (substrate inhibition). This is the first report on the key amino acid residues responsible for taurocyamine binding in mitochondrial TK.

The structure of lombricine kinase: implications for phosphagen kinase conformational changes.

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His(178). Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.

Deuteration of Escherichia coli enzyme I(Ntr) alters its stability.

Enzyme I(Ntr) is the first protein in the nitrogen phosphotransferase pathway. Using an array of biochemical and biophysical tools, we characterized the protein, compared its properties to that of EI of the carbohydrate PTS and, in addition, examined the effect of substitution of all nonexchangeable protons by deuterium (perdeuteration) on the properties of EI(Ntr). Notably, we find that the catalytic function (autophosphorylation and phosphotransfer to NPr) remains unperturbed while its stability is modulated by deuteration. In particular, the deuterated form exhibits a reduction of approximately 4°C in thermal stability, enhanced oligomerization propensity, as well as increased sensitivity to proteolysis in vitro. We investigated tertiary, secondary, and local structural changes, both in the absence and presence of PEP, using near- and far-UV circular dichroism and Trp fluorescence spectroscopy. Our data demonstrate that the aromatic residues are particularly sensitive probes for detecting effects of deuteration with an enhanced quantum yield upon PEP binding and apparent decreases in tertiary contacts for Tyr and Trp side chains. Trp mutagenesis studies showed that the region around Trp522 responds to binding of both PEP and NPr. The significance of these results in the context of structural analysis of EI(Ntr) are evaluated.