Structural basis for the dynamics of human methionyl-tRNA synthetase in multi-tRNA synthetase complexes.

In mammals, eight aminoacyl-tRNA synthetases (AARSs) and three AARS-interacting multifunctional proteins (AIMPs) form a multi-tRNA synthetase complex (MSC). MSC components possess extension peptides for MSC assembly and specific functions. Human cytosolic methionyl-tRNA synthetase (MRS) has appended peptides at both termini of the catalytic main body. The N-terminal extension includes a glutathione transferase (GST) domain responsible for interacting with AIMP3, and a long linker peptide between the GST and catalytic domains. Herein, we determined crystal structures of the human MRS catalytic main body, and the complex of the GST domain and AIMP3. The structures reveal human-specific structural details of the MRS, and provide a dynamic model for MRS at the level of domain orientation. A movement of zinc knuckles inserted in the catalytic domain is required for MRS catalytic activity. Depending on the position of the GST domain relative to the catalytic main body, MRS can either block or present its tRNA binding site. Since MRS is part of a huge MSC, we propose a dynamic switching between two possible MRS conformations; a closed conformation in which the catalytic domain is compactly attached to the MSC, and an open conformation with a free catalytic domain dissociated from other MSC components.

X-ray Crystal Structure-Guided Design and Optimization of 7H-Pyrrolo[2,3-d]pyrimidine-5-carbonitrile Scaffold as a Potent and Orally Active Monopolar Spindle 1 Inhibitor.

Triple-negative breast cancer (TNBC) is an aggressive breast-cancer subtype associated with poor prognosis and high relapse rates. Monopolar spindle 1 kinase (MPS1) is an apical dual-specificity protein kinase that is over-expressed in TNBC. We herein report a highly selective MPS1 inhibitor based on a 7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile scaffold. Our lead optimization was guided by key X-ray crystal structure analysis. In vivo evaluation of candidate (9) is shown to effectively mitigate human TNBC cell proliferation.

Symmetric Assembly of a Decameric Subcomplex in Human Multi-tRNA Synthetase Complex Via Interactions between Glutathione Transferase-Homology Domains and Aspartyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (AARSs) ligate amino acids to their cognate tRNAs during protein synthesis. In humans, eight AARSs and three non-enzymatic AARS-interacting multifunctional proteins (AIMP1-3), which are involved in various biological processes, form a multi-tRNA synthetase complex (MSC). Elucidation of the structures and multiple functions of individual AARSs and AIMPs has aided current understanding of the structural arrangement of MSC components and their assembly processes. Here, we report the crystal structure of a complex comprising a motif from aspartyl-tRNA synthetase (DRS) and the glutathione transferase (GST)-homology domains of methionyl-tRNA synthetase (MRS), glutamyl-prolyl-tRNA synthetase (EPRS), AIMP2, and AIMP3. In the crystal structure, the four GST domains are assembled in the order of MRS-AIMP3-EPRS-AIMP2, and the GST domain of AIMP2 binds DRS through the ß-sheet in the GST domain. The C-terminus of AIMP3 enhances the binding of DRS to the tetrameric GST complex. A DRS dimer and two GST tetramers binding to the dimer with 2-fold symmetry complete a decameric complex. The formation of this complex enhances the stability of DRS and enables it to retain its reaction intermediate, aspartyl adenylate. Since the catalytic domains of MRS and EPRS are connected to the decameric complex through their flexible linker peptides, and lysyl-tRNA synthetase and AIMP1 are also linked to the complex via the N-terminal region of AIMP2, the DRS-GST tetramer complex functions as a frame in the MSC.

Structural basis for substrate binding to human pyridoxal 5'-phosphate phosphatase/chronophin by a conformational change.

Human pyridoxal 5'-phosphate phosphatase (PLPP), also known as a chronophin, is a phosphatase belonging to subfamily II of the HAD phosphatases, characterized by a large cap domain. As a member of the subfamily, its cap-open conformation is expected for substrate binding. We determined apo and PLP-bound PLPP/chronophin structures showing a cap-closed conformation. The active site, in which a PLP molecule was found, is too small to accommodate a phospho-cofilin peptide, the substrate of chronophin. A conformational change to a cap-open conformation may be required for substrate binding. The core and cap domains are joined through linker peptide hinges that change conformation to open the active site. The crystal structures reveal that a disulphide bond between the cap and core domains restricts the hinge motion. The enzyme displays PLP dephosphorylation activity in the cap-closed conformation with the disulphide bond and even in the crystal state, in which repositioning of the cap and core domains is restricted. Structural analysis suggests that a small substrate such as PLP can bind to the active site through a small movement of a local motif. However, a change to the cap-open conformation is required for binding of larger substrates such as phosphopeptides to the active site.

NADP+-dependent cytosolic isocitrate dehydrogenase provides NADPH in the presence of cadmium due to the moderate chelating effect of glutathione.

Cadmium (Cd2+) is toxic to living organisms because it causes the malfunction of essential proteins and induces oxidative stress. NADP+-dependent cytosolic isocitrate dehydrogenase (IDH) provides reducing energy to counteract oxidative stress via oxidative decarboxylation of isocitrate. Intriguingly, the effects of Cd2+ on the activity of IDH are both positive and negative, and to understand the molecular basis, we determined the crystal structure of NADP+-dependent cytosolic IDH in the presence of Cd2+. The structure includes two Cd2+ ions, one coordinated by active site residues and another near a cysteine residue. Cd2+ presumably inactivates IDH due to its high affinity for thiols, leading to a covalent enzyme modification. However, Cd2+ also activates IDH by providing a divalent cation required for catalytic activity. Inactivation of IDH by Cd2+ is less effective when the enzyme is activated with Cd2+ than Mg2+. Although reducing agents cannot restore activity following inactivation by Cd2+, they can maintain IDH activity by chelating Cd2+. Glutathione, a cellular sulphydryl reductant, has a moderate affinity for Cd2+, allowing IDH to be activated with residual Cd2+, unlike dithiothreitol, which has a much higher affinity. In the presence of Cd2+-consuming cellular antioxidants, cells must continually supply reductants to protect against oxidative stress. The ability of IDH to utilise Cd2+ to generate NADPH could allow cells to protect themselves against Cd2+.

Oncogenic Mutation of AIMP2/p38 Inhibits Its Tumor-Suppressive Interaction with Smurf2.

AIMP2/p38 is a multifunctional tumor suppressor that normally resides in the cytosol as a scaffold protein of the multi-tRNA synthetase complex (MSC). One of the tumor-suppressive functions of AIMP2 is to facilitate ubiquitin-mediated degradation of FUSE-binding protein (FBP, FUBP1), a transcriptional activator of c-Myc. However, the mechanism by which AIMP2 functions within this pathway and its significance in tumorigenesis are uncertain. Here, we report that Smurf2 is responsible for AIMP2-mediated ubiquitination of FBP, and a mutation in AIMP2 that inhibited its nuclear interaction with Smurf2 enhanced cellular transformation and tumorigenesis in vivo Treatment of HeLa cells with TGFß resulted in the phosphorylation of AIMP2 on S156, a residue that is exposed on the embedded GST domain of AIMP2. We further found that phospho-AIMP2 dissociated from the MSC and translocated to the nucleus, where it bound to Smurf2, enhancing ubiquitination of FBP. AIMP2 also inhibited nuclear export of Smurf2 to sustain TGFß signaling. Collectively, these findings present a novel tumor-suppressive interaction between AIMP2 and Smurf2 and suggest that the disruption of this interaction can lead to oncogenic transformation. Cancer Res; 76(11); 3422-36. ©2016 AACR.

Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains.

Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors.

Optimal difference test sequence and power for discriminating soups of varying sodium content: DTFM version of dual-reference duo-trio with unspecified tetrad tests.

As sodium reduction has become very important in the food industry, various types of unspecified duo-trio tests have been studied to improve its efficiency for studying samples with high sodium content, and a constant-saltier-reference duo-trio test with dual reference, one reference in the first position and the second reference in the middle between the two test stimuli (DTFM), has been recommended. For the duo-trio test, a 'comparison of distance' (COD) strategy has been generally assumed. Yet, theoretically for DTFM, the 2-AFC reminder (2-AFCR) τ-strategy is also possible, which would make DTFM more efficient than the unspecified tetrad test. In this study, the hypothesis was that when subjects are pre-exposed to two types of samples, the 2-AFCR τ-strategy can be adopted in a constant-reference DTFM using a fixed design experiment. In order to test this hypothesis, unspecified tetrad tests involving categorization tasks were used as a means of pre-exposure to the two types of samples for DTFM, and a performance comparison was conducted. Two groups of 39 untrained/naive subjects performed both the unspecified tetrad and DTFM tests in varying orders for the purpose of discriminating two different soup samples of varying sodium content. A comparison of the d' estimate across different methods supported the hypothesis that the more efficient 2-AFCR τ-strategy was appropriate when the tetrad test preceded DTFM, while when DTFM was performed first without pre-categorization of samples, the conventional duo-trio COD strategy was appropriate for the constant-reference DTFM.

Serine 83 in DosR, a response regulator from Mycobacterium tuberculosis, promotes its transition from an activated, phosphorylated state to an inactive, unphosphorylated state.

A sensor kinase, DosS, and its corresponding response regulator, DosR, constitute a two component system for regulating gene expression under hypoxic conditions in Mycobacterium tuberculosis. Among response regulators in M. tuberculosis, NarL has high sequence similarity to DosR, and autophosphorylated DosS transfers its phosphate group not only to DosR but also to NarL. Phosphorylated DosR is more rapidly dephosphorylated than phosphorylated NarL. DosR and NarL differ with respect to the amino acids at positions T+1 and T+2 around the phosphorylation sites in the N-terminal phosphoacceptor domain; NarL has S83 and Y84, whereas DosR has A90 and H91. A DosR S83A mutant shows prolonged phosphorylation. Structural comparison with a histidinol phosphate phosphatase suggests that the hydroxyl group of DosR S83 could play a role in activating the water molecule involved in the triggering of autodephosphorylation.

Activation of ATP binding for the autophosphorylation of DosS, a Mycobacterium tuberculosis histidine kinase lacking an ATP lid motif.

The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity.

Blockage of the channel to heme by the E87 side chain in the GAF domain of Mycobacterium tuberculosis DosS confers the unique sensitivity of DosS to oxygen.

Two sensor kinases, DosS and DosT, are responsible for recognition of hypoxia in Mycobacterium tuberculosis. Both proteins are structurally similar to each other, but DosS is a redox sensor while DosT binds oxygen. The primary difference between the two proteins is the channel to the heme present in their GAF domains. DosS has a channel that is blocked by E87 while DosT has an open channel. Absorption spectra of DosS mutants with an open channel show that they bind oxygen as DosT does when they are exposed to air, while DosT G85E mutant is oxidized similarly to DosS without formation of an oxy-ferrous form. This suggests that oxygen accessibility to heme is the primary factor governing the oxygen-binding properties of these proteins.

Structural and functional analysis of bacterial flavin-containing monooxygenase reveals its ping-pong-type reaction mechanism.

A bacterial flavin-containing monooxygenase (bFMO) catalyses the oxygenation of indole to produce indigoid compounds. In the reductive half of the indole oxygenation reaction, NADPH acts as a reducing agent, and NADP(+) remains at the active site, protecting bFMO from reoxidation. Here, the crystal structures of bFMO and bFMO in complex with NADP(+), and a mutant bFMO(Y207S), which lacks indole oxygenation activity, with and without indole are reported. The crystal structures revealed overlapping binding sites for NADP(+) and indole, suggestive of a double-displacement reaction mechanism for bFMO. In biochemical assays, indole inhibited NADPH oxidase activity, and NADPH in turn inhibited the binding of indole and decreased indoxyl production. Comparison of the structures of bFMO with and without bound NADP(+) revealed that NADPH induces conformational changes in two active site motifs. One of the motifs contained Arg-229, which participates in interactions with the phosphate group of NADPH and appears be a determinant of the preferential binding of bFMO to NADPH rather than NADH. The second motif contained Tyr-207. The mutant bFMO(Y207S) exhibited very little indoxyl producing activity; however, the NADPH oxidase activity of the mutant was higher than the wild-type enzyme. It suggests a role for Y207, in the protection of hydroperoxyFAD. We describe an indole oxygenation reaction mechanism for bFMO that involves a ping-pong-like interaction of NADPH and indole.

Substrate binding mechanism of a type I extradiol dioxygenase.

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a ß-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and ß-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.

Structural insight into the heme-based redox sensing by DosS from Mycobacterium tuberculosis.

Mycobacterium tuberculosis is thought to undergo transformation into its non-replicating persistence state under the influence of hypoxia or nitric oxide (NO). This transformation is thought to be mediated via two sensor histidine kinases, DosS and DosT, each of which contains two GAF domains that are responsible for detecting oxygen tension. In this study we determined the crystal structures of the first GAF domain (GAF-A) of DosS, which shows an interaction with a heme. A b-type heme was embedded in a hydrophobic cavity of the GAF-A domain and was roughly perpendicular to the beta-sheet of the GAF domain. The heme iron was liganded by His-149 at the proximal heme axial position. The iron, in the oxidized form, was six-coordinated with a water molecule at the distal position. Upon reduction, the iron, in ferrous form, was five-coordinated, and when the GAF domain was exposed to atmospheric O(2), the ferrous form was oxidized to generate the Met form rather than a ferrous O(2)-bound form. Because the heme is isolated inside the GAF domain, its accessibility is restricted. However, a defined hydrogen bond network found at the heme site could accelerate the electron transferability and would explain why DosS was unable to bind O(2). Flavin nucleotides were shown to reduce the heme iron of DosS while NADH was unable to do so. These results suggest that DosS is a redox sensor and detects hypoxic conditions by its reduction.

O2- and NO-sensing mechanism through the DevSR two-component system in Mycobacterium smegmatis.

The DevS histidine kinase of Mycobacterium smegmatis contains tandem GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The heme iron of DevS is in the ferrous state when purified and is resistant to autooxidation from a ferrous to a ferric state in the presence of O(2). The redox property of the heme and the results of sequence comparison analysis indicate that DevS of M. smegmatis is more closely related to DosT of Mycobacterium tuberculosis than DevS of M. tuberculosis. The binding of O(2) to the deoxyferrous heme led to a decrease in the autokinase activity of DevS, whereas NO binding did not. The regulation of DevS autokinase activity in response to O(2) and NO was not observed in the DevS derivatives lacking its heme, indicating that the ligand-binding state of the heme plays an important role in the regulation of DevS kinase activity. The redox state of the quinone/quinol pool of the respiratory electron transport chain appears not to be implicated in the regulation of DevS activity. Neither cyclic GMP (cGMP) nor cAMP affected DevS autokinase activity, excluding the possibility that the cyclic nucleotides serve as the effector molecules to modulate DevS kinase activity. The three-dimensional structure of the putative GAF-B domain revealed that it has a GAF folding structure without cyclic nucleotide binding capacity.

Crystallization and preliminary crystallographic analysis of the second GAF domain of DevS from Mycobacterium smegmatis.

Mycobacterium tuberculosis is known to transform into the nonreplicating persistence state under the influence of hypoxia or nitric oxide. DevS-DevR is a two-component regulatory system that mediates the genetic response for the transformation. DevS is a histidine kinase that contains two GAF domains for sensing hypoxia or nitric oxide. The second GAF from M. smegmatis DevS was crystallized using the sitting-drop vapour-diffusion method in the presence of sodium citrate and 2-propanol as precipitants. X-ray diffraction data were collected from crystals containing selenomethionine to a maximum resolution of 2.0 A on a synchrotron beamline. The crystals belong to the hexagonal space group P6(1). The asymmetric unit contains one molecule, corresponding to a packing density of 2.5 A(3) Da(-1). The selenium substructure was determined by the single anomalous dispersion method and structure refinement is in progress.