Secreted tryptophanyl-tRNA synthetase as a primary defence system against infection.

The N-terminal truncated form of a protein synthesis enzyme, tryptophanyl-tRNA synthetase (mini-WRS), is secreted as an angiostatic ligand. However, the secretion and function of the full-length WRS (FL-WRS) remain unknown. Here, we report that the FL-WRS, but not mini-WRS, is rapidly secreted upon pathogen infection to prime innate immunity. Blood levels of FL-WRS were increased in sepsis patients, but not in those with sterile inflammation. FL-WRS was secreted from monocytes and directly bound to macrophages via a toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex to induce phagocytosis and chemokine production. Administration of FL-WRS into Salmonella typhimurium-infected mice reduced the levels of bacteria and improved mouse survival, whereas its titration with the specific antibody aggravated the infection. The N-terminal 154-amino-acid eukaryote-specific peptide of WRS was sufficient to recapitulate FL-WRS activity and its interaction mode with TLR4-MD2 is now suggested. Based on these results, secretion of FL-WRS appears to work as a primary defence system against infection, acting before full activation of innate immunity.

Chemical inhibition of prometastatic lysyl-tRNA synthetase-laminin receptor interaction.

Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds KRS, impinged on the interaction of KRS with 67LR and suppressed metastasis in three different mouse models. The compound inhibited the KRS-67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS-67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS.

Crystal structure of human cytosolic aspartyl-tRNA synthetase, a component of multi-tRNA synthetase complex.

Human cytosolic aspartyl-tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi-tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Å(2) which comprises 16.6% of the monomeric surface area. Our structure reveals the C-terminal end of the N-helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N-helix for the transfer of tRNA(Asp) to elongation factor 1α. From our analyses of the crystal structure and post-translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions.

Structural characterization of the bifunctional glucanase-xylanase CelM2 reveals the metal effect and substrate-binding moiety.

The bifunctional glycoside hydrolase enzyme, CelM2, is able to hydrolyze glucan and xylan effectively. The crystal structure of this protein has been determined, providing useful sequential and structural information [K.H. Nam, S.J. Kim, K.Y. Hwang, Crystal structure of CelM2, a bifunctional glucanase-xylanase protein from a metagenome library, Biochem. Biophys. Res. Commun. 383 (2009) 183-186]. In addition, this protein is a good model for understanding bifunctional enzymes, and it will provide information relevant for genetic engineering that will be useful in the design of bifunctional proteins. However, previous structural characterization was not sufficient to develop an understanding of the metal ion and substrate-binding moiety. Herein, we determined the metal-binding site of CelM2 using zinc ions. Our results revealed that the zinc ions participate in the crystallographic packing and enzyme folding of the external region of the TIM-like barrel domain. Based on our structure, zinc ions induce the passive form of the CAP region at the catalytic cleft of the CelM2 protein. Moreover, glucose was bound to the CelM2 structure at the catalytic site. This structure provides the binding moiety that binds to the hydroxyl group of substrates such as cellulose. In addition, a structural comparison of celM2 with Cel44 provides a good model of the binding mode of CelM2. Thus, our study represents a novel structural characterization of the metal-binding site and the structure of the complex formed between CelM2 and its substrate.

Structural and mutational analysis of tRNA intron-splicing endonuclease from Thermoplasma acidophilum DSM 1728: catalytic mechanism of tRNA intron-splicing endonucleases.

In archaea, RNA endonucleases that act specifically on RNA with bulge-helix-bulge motifs play the main role in the recognition and excision of introns, while the eukaryal enzymes use a measuring mechanism to determine the positions of the universally positioned splice sites relative to the conserved domain of pre-tRNA. Two crystallographic structures of tRNA intron-splicing endonuclease from Thermoplasma acidophilum DSM 1728 (EndA(Ta)) have been solved to 2.5-A and 2.7-A resolution by molecular replacement, using the 2.7-A resolution data as the initial model and the single-wavelength anomalous-dispersion phasing method using selenomethionine as anomalous signals, respectively. The models show that EndA(Ta) is a homodimer and that it has overall folding similar to that of other archaeal tRNA endonucleases. From structural and mutational analyses of H236A, Y229F, and K265I in vitro, we have demonstrated that they play critical roles in recognizing the splice site and in cleaving the pre-tRNA substrate.

Expression, purification, and preliminary X-ray crystallographic analysis of the complex of G(alphai3)-RGS5 from human with GDP/Mg2+)/AlF4-.

Regulator of G-protein signaling 5 (RGS5), an inhibitor of Gq and Gi activation, is a member of the small RGS protein subfamily. However, despite significant process in the investigation of RGS5, no structure is yet available. In order to elucidate the mechanism of the RGS5 in G protein signaling pathway, we have overexpressed the RGS5 and Galphai(3) from human in Escherichia coli and crystallized the complex of RGS5 and Galphai(3) proteins with GDP/Mg(2+)/AlF(4)(-) at 3.0 A resolution using a synchrotron radiation source. The complex crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit cell parameters a=b=95.9 A, and c=138.8 A. Assuming one complex protein in the crystallographic asymmetric unit, the calculated Matthews parameter (V(M)) is 2.57 A(3)/Da and solvent content is 52.2 %.

Crystallization and preliminary X-ray crystallographic analysis of human RGS10 complexed with Galphai3.

G-protein-coupled receptors, which are major targets for drug discovery, play a major role in diverse physiological processes by relating changes in the extracellular environment to intracellular functions via activation of heterotrimeric G-proteins. However, G-protein activity is also modulated by a family of proteins called regulators of G-protein signalling (RGS), which are classified into six subfamilies. RGS10 belongs to the subgroup D/R12 and is known to act specifically on activated forms of three Galpha proteins (Galphai3, Galphaz and Galphao but not Galphas). It is abundantly expressed in brain and immune tissues and has been implicated in the pathophysiology of schizophrenia. The RGS domain of RGS10 was cloned, purified, complexed with human Galphai3 and crystallized. The crystals containing both RGS and Galphai3 belong to space group P4(3)2(1)2 (or P4(1)2(1)2), with unit-cell parameters a = 99.88, b = 99.88, c = 144.59 A, alpha = beta = gamma = 90 degrees . A full set of diffraction data were collected to 2.5 A resolution at 100 K using synchrotron radiation at Pohang beamline 4A.

Crystallization and preliminary X-ray crystallographic studies of fatty acid-CoA racemase from Mycobacterium tuberculosis H37Rv.

Fatty acid-CoA racemase plays an important role in the beta-oxidation of branched-chain fatty acids and fatty-acid derivatives as it catalyzes the conversion of several (2R)-branched-chain fatty acid-CoAs to their (2S)-stereoisomers. Fatty acid-CoA racemase from Mycobacterium tuberculosis H37Rv has been purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method with polyethylene glycol 4000 as precipitant. The crystals belong to the trigonal space group P3(1) or P3(2), with unit-cell parameters a = b = 109.56, c = 147.97 A. The asymmetric unit contains six monomers, corresponding to a VM value of 2.15 A3 Da(-1). A complete native data set has been collected at 2.7 A resolution using a synchrotron-radiation source.