Structure of Leishmania major methionyl-tRNA synthetase in complex with intermediate products methionyladenylate and pyrophosphate

Abstract

Leishmania parasites cause two million new cases of leishmaniasis each year with several hundreds of millions people at risk. Due to the paucity and shortcomings of available drugs, we have undertaken the crystal structure determination of a key enzyme from Leishmania major in hopes of creating a platform for the rational design of new therapeutics. Crystals of the catalytic core of methionyl-tRNA synthetase from L. major (LmMetRS) were obtained with the substrates MgATP and methionine present in the crystallization medium. These crystals yielded the 2.0 Å resolution structure of LmMetRS in complex with two products, methionyladenylate and pyrophosphate, along with a Mg 2+ ion that bridges them. This is the first class I aminoacyl-tRNA synthetase (aaRS) structure with pyrophosphate bound. The residues of the class I aaRS signature sequence motifs, KISKS and HIGH, make numerous contacts with the pyrophosphate. Substantial differences between the LmMetRS structure and previously reported complexes of E. coli MetRS (EcMetRS) with analogs of the methionyladenylate intermediate product are observed, even though one of these analogs only differs by one atom from the intermediate. The source of these structural differences is attributed to the presence of the product pyrophosphate in LmMetRS. Analysis of the LmMetRS structure in light of the Aquifex aeolicus MetRS-tRNA Met complex shows that major rearrangements of multiple structural elements of enzyme and/or tRNA are required to allow the CCA acceptor triplet to reach the methionyladenylate intermediate in the active site. Comparison with sequences of human cytosolic and mitochondrial MetRS reveals interesting differences near the ATP-and methionine-binding regions of LmMetRS, suggesting that it should be possible to obtain compounds that selectively inhibit the parasite enzyme.

Selective Inhibitors of Methionyl-tRNA Synthetase Have Potent Activity against Trypanosoma brucei Infection in Mice

“…The structures in the Protein Data Bank (PDB) originate from Aquifex aeolicus, Thermus thermophilus, Pyrococcus abyssi, Escherichia coli, and Mycobacterium smegmatis. In addition, the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) consortium recently solved the structure of the Leishmania major enzyme, the first eukaryotic MetRS structure to be solved (18). Sequence comparisons of Table S2 in the supplemental material).…”

Highlights on Trypanosomatid Aminoacyl-tRNA Synthesis

“…The crystal structures of these enzymes in complex with their intermediates have been published and are very similar (Larson et al 2011b ;Koh et al 2012 ). They differ only in the degree of closing of the active site in relation to the fl exible CP knuckle and KISKS loop.…”

“…When compared to T. brucei and T. cruzi , the leishmanial enzymes show an identity of 68 %, but if we consider only the active sites, they are 100 % identical. MetRSs from both L. major and T. brucei are characterized by several key structural features: (1) a Rossmann fold catalytic core with an inserted connective peptide (CP) domain; (2) a stem-contact fold (SCF) domain containing the conserved KMSKS signature (KISKS in trypanosomatids); and (3) an anticodon binding α helix bundle (Larson et al 2011b ;Koh et al 2012 ).…”

“…The carbonyl oxygen of the Gly514 points towards the ligand while the Ser side chain in the human mitochondrial enzyme points away from the ligand in the direction of a hole between the side chains of Phe238, Lys310, and Asp383, as deduced from the L. major structure for which the corresponding residues are respectively, Arg401, Lys485, and Asp558. Among the residues surrounding the latter, fi ve amino acids differ: Val482 and Val483 in L. major MetRS, which are both an Ile in human mitochondrial MetRS, and the tripeptide Ser553-Asn554-Phe555, which is Gly-Val-Pro in the human mitochondrial enzyme (Larson et al 2011b ).…”

“…This group forms a clade in the TyrRS phylogenetic tree where we fi nd the trypanosomatids, plants, other basal eukaryotic organisms and mimivirus, which have their origin on the euryarchaeota branch (Bonnefond et al 2005 ;Larson et al 2011b ).…”