Crystal structures of three protozoan homologs of tryptophanyl-tRNA synthetase

Abstract

Tryptophanyl-tRNA synthetase (TrpRS) is an essential enzyme that is recognizably conserved across all forms of life. It is responsible for activating and attaching tryptophan to a cognate tRNATrp molecule for use in protein synthesis. In some eukaryotes this original core function has been supplemented or modified through the addition of extra domains or the expression of variant TrpRS isoforms. The three TrpRS structures from pathogenic protozoa described here represent three illustrations of this malleability in eukaryotes. The Cryptosporidium parvum genome contains a single TrpRS gene, which codes for an N-terminal domain of uncertain function in addition to the conserved core TrpRS domains. Sequence analysis indicates that this extra domain, conserved among several apicomplexans, is related to the editing domain of some AlaRS and ThrRS. The C. parvum enzyme remains fully active in charging tRNATrp after truncation of this extra domain. The crystal structure of the active, truncated enzyme is presented here at 2.4 Å resolution. The Trypanosoma brucei genome contains separate cytosolic and mitochondrial isoforms of TrpRS that have diverged in their respective tRNA recognition domains. The crystal structure of the T. brucei cytosolic isoform is presented here at 2.8 Å resolution. The Entamoeba histolytica genome contains three sequences that appear to be TrpRS homologs. However one of these, whose structure is presented here at 3.0 Å resolution, has lost the active site motifs characteristic of the Class I aminoacyl-tRNA synthetase catalytic domain while retaining the conserved features of a fully formed tRNATrp recognition domain. The biological function of this variant E. histolytica TrpRS remains unknown, but, on the basis of a completely conserved tRNA recognition region and evidence for ATP but not tryptophan binding, it is tempting to speculate that it may perform an editing function. Together with a previously reported structure of an unusual TrpRS from Giardia, these protozoan structures broaden our perspective on the extent of structural variation found in eukaryotic TrpRS homologs.

An Appended Domain Results in an Unusual Architecture for Malaria Parasite Tryptophanyl-tRNA Synthetase

“…Crystal structures of AlaX-S (specific to Ser-tRNA Ala ) and AlaX-M (specific to Ser-tRNA Ala and Gly-tRNA Ala ) from archaeon Pyrococcus horikoshii have been reported [20], [21]. Our earlier computational investigations had revealed that P. falciparum contains a variant WRS where the catalytic domain is fused to a distant homolog of the free-standing, proofreading factor AlaX [22], [23]. Cryptosporidium parvum genome also contains a single WRS gene that possesses an N-terminal AlaX like domain in addition to the conserved catalytic WRS domains [23].…”

Crystal structures of Plasmodium falciparum cytosolic tryptophanyl-tRNA synthetase and its potential as a target for structure-guided drug design

“…1A). While not tested for Pf -cTrpRS, the deletion of the first part of N-terminal extension did not affect the aminoacylation activity of the homologous apicomplexan Cryptosporidium parvum TrpRS [25]. The construct was cloned into the AVA0421 vector for expression in E. coli [27].…”

“…As part of our ongoing efforts to explore aaRSs as anti-parasitic drug targets [14, 19–25], we have determined the crystal structure of one of the two tryptophanyl-tRNA synthetases from P. falciparum . Like many other higher eukaryotes, P. falciparum has two genes encoding for TrpRS.…”

Highlights on Trypanosomatid Aminoacyl-tRNA Synthesis

“…The crystal structure of the T. brucei cytosolic TrpRS has been partially obtained, sharing a 56 % identity with the human enzyme (Merritt et al 2011 ). Although the portion of the enzyme expected to interact with the tRNA anticodon arm was not solved, a good superposition with the helical regions that contain residues involved in binding the anticodon in the human (Lys 431, Ser 378, and Thr 427) and yeast TrpRS-tRNA…”

“…The overall error rate in translation is approximately 10 −4 , indicating that protein synthesis is accomplished through a high-fi delity process, necessary for the cell's life maintenance (Loftfi eld and Vanderjagt 1972 ;Ibba and Soll 1999 ;Crain et al 2002 ;Charrière et al 2006 ;Bruske et al 2009 ). Although previously believed that in all organisms there were 20 aaRSs (one for each amino acid known at the time) and only one route for tRNA aminoacylation, we now know it is not the case, and indirect pathways of aa-tRNA formation as well as new amino acids have been described (Hao et al 2002 ;Sheppard et al 2008 ;Merritt et al 2011 ). Nevertheless, in the eukaryotic nucleus there are at least 20 of these enzymes (encoded by one or more genes) and the tRNAs are charged through a direct route, with the exception of the selenocysteinyl-tRNA Sec .…”