Methionyl-tRNA synthetase from Caenorhabditis elegans: a specific multidomain organization for convergent functional evolution

Abstract

Methionyl-tRNA synthetase (MetRS) is a multidomain protein that specifically binds tRNAMet and catalyzes the synthesis of methionyl-tRNAMet. The minimal, core enzyme found in Aquifex aeolicus is made of a catalytic domain, which catalyzes the aminoacylation reaction, and an anticodon-binding domain, which promotes tRNA-protein association. In eukaryotes, additional domains are appended in cis or in trans to the core enzyme and increase the stability of the tRNA-protein complexes. Eventually, as observed for MetRS from Homo sapiens, the C-terminal appended domain causes a slow release of aminoacyl-tRNA and establishes a limiting step in the global aminoacylation reaction. Here, we report that MetRS from the nematode Caenorhabditis elegans displays a new type of structural organization. Its very C-terminal appended domain is related to the oligonucleotide binding-fold-based tRNA-binding domain (tRBD) recovered at the C-terminus of MetRS from plant, but, in the nematode enzyme, this domain is separated from the core enzyme by an insertion domain. Gel retardation and tRNA aminoacylation experiments show that MetRS from nematode is functionally related to human MetRS despite the fact that their appended tRBDs have distinct structural folds, and are not orthologs. Thus, functional convergence of human and nematode MetRS is the result of parallel and convergent evolution that might have been triggered by the selective pressure to invent processivity of tRNA handling in translation in higher eukaryotes.

Figure 1

Domain organization of MetRS from nematode and comparison with other known MetRS. The smallest MetRS, made of a catalytic (CAT) and an anticodon-binding domain (ABD), is known in Aquifex aeolicus. Its crystal structure in complex with tRNA^Met is shown; the arrow points to the C-terminus of the protein (Ct). In the plant Oriza sativa, a tRNA-binding domain (tRBD) is appended in cis at the C-terminus of the protein. The crystal structure of the C-terminal domain of human p43, also known as EMAPII, a domain homologous to the tRBD of plant MetRS, is shown. In the yeast Saccharomyces cerevisiae, a similar tRBD is associated to MetRS in trans. An N-terminal polypeptide extension contributes a protein-binding domain (PBD) that mediates association of MetRS with Arc1p, a factor containing this tRBD. The crystal structure of a complex between the PBD of Arc1p and of MetRS is shown. In the nematode Caenorhabditis elegans, a similar tRBD is appended in cis at the very C-terminus of MetRS, but a putative PBD is inserted between the ABD and the tRBD. In Homo sapiens, MetRS contains an N-terminal PBD that mediates its association within the multisynthetase complex MARS and a specific C-terminal tRBD. The solution structure of the human tRBD is shown.

Figure 2

Multiple alignment of the tRBD homologous to the C-terminus of MetRS-Ce. (A) The C-terminal domain of nematode MetRS is aligned with the C-terminal domains of human p43 (p43-Hs), of yeast Arc1p (Arc1p-Sc), and of rice MetRS (MetRS-Os). Residues that are conserved with the nematode sequence are indicated by a black box. The best-conserved signature sequence for tRNA binding (KKKφWE) reported previously for p43-Hs is indicated. (B) Antibodies raised against the C-terminal domain of human p43 cross reacted with the C-domain of nematode MetRS. Purified p43-Hs (25 ng) (lane 1) and a 5M excess of purified MetRSΔC-Ce (250 ng) (lane 2) or MetRS-Ce (375 ng) (lane 3) were analyzed by Western blotting.

Figure 3

Structure of the mrs-1 gene and expression of native MetRS-Ce and of a C-terminally truncated derivative. (A) The mrs-1 gene is composed of six exons. The 5′ and 3′ noncoding sequences are indicated by dark gray boxes. The first three exons encode the core domain of MetRS-Ce (white box), made of 595 residues, containing the catalytic and anticodon-binding domains. The C-terminal appended domain, from residues 596 to 917 (gray and black boxes corresponding to the PBD and tRBD indicated in Fig. 1, respectively), is encoded by the last three exons of the longest splice variant (L). The C-terminus of the putative short variant (S) would be encoded by the intron located between exons 3 and 4. (B) Two derivatives, with (MetRS-Ce, lane 2, 104 kDa with the appended His-tag) or without (MetRSΔC-Ce, lane 3, 69 kDa with His-tag) the C-terminal domain, were expressed in E.coli, purified to homogeneity, analyzed by SDS-PAGE, and visualized by Coomassie staining. The multisynthetase complex from rabbit, containing MetRS at 101 kDa, is shown as a size marker (lane 1).

Figure 4

Nematode MetRS is a monomer in solution. (A) The full-length (MetRS) and C-terminally truncated derivative (MetRSΔC) of MetRS from C. elegans were subjected to size exclusion chromatography on a Superose 12 column. (B) The apparent molecular masses of the two proteins were deduced from their relative elution volumes K_av. The molecular mass standards included (1) yeast phenylalanyl-tRNA synthetase (250 kDa), (2) the dimer (136 kDa) and (3) the monomer (68 kDa) of bovine serum albumin, and (4) ovalbumin (45 kDa).

Figure 5

Analysis of the MetRS species expressed in C. elegans during development. Antibodies were raised against the core domain of MetRS from the nematode (MetRSΔC-Ce) and used to identify the MetRS species expressed in the worm. (A) Equimolar amounts of purified MetRS-Ce (15 ng) and MetRSΔC-Ce (10 ng) (lane 3), and a SDS (lane 1) or soluble (lane 2) extract of a nonsynchronized culture of C. elegans were analyzed by Western blotting. (B) SDS extracts of embryos (E), of larvae at the L1, L2, L3, or L4 stages, and of young adults (A0) and 1-day adults (A1) of the nematode were analyzed by Western blotting using antibodies directed to the core protein MetRSΔC-Ce or to tubulin.

Figure 6

The C-domain confers on nematode MetRS tRNA-binding properties. ³²P-labeled in vitro transcribed tRNA^Met was incubated with native (MetRS-Ce) (left) or C-terminally truncated (MetRSΔC-Ce) (right) nematode MetRS at the concentrations indicated. After electrophoresis at 4°C on a 6% native polyacrylamide gel, the mobility shift of tRNA was visualized by autoradiography. The arrowheads point to free tRNA species.

Figure 7

The C-terminal domain of nematode MetRS restricts tRNA turnover in the aminoacylation reaction. The tRNA saturation kinetics in the tRNA^Met aminoacylation reaction were determined with partially purified rabbit elongator tRNA^Met (methionine acceptance of 660 pmol/A₂₆₀ unit) in the presence of 6.5 nM MetRS-Ce (•) or MetRSΔC-Ce (▪). Experimental values (symbols) were fitted to the Michaelis–Menten equation (curves).