Ivana Weygand-Durasevic

Structure of the unusual seryl-tRNA synthetase reveals a distinct zinc-dependent mode of substrate recognition.

Methanogenic archaea possess unusual seryl‐tRNA synthetase (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. The two types of SerRSs show only minimal sequence similarity, primarily within class II conserved motifs 1, 2 and 3. Here, we report a 2.5 Å resolution crystal structure of the atypical methanogenic Methanosarcina barkeri SerRS and its complexes with ATP, serine and the nonhydrolysable seryl‐adenylate analogue 5′‐O‐(N‐serylsulfamoyl)adenosine. The structures reveal two idiosyncratic features of methanogenic SerRSs: a novel N‐terminal tRNA‐binding domain and an active site zinc ion. The tetra‐coordinated Zn2+ ion is bound to three conserved protein ligands (Cys306, Glu355 and Cys461) and binds the amino group of the serine substrate. The absolute requirement of the metal ion for enzymatic activity was confirmed by mutational analysis of the direct zinc ion ligands. This zinc‐dependent serine recognition mechanism differs fundamentally from the one employed by the bacterial‐type SerRSs. Consequently, SerRS represents the only known aminoacyl‐tRNA synthetase system that evolved two distinct mechanisms for the recognition of the same amino‐acid substrate.

Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.

An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl‐tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity. Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzymes active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated. Aminoacylation of tRNA(CUA)Tyr [tyrT (UAG)] by GlnRS‐D235H resulted in a 4‐fold increase in the Km for the Gln, which was reduced to a 2‐fold increase when A73 was replaced with G73. These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3′ terminus. Disruption of these interactions can change the Km for Gln over a 30‐fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln‐GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein‐RNA interactions facilitate catalysis.

Hydrolysis of non-cognate aminoacyl-adenylates by a class II aminoacyl-tRNA synthetase lacking an editing domain

Aminoacyl‐tRNA synthetases, a group of enzymes catalyzing aminoacyl‐tRNA formation, may possess inherent editing activity to clear mistakes arising through the selection of non‐cognate amino acid. It is generally assumed that both editing substrates, non‐cognate aminoacyl‐adenylate and misacylated tRNA, are hydrolyzed at the same editing domain, distant from the active site. Here, we present the first example of an aminoacyl‐tRNA synthetase (seryl‐tRNA synthetase) that naturally lacks an editing domain, but possesses a hydrolytic activity toward non‐cognate aminoacyl‐adenylates. Our data reveal that tRNA‐independent pre‐transfer editing may proceed within the enzyme active site without shuttling the non‐cognate aminoacyl‐adenylate intermediate to the remote editing site.

Homologs of aminoacyl-tRNA synthetases acylate carrier proteins and provide a link between ribosomal and nonribosomal peptide synthesis

Aminoacyl-tRNA synthetases (aaRSs) are ancient and evolutionary conserved enzymes catalyzing the formation of aminoacyl-tRNAs, that are used as substrates for ribosomal protein biosynthesis. In addition to full length aaRS genes, genomes of many organisms are sprinkled with truncated genes encoding single-domain aaRS-like proteins, which often have relinquished their canonical role in genetic code translation. We have identified the genes for putative seryl-tRNA synthetase homologs widespread in bacterial genomes and characterized three of them biochemically and structurally. The proteins encoded are homologous to the catalytic domain of highly diverged, atypical seryl-tRNA synthetases (aSerRSs) found only in methanogenic archaea and are deprived of the tRNA-binding domain. Remarkably, in comparison to SerRSs, aSerRS homologs display different and relaxed amino acid specificity. aSerRS homologs lack canonical tRNA aminoacylating activity and instead transfer activated amino acid to phosphopantetheine prosthetic group of putative carrier proteins, whose genes were identified in the genomic surroundings of aSerRS homologs. Detailed kinetic analysis confirmed that aSerRS homologs aminoacylate these carrier proteins efficiently and specifically. Accordingly, aSerRS homologs were renamed amino acid:[carrier protein] ligases (AMP forming). The enzymatic activity of aSerRS homologs is reminiscent of adenylation domains in nonribosomal peptide synthesis, and thus they represent an intriguing link between programmable ribosomal protein biosynthesis and template-independent nonribosomal peptide synthesis.

Detection of Noncovalent tRNA·Aminoacyl-tRNA Synthetase Complexes by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) was used for the study of complexes formed by yeast seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) with tRNASer and tRNATyr. Cognate and noncognate complexes were easily distinguished due to a large mass difference between the two tRNAs. Both homodimeric synthetases gave MS spectra indicating intact desorption of dimers. The spectra of synthetase-cognate tRNA mixtures showed peaks of free components and peaks assigned to complexes. Noncognate complexes were also detected. In competition experiments, where both tRNA species were mixed with each enzyme only cognate α2·tRNA complexes were observed. Only cognate α2·tRNA2 complexes were detected with each enzyme. These results demonstrate that MALDI-MS can be used successfully for accurate mass and, thus, stoichiometry determination of specific high molecular weight noncovalent protein-nucleic acid complexes.

Defining the Active Site of Yeast Seryl-tRNA Synthetase MUTATIONS IN MOTIF 2 LOOP RESIDUES AFFECT tRNA-DEPENDENT AMINO ACID RECOGNITION

The active site of class II aminoacyl-tRNA synthetases contains the motif 2 loop, which is involved in binding of ATP, amino acid, and the acceptor end of tRNA. In order to characterize the active site of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS), we performed in vitro mutagenesis of the portion of the SES1 gene encoding the motif 2 loop. Substitutions of amino acids conserved in the motif 2 loop of seryl-tRNA synthetases from other sources led to loss of complementation of a yeast SES1 null allele strain by the mutant yeast SES1 genes. Steady-state kinetic analyses of the purified mutant SerRS proteins revealed elevated Km values for serine and ATP, accompanied by decreases in kcat (as expected for replacement of residues involved in aminoacyl-adenylate formation). The differences in the affinities for serine and ATP, in the absence and presence of tRNA are consistent with the proposed conformational changes induced by positioning the 3′-end of tRNA into the active site, as observed recently in structural studies of Thermus thermophilus SerRS (Cusack, S., Yaremchuk, A., and Tukalo, M. (1996) EMBO J. 15, 2834-2842). The crystal structure of this moderately homologous prokaryotic counterpart of the yeast enzyme allowed us to produce a model of the yeast SerRS structure and to place the mutations in a structural context. In conjunction with structural data for T. thermophilus SerRS, the kinetic data presented here suggest that yeast seryl-tRNA synthetase displays tRNA-dependent amino acid recognition.

The C-terminal extension of yeast seryl-tRNA synthetase affects stability of the enzyme and its substrate affinity.

Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) contains a 20-amino acid C-terminal extension, which is not found in prokaryotic SerRS enzymes. A truncated yeast SES1 gene, lacking the 60 base pairs that encode this C-terminal domain, is able to complement a yeast SES1 null allele strain; thus, the C-terminal extension in SerRS is dispensable for the viability of the cell. However, the removal of the C-terminal peptide affects both stability of the enzyme and its affinity for the substrates. The truncation mutant binds tRNA with 3.6-fold higher affinity, while the K for serine is 4-fold increased relative to the wild-type SerRS. This indicates the importance of the C-terminal extension in maintaining the overall structure of SerRS.

New Roles for Codon Usage

Nucleotide coding of actin is linked to the rate of translation, polypeptide modification, and stability. How do two proteins with almost indistinguishable amino acid sequences have different functions in the cell? A single amino acid change can dictate a property as complex as the topology of a membrane protein (1), but other examples are less easy to explain. One such puzzle relates to the mammalian cytoskeletal proteins β- and γ-actin, whose amino acid sequences are 98% identical. β-Actin is modified by the addition of arginine, whereas γ-actin is not, resulting in distinct roles for each in the cell. Identifying what exactly distinguishes β- from γ-actin has proved perplexing because the marginal differences in their amino-terminal sequences are not sufficient to explain why one is modified and the other is not. On page 1534 of this issue, Zhang et al. (2) show that the modification of β- and γ-actin is dictated by the codons—the triplets of nucleic acids that encode amino acids— rather than the specific amino acids themselves in the amino termini of these proteins. This is an unexpected example of proteins whose properties are determined at the nucleotide rather than the amino acid level, forcing a reassessment of what defines a synonymous change in a gene sequence.

Peroxin Pex21p interacts with the C‐terminal noncatalytic domain of yeast seryl‐tRNA synthetase and forms a specific ternary complex with tRNASer

The seryl‐tRNA synthetase from Saccharomyces cerevisiae interacts with the peroxisome biogenesis‐related factor Pex21p. Several deletion mutants of seryl‐tRNA synthetase were constructed and inspected for their ability to interact with Pex21p in a yeast two‐hybrid assay, allowing mapping of the synthetase domain required for complex assembly. Deletion of the 13 C‐terminal amino acids abolished Pex21p binding to seryl‐tRNA synthetase. The catalytic parameters of purified truncated seryl‐tRNA synthetase, determined in the serylation reaction, were found to be almost identical to those of the native enzyme. In vivo loss of interaction with Pex21p was confirmed in vitro by coaffinity purification. These data indicate that the C‐terminally appended domain of yeast seryl‐tRNA synthetase does not participate in substrate binding, but instead is required for association with Pex21p. We further determined that Pex21p does not directly bind tRNA, and nor does it possess a tRNA‐binding motif, but it instead participates in the formation of a specific ternary complex with seryl‐tRNA synthetase and tRNASer, strengthening the interaction of seryl‐tRNA synthetase with its cognate tRNASer.

Dual targeting of organellar seryl-tRNA synthetase to maize mitochondria and chloroplasts

Aminoacyl-tRNA synthetases (AARSs) play a critical role in translation and are thus required in three plant protein-synthesizing compartments: cytosol, mitochondria and plastids. A systematic study had previously shown extensive sharing of organellar AARSs from Arabidopsis thaliana, mostly between mitochondria and chloroplasts. However, distribution of AARSs from monocot species, such as maize, has never been experimentally investigated. Here we demonstrate dual targeting of maize seryl-tRNA synthetase, SerZMo, into both mitochondria and chloroplasts using combination of complementary methods, including in vitro import assay, transient expression analysis of green fluorescent protein (GFP) fusions and immunodetection. We also show that SerZMo dual localization is established by the virtue of an ambiguous targeting peptide. Full-length SerZMo protein fused to GFP is targeted to chloroplast stromules, indicating that SerZMo protein performs its function in plastid stroma. The deletion mutant lacking N-terminal region of the ambiguous SerZMo targeting peptide was neither targeted into mitochondria nor chloroplasts, indicating the importance of this region in both mitochondrial and chloroplastic import.