Silvija Bilokapic

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.

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.

Structural flexibility of the methanogenic-type seryl-tRNA synthetase active site and its implication for specific substrate recognition

Seryl‐tRNA synthetase (SerRS) is a class II aminoacyl‐tRNA synthetase that catalyzes serine activation and its transfer to cognate tRNASer. Previous biochemical and structural studies have revealed that bacterial‐ and methanogenic‐type SerRSs employ different strategies of substrate recognition. In addition to other idiosyncratic features, such as the active site zinc ion and the unique fold of the N‐terminal tRNA‐binding domain, methanogenic‐type SerRS is, in comparison with bacterial homologues, characterized by a notable shortening of the motif 2 loop. Mutational analysis of Methanosarcina barkeri SerRS (mMbSerRS) was undertaken to identify the active site residues that ensure the specificity of amino acid and tRNA 3′‐end recognition. Residues predicted to contribute to the amino acid specificity were selected for mutation according to the crystal structure of mMbSerRS complexed with its cognate aminoacyl‐adenylate, whereas those involved in binding of the tRNA 3′‐end were identified and mutagenized on the basis of modeling the mMbSerRS:tRNA complex. Although mMbSerRSs variants with an altered serine‐binding pocket (W396A, N435A, S437A) were more sensitive to inhibition by threonine and cysteine, none of the mutants was able to activate noncognate amino acids to greater extent than the wild‐type enzyme. In vitro kinetics results also suggest that conformational changes in the motif 2 loop are required for efficient serylation.

Identification of Amino Acids in the N-terminal Domain of Atypical Methanogenic-type Seryl-tRNA Synthetase Critical for tRNA Recognition

Seryl-tRNA synthetase (SerRS) from methanogenic archaeon Methanosarcina barkeri, contains an idiosyncratic N-terminal domain, composed of an antiparallel β-sheet capped by a helical bundle, connected to the catalytic core by a short linker peptide. It is very different from the coiled-coil tRNA binding domain in bacterial-type SerRS. Because the crystal structure of the methanogenic-type SerRS·tRNA complex has not been obtained, a docking model was produced, which indicated that highly conserved helices H2 and H3 of the N-terminal domain may be important for recognition of the extra arm of tRNASer. Based on structural information and the docking model, we have mutated various positions within the N-terminal region and probed their involvement in tRNA binding and serylation. Total loss of activity and inability of the R76A variant to form the complex with cognate tRNA identifies Arg76 located in helix H2 as a crucial tRNA-interacting residue. Alteration of Lys79 positioned in helix H2 and Arg94 in the loop between helix H2 and β-strand A4 have a pronounced effect on SerRS·tRNASer complex formation and dissociation constants (KD) determined by surface plasmon resonance. The replacement of residues Arg38 (located in the loop between helix H1 and β-strand A2), Lys141 and Asn142 (from H3), and Arg143 (between H3 and H4) moderately affect both the serylation activity and the KD values. Furthermore, we have obtained a striking correlation between these results and in vivo effects of these mutations by quantifying the efficiency of suppression of bacterial amber mutations, after coexpression of the genes for M. barkeri suppressor tRNASer and a set of mMbSerRS variants in Escherichia coli.

Idiosyncratic Helix-Turn-Helix Motif in Methanosarcina barkeri Seryl-tRNA Synthetase Has a Critical Architectural Role

All seryl-tRNA synthetases (SerRSs) are functional homodimers with a C-terminal active site domain typical for class II aminoacyl-tRNA synthetases and an N-terminal domain involved in tRNA binding. The recently solved three-dimensional structure of Methanosarcina barkeri SerRS revealed the idiosyncratic features of methanogenic-type SerRSs; that is, an active site zinc ion, a unique tRNA binding domain, and an insertion of ∼30 residues in the catalytic domain, which adopt a helix-turn-helix (HTH) fold. Here, we present biochemical evidence for multiple roles of the HTH motif; it is important for dimerization of the enzyme, contributes to the overall stability, and is critical for the proper positioning of the tRNA binding domain relative to the catalytic domain. The changes in intrinsic fluorescence during denaturation of the wild-type M. barkeri SerRS and of the mutated variant lacking the HTH motif combined with cross-linking and gel analysis of protein subunits during various stages of the unfolding process revealed significantly reduced stability of the mutant dimers. In vitro kinetic analysis of enzymes, mutated in one of the N-terminal helices and the HTH motif, shows impaired tRNA binding and aminoacylation and emphasizes the importance of this domain for the overall architecture of the enzyme. The role of the idiosyncratic HTH motif in dimer stabilization and association between the catalytic and tRNA binding domain has been additionally confirmed by a yeast two-hybrid approach. Furthermore, we provide experimental evidence that tRNA binds across the dimer.