A Single Substitution in the Motif 1 of Escherichia coli Lysyl-tRNA Synthetase Induces Cooperativity toward Amino Acid Binding

Commans, Stéphane; Blanquet, Sylvain; Plateau, Pierre

doi:10.1021/bi00025a025

Cited by 19 publications

(20 citation statements)

References 48 publications

(47 reference statements)

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“…However, it is unclear how the two dimeric proteins interact with each other to make an ␣ 2 ␤ 2 heterotetramer, and, in particular, the location of the surfaces on LysRS that can accommodate the p38 interactions. For both human and E. coli LysRS, the ␣ 2 dimer is required for charging tRNA Lys (23,24). Given that LysRS within the MSC is fully functional for aminoacylation (22,25), it is most likely that the ␣ 2 form of LysRS interacts with the dimeric p38 in the MSC.…”

Section: Prediction Of Tetramer Interface Of Lysrs For P38 Interactionmentioning

confidence: 99%

Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation

Guo

Ignatov

Musier‐Forsyth

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

114

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In mammals, many aminoacyl-tRNA synthetases are bound together in a multisynthetase complex (MSC) as a reservoir of procytokines and regulation molecules for functions beyond aminoacylation. The ␣2 homodimeric lysyl-tRNA synthetase (LysRS) is tightly bound in the MSC and, under specific conditions, is secreted to trigger a proinflammatory response. Results by others suggest that ␣2 LysRS is tightly bound into the core of the MSC with homodimeric ␤2 p38, a scaffolding protein that itself is multifunctional. Not understood is how the two dimeric proteins combine to make a presumptive ␣2␤2 heterotetramer and, in particular, the location of the surfaces on LysRS that would accommodate the p38 interactions. Here we present a 2.3-Å crystal structure of a tetrameric form of human LysRS. The relatively loose (as seen in solution) tetramer interface is assembled from two eukaryotespecific sequences, one in the catalytic-and another in the anticodon-binding domain. This same interface is predicted to provide unique determinants for interaction with p38. The analyses suggest how the core of the MSC is assembled and, more generally, that interactions and functions of synthetases can be built and regulated through dynamic protein-protein interfaces. These interfaces are created from small adaptations to what is otherwise a highly conserved (through evolution) polypeptide sequence.AIMP2 ͉ aminoacyl-tRNA synthetase ͉ p38

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Section: Prediction Of Tetramer Interface Of Lysrs For P38 Interactionmentioning

confidence: 99%

Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation

Guo

Ignatov

Musier‐Forsyth

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

114

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“…Accordingly, one would expect that the two active sites are functionally related. The finding that a LystRNA synthetase mutant affected in its dimeric interface displays cooperativity toward lysine binding strongly supports this hypothesis (14).…”

Section: Discussionmentioning

confidence: 64%

“…Because the corresponding residues belong to a long ␣-helix and a ␤-strand located at the dimeric interface, it was first believed that motif 1 is a key element of oligomerization. However, recent data suggest that this motif is predominantly involved in stabilizing the conformation of the active sites of the two monomers and participates in intersubunit communication (9,13,14). Furthermore, a canonical motif 1 was also identified in two monomeric class II enzymes: Ala-tRNA synthetase from yeast, insect, or human (15) and Phe-tRNA synthetase from yeast mitochondria (16).…”

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confidence: 99%

Expression of Rat Aspartyl-tRNA Synthetase in Saccharomyces cerevisiae

Agou¹,

Waller

Mirande

1996

Journal of Biological Chemistry

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Cytoplasmic aspartyl-tRNA synthetase from mammals is one of the components of a multienzyme complex comprising nine synthetase activities. The presence of an amino-terminal extension composed of about 40 residues is a characteristic of the eukaryotic enzyme. We report here the expression in the yeast Saccharomyces cerevisiae of a native form of rat aspartyl-tRNA synthetase and of two truncated derivatives lacking 20 or 36 amino acid residues from their amino-terminal polypeptide extension. The three recombinant enzyme species were purified to homogeneity. They behave as ␣ 2 dimers and display catalytic parameters in the tRNA aminoacylation reaction identical to those determined for the native, complex-associated form of aspartyl-tRNA synthetase isolated from rat liver. Because the dimer dissociation constant of rat AspRS is much higher than that of its bacterial and yeast counterparts, we could establish a direct correlation between dissociation of the dimer and inactivation of the enzyme. Our results clearly show that the monomer is devoid of amino acid activation and tRNA aminoacylation activities, indicating that dimerization is essential to confer an active conformation on the catalytic site. The two NH 2 -terminal truncated derivatives were fully active, but proved to be more unstable than the recombinant native enzyme, suggesting that the polypeptide extension fulfills structural rather than catalytic requirements.Aminoacyl-tRNA synthetases are essential enzymes that catalyze a key step during the process of protein biosynthesis: activation of a specific amino acid and the charging of the cognate tRNA (1). On the basis of the comparison of the primary structures of the 20 enzymes from Escherichia coli, it was shown that this family is composed of two phylogenetically distinct protein groups harboring mutually exclusive sets of conserved sequence motifs (2). Accordingly, the topological arrangement of the active site domain, as well as the mode of tRNA:enzyme recognition, are essentially different for these two enzyme classes (3). Class I enzymes are characterized by the two consensus sequences HIGH and KMSKS, which are arranged within a structural fold of the Rossmann type: a six-stranded parallel ␤-sheet. On the other hand, the structural base for the catalytic domain of class II enzymes is a sixstranded anti-parallel ␤-sheet that supports three conserved sequence motifs.Crystallographic studies designate motifs 2 and 3 of class II aminoacyl-tRNA synthetases as essential pieces of their active site (4 -9). This was confirmed by site-directed mutagenesis of presumed key amino acids within these conserved sequences (4, 10 -12). Motif 1 was originally described as a prominent structural element building the subunit interface of class II enzymes that form ␣ 2 dimers. Because the corresponding residues belong to a long ␣-helix and a ␤-strand located at the dimeric interface, it was first believed that motif 1 is a key element of oligomerization. However, recent data suggest that this motif is predominantly inv...

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“…Bacteria and Plasmids-The following E. coli K12 derivatives were used in this study: JM101TR (⌬(lac-pro) supE thi recA56 srl-300::Tn10 (FЈ traD36 proAB lacI q lacZ⌬M15)) (25) and PAL⌬S⌬UTR (F Ϫ ⌬(lacpro) gyrA rpoB metB argE(Am) ara supF ⌬lysS::kan ⌬lysU srl-300::Tn10 recA56 (pMAK705 lysU)) (26). Plasmid pLysSH, harboring the wild-type E. coli lysS gene, was obtained by inserting the 1-kbp PstI-HindII fragment from M13PhlysS (26) into the corresponding sites of plasmid pC1 (26).…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid pLysSH, harboring the wild-type E. coli lysS gene, was obtained by inserting the 1-kbp PstI-HindII fragment from M13PhlysS (26) into the corresponding sites of plasmid pC1 (26). Plasmid pBluescript KSϩ was from Stratagene, and plasmid pQE70 was from Qiagen.…”

Section: Methodsmentioning

confidence: 99%

Anticodon Recognition in Evolution

Brevet

Chen

Commans

et al. 2003

Journal of Biological Chemistry

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The highly conserved aspartyl-, asparaginyl-, and lysyl-tRNA synthetases compose one subclass of aminoacyl-tRNA synthetases, called IIb. The three enzymes possess an OB-folded extension at their N terminus. The function of this extension is to specifically recognize the anticodon triplet of the tRNA. Three-dimensional models of bacterial aspartyl-and lysyl-tRNA synthetases complexed to tRNA indicate that a rigid scaffold of amino acid residues along the five ␤-strands of the OBfold accommodates the base U at the center of the anticodon. The binding of the adjacent anticodon bases occurs through interactions with a flexible loop joining strands 4 and 5 (L 45 ). As a result, a switching of the specificity of lysyl-tRNA synthetase from tRNA Lys (anticodon UUU) toward tRNA Asp (GUC) could be attempted by transplanting the small loop L 45 of aspartyl-tRNA synthetase inside lysyl-tRNA synthetase. Upon this transplantation, lysyl-tRNA synthetase loses its capacity to aminoacylate tRNA Lys . In exchange, the chimeric enzyme acquires the capacity to charge tRNA Asp with lysine. Upon giving the tRNA Asp substrate the discriminator base of tRNA Lys , the specificity shift is improved. The change of specificity was also established in vivo. Indeed, the transplanted lysyl-tRNA synthetase succeeds in suppressing a missense Lys 3 Asp mutation inserted into the ␤-lactamase gene. These results functionally establish that sequence variation in a small peptide region of subclass IIb aminoacyl-tRNA synthetases contributes to specification of nucleic acid recognition. Because this peptide element is not part of the core catalytic structure, it may have evolved independently of the active sites of these synthetases.In protein biosynthesis, aminoacyl-tRNA synthetases (aaRS) 1 establish physical relations between each amino acid and the corresponding tRNAs. Therefore, the accuracy of this family of enzymes is crucial to guarantee the fidelity of translation of mRNA. To ensure a correct matching between substrates, each synthetase recognizes a small number of nucleotide positions inside its cognate tRNA(s). These positions are also called the identity elements of tRNAs. The major identity elements in all Escherichia coli aminoacylation systems have now been identified (reviewed in Ref. 1). In most cases (17 of 20), the anticodon sequence itself behaves as a major identity element. Additional identity elements are frequently found in the acceptor stem. They often include the so-called discriminator base 73 (2).From primary sequence and structural analyses, a partition of the 20 synthetases into two classes has been proposed (3, 4). In the first class, the catalytic domain adopts a nucleotide binding fold (Rossmann fold). In class II synthetases, the enzyme center is built around an antiparallel ␤-sheet. Additional domains, the three-dimensional structures of which are highly variable, contribute to the specificity and the strength of cognate tRNA binding to each synthetase. Among these domains, some are believed to have been added late i...

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A Single Substitution in the Motif 1 of Escherichia coli Lysyl-tRNA Synthetase Induces Cooperativity toward Amino Acid Binding

Cited by 19 publications

References 48 publications

Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation

Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation

Expression of Rat Aspartyl-tRNA Synthetase in Saccharomyces cerevisiae

Anticodon Recognition in Evolution

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