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Leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase.

Yan W, Tan M, Eriani G, Wang ED - Nucleic Acids Res. (2013)

Bottom Line: Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding.Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD.The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology, Center for RNA Research, Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China.

ABSTRACT
The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNA(Leu) recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

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Editing and mischarging properties of MmLeuRS-CP1/LSD-K598A. (A) Total editing activity was measured by the AMP formation assay with 1 µM of MmLeuRS-CP1/LSD-K598A and 15 mM Nva in the absence or presence of 5 µM EctRNALeuGAG or MmtRNALeuUAG. (B) Deacylation of [3H]-Ile-EctRNALeu (1 µM) by 20 nM of MmLeuRS (black circle), MmLeuRS-CP1/LSD-K598A (inverted black triangle), MmLeuRS-CP1/LSD (black square) and MmLeuRS-CP1 (black triangle). (C) Mischarging of EctRNALeuGAG (20 µM) with Ile catalyzed by 1 µM of MmLeuRS (black circle), MmLeuRS-CP1/LSD-K598A (inverted black triangle), MmLeuRS-CP1/LSD (black square) and MmLeuRS-CP1(black triangle). (D) Crystal structure of tRNALeu (light blue in the cartoon model) in complex with EcLeuRS (grey) during the editing conformation (PDB ID code 4ARC, Ref.4). Residues R595, K598 and R600 of LSD (green) are numbered and shown in stick representation with labelling. Both G10 and G46 of tRNALeu were also highlighted with the stick model with their distances to K598 labelled.
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gkt185-F4: Editing and mischarging properties of MmLeuRS-CP1/LSD-K598A. (A) Total editing activity was measured by the AMP formation assay with 1 µM of MmLeuRS-CP1/LSD-K598A and 15 mM Nva in the absence or presence of 5 µM EctRNALeuGAG or MmtRNALeuUAG. (B) Deacylation of [3H]-Ile-EctRNALeu (1 µM) by 20 nM of MmLeuRS (black circle), MmLeuRS-CP1/LSD-K598A (inverted black triangle), MmLeuRS-CP1/LSD (black square) and MmLeuRS-CP1 (black triangle). (C) Mischarging of EctRNALeuGAG (20 µM) with Ile catalyzed by 1 µM of MmLeuRS (black circle), MmLeuRS-CP1/LSD-K598A (inverted black triangle), MmLeuRS-CP1/LSD (black square) and MmLeuRS-CP1(black triangle). (D) Crystal structure of tRNALeu (light blue in the cartoon model) in complex with EcLeuRS (grey) during the editing conformation (PDB ID code 4ARC, Ref.4). Residues R595, K598 and R600 of LSD (green) are numbered and shown in stick representation with labelling. Both G10 and G46 of tRNALeu were also highlighted with the stick model with their distances to K598 labelled.

Mentions: Consistently, in the chimeric MmLeuRS-CP1/LSD, the K598A mutation controlled post-transfer editing, as there was a drop in the AMP synthesis rate (Figure 4A, Table 2) and an absence of deacylation activity towards Ile-EctRNALeuGAG (Figure 4B). The loss of deacylation properties was further confirmed by a loss of aminoacylation specificity as illustrated by the Ile mischarging of EctRNALeuGAG (Figure 4C). Both MmLeuRS-CP1/LSD-K598A and MmLeuRS were able to mischarge Ile in contrast to MmLeuRS-CP1/LSD and MmLeuRS-CP1 that could not catalyze this substrate. Taken together, these results suggested that the crucial K598 residue of LSD mediated the interaction with tRNA and involved in tRNA recognition.Figure 4.


Leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase.

Yan W, Tan M, Eriani G, Wang ED - Nucleic Acids Res. (2013)

Editing and mischarging properties of MmLeuRS-CP1/LSD-K598A. (A) Total editing activity was measured by the AMP formation assay with 1 µM of MmLeuRS-CP1/LSD-K598A and 15 mM Nva in the absence or presence of 5 µM EctRNALeuGAG or MmtRNALeuUAG. (B) Deacylation of [3H]-Ile-EctRNALeu (1 µM) by 20 nM of MmLeuRS (black circle), MmLeuRS-CP1/LSD-K598A (inverted black triangle), MmLeuRS-CP1/LSD (black square) and MmLeuRS-CP1 (black triangle). (C) Mischarging of EctRNALeuGAG (20 µM) with Ile catalyzed by 1 µM of MmLeuRS (black circle), MmLeuRS-CP1/LSD-K598A (inverted black triangle), MmLeuRS-CP1/LSD (black square) and MmLeuRS-CP1(black triangle). (D) Crystal structure of tRNALeu (light blue in the cartoon model) in complex with EcLeuRS (grey) during the editing conformation (PDB ID code 4ARC, Ref.4). Residues R595, K598 and R600 of LSD (green) are numbered and shown in stick representation with labelling. Both G10 and G46 of tRNALeu were also highlighted with the stick model with their distances to K598 labelled.
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Related In: Results  -  Collection

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Show All Figures
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gkt185-F4: Editing and mischarging properties of MmLeuRS-CP1/LSD-K598A. (A) Total editing activity was measured by the AMP formation assay with 1 µM of MmLeuRS-CP1/LSD-K598A and 15 mM Nva in the absence or presence of 5 µM EctRNALeuGAG or MmtRNALeuUAG. (B) Deacylation of [3H]-Ile-EctRNALeu (1 µM) by 20 nM of MmLeuRS (black circle), MmLeuRS-CP1/LSD-K598A (inverted black triangle), MmLeuRS-CP1/LSD (black square) and MmLeuRS-CP1 (black triangle). (C) Mischarging of EctRNALeuGAG (20 µM) with Ile catalyzed by 1 µM of MmLeuRS (black circle), MmLeuRS-CP1/LSD-K598A (inverted black triangle), MmLeuRS-CP1/LSD (black square) and MmLeuRS-CP1(black triangle). (D) Crystal structure of tRNALeu (light blue in the cartoon model) in complex with EcLeuRS (grey) during the editing conformation (PDB ID code 4ARC, Ref.4). Residues R595, K598 and R600 of LSD (green) are numbered and shown in stick representation with labelling. Both G10 and G46 of tRNALeu were also highlighted with the stick model with their distances to K598 labelled.
Mentions: Consistently, in the chimeric MmLeuRS-CP1/LSD, the K598A mutation controlled post-transfer editing, as there was a drop in the AMP synthesis rate (Figure 4A, Table 2) and an absence of deacylation activity towards Ile-EctRNALeuGAG (Figure 4B). The loss of deacylation properties was further confirmed by a loss of aminoacylation specificity as illustrated by the Ile mischarging of EctRNALeuGAG (Figure 4C). Both MmLeuRS-CP1/LSD-K598A and MmLeuRS were able to mischarge Ile in contrast to MmLeuRS-CP1/LSD and MmLeuRS-CP1 that could not catalyze this substrate. Taken together, these results suggested that the crucial K598 residue of LSD mediated the interaction with tRNA and involved in tRNA recognition.Figure 4.

Bottom Line: Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding.Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD.The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology, Center for RNA Research, Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, PR China.

ABSTRACT
The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNA(Leu) recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

Show MeSH
Related in: MedlinePlus