Figure - available from: Nature Communications
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Comparison of crystal structures of PmIleRS2 and PmIleRS1 in complex with the non-hydrolysable analog of the reaction intermediate Ile-AMS a, b The canonical structures with the HUP catalytic domain (colored gray in IleRS2 and yellow in IleRS1), the CP1 and CP2 domains (CP refers to connective peptide, colored purple and raspberry, respectively) and the editing domains (ED, colored red) inserted into CP1 are visualized. The full-length C-terminal domain of type 2 IleRS is resolved, revealing three subdomains (SD1-SD3, colored blue, green and pink, respectively), among which SD3 differs in size, fold and lack of the zinc-binding motif relative to SD3 in IleRS1. An insertion (colored cyan) into SD2 is observed in IleRS2. For details see Supplementary Fig. 6. c Structural overlay of the IleRS1 and IleRS2 HUP catalytic cores (residues 50–174 and 523–635 in IleRS1 and 40–168 and 513–632 in IleRS2) bound to Ile-AMS revealed overlapping positions of Ile-AMS and a conformational rearrangement of the active site in IleRS2 relative to IleRS1. The tip of helix α1 comprising the HXGH motif (blue in IleRS2 and orange in IleRS1) and the KMSKS loop move towards each other in IleRS2. This repositions both the first and fourth histidine residues as well as the glycine α-carbon (insets).
Source publication
Antibiotics target key biological processes that include protein synthesis. Bacteria respond by developing resistance, which increases rapidly due to antibiotics overuse. Mupirocin, a clinically used natural antibiotic, inhibits isoleucyl-tRNA synthetase (IleRS), an enzyme that links isoleucine to its tRNAIle for protein synthesis. Two IleRSs, mupi...
Citations
Mupirocin is a broad-spectrum antibiotic that acts predominantly against Gram-positive bacteria. It is produced by Pseudomonas fluorescens NCIMB 10586 and has been clinically used to treat primary and secondary skin infections and to eradicate nasal colonisation of methicillin-resistant Staphylococcus aureus strains. Mupirocin inhibits protein synthesis by blocking the active site of isoleucyl-tRNA synthetase (IleRS), which prevents the enzyme from binding isoleucine and ATP for Ile-tRNAIle synthesis. Two types of IleRS are found in bacteria — while IleRS1 is susceptible to mupirocin inhibition, IleRS2 provides resistance to cells. These two types belong to distinct evolutionary clades which likely emerged from an early gene duplication in bacteria. Resistance in IleRS2 is based on the loss of interactions that govern mupirocin binding to IleRS1, such as hydrogen bonding to the carboxylate moiety of mupirocin. IleRS2 enzymes with Ki in the millimolar range have recently been discovered. These hyper-resistant IleRS2 variants surprisingly have a non-canonical version of the catalytic motif, which serves as a signature motif of class I aminoacyl-tRNA synthetases to which IleRS belongs. The non-canonical motif, in which the 1st and 3rd positions are swapped, is key for hyper-resistance and can be accommodated without abolishing enzyme activity in IleRS2 but not in IleRS1. Clinical use of mupirocin led to the emergence of resistance in S. aureus. Low-level resistance arises by mutations of the housekeeping IleRS1, while high-level resistance develops by the acquisition of the resistant IleRS2 on a plasmid. There is no evidence that hyper-resistant variants have been found in clinical isolates.
Helicobacter pylori infection is a global health concern, affecting over half of the world's population. Acquiring structural information on pharmacological targets is crucial to facilitate inhibitor design. Here, we have determined the crystal structures of H. pylori isoleucyl‐tRNA synthetase ( Hp IleRS) in apo form as well as in complex with various substrates (Ile, Ile‐AMP, Val, and Val‐AMP) or an inhibitor (mupirocin). Our results provide valuable insights into substrate specificity, recognition, and the mechanism by which Hp IleRS is inhibited by an antibiotic. Moreover, we identified Asp641 as a prospective regulatory site and conducted biochemical analyses to investigate its regulatory mechanism. The detailed structural information acquired from this research holds promise for the development of highly selective and effective inhibitors against H. pylori infection.
