Substrate recognition by a peptide-aminoacyl-tRNA ligase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Peptide-aminoacyl-tRNA ligases (PEARLs) are enzymes that attach amino acids to peptide C-termini.
• The recognition mechanisms for peptide substrates and aminoacyl-tRNAs by PEARLs are not well understood.
• Previous research has identified PEARLs capable of conjugating various amino acids, including Cys, Ala, Trp, Gly, Leu, Asn, and Thr.

Purpose of the Study:

• To investigate the peptide substrate specificity of a specific PEARL enzyme responsible for tryptophan incorporation.
• To elucidate the minimal sequence requirements for peptide substrate recognition by this PEARL.
• To explore the potential of this PEARL as a tool for C-terminal protein labeling.

Main Methods:

• Utilized cell-free expression (CFE) to rapidly assay peptide substrate variants.
• Performed truncation and replacement experiments to determine minimal sequence requirements.
• Employed AlphaFold3 modeling to predict binding interactions.
• Conducted site-directed mutagenesis to validate predicted binding modes for tRNA, ATP, and Mg²⁺.
• Applied the identified minimal sequence as a protein tag for C-terminal labeling.

Main Results:

• The studied PEARL demonstrated remarkable tolerance for changes to the C-terminal amino acid of its peptide substrate.
• Minimal sequence requirements for peptide substrate recognition were identified.
• AlphaFold3 modeling provided insights into substrate peptide, tRNA, ATP, and Mg²⁺ binding.
• Mutagenesis experiments supported the role of conserved residues in recognizing the tRNA 3'-CCA sequence.
• The enzyme was successfully used to C-terminally label eGFP, lysozyme, and MBP with tryptophan and 5-bromo-tryptophan.

Conclusions:

• The findings reveal key aspects of peptide and tRNA recognition by a tryptophan-incorporating PEARL.
• The study establishes a minimal sequence requirement for substrate peptides, facilitating enzyme engineering.
• The developed PEARL-based protein labeling system offers a novel method for site-specific C-terminal modification.