Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA...

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Pyrrolysyl-tRNA synthetase (PylRS) and tRNA^Pyl are key tools for genetic-code expansion.
• A specific PylRS mutant, PylRS(Y306A/Y384F), from Methanosarcina mazei, enables the incorporation of various bulky non-natural lysine derivatives via the UAG codon.

Purpose of the Study:

• To investigate the substrate recognition mechanisms of PylRS(Y306A/Y384F) for diverse non-natural amino acids.
• To understand how this mutant enzyme accommodates different lysine derivatives for efficient protein incorporation.

Main Methods:

• Utilized PylRS(Y306A/Y384F) to ligate and incorporate 17 non-natural lysine derivatives, including N^ɛ-(benzyloxycarbonyl)lysine (ZLys) and its substituted variants, into proteins.
• Determined crystal structures of 14 non-natural lysine derivatives bound to the catalytic fragment of PylRS(Y306A/Y384F).
• Assessed the aminoacylation rates of PylRS(Y306A/Y384F) with ZLys.

Main Results:

• PylRS(Y306A/Y384F) efficiently ligated and incorporated ZLys and 10 ortho/meta/para-substituted ZLys derivatives.
• Crystal structures revealed that meta- and para-substituted ZLys derivatives bind in a productive mode.
• Unsubstituted and ortho-substituted ZLys derivatives exhibited both productive and alternative binding modes.
• The double-binding mode for ZLys minimally impacted the high aminoacylation rate.

Conclusions:

• PylRS(Y306A/Y384F) demonstrates precise substrate recognition for bulky non-natural lysine derivatives.
• The observed binding modes explain the enzyme's efficiency and specificity.
• These findings provide a foundation for structure-based design of novel non-natural amino acids for expanding the genetic code.