Probing the Active Site of Deubiquitinase USP30 with Noncanonical Tryptophan Analogues

Methanosarcina mazei pyrrolysyl-tRNA synthetase
(PylRS) and its cognate tRNA have been evolved to generate genetically
encoded noncanonical amino acids (ncAAs). Use of tryptophan (Trp)
analogues with pyrrole ring modification for their spatial and polarity
tuning in enzyme activity and substrate specificity is still limited.
Herein, we report the application of an evolved PylRS, FOWRS2, for
efficient incorporation of five Trp analogues into the deubiquitinase
USP30 to decipher the role of W475 for diubiquitin selectivity. Structures
of the five FOWRS-C/Trp analogue complexes at 1.7–2.5 Å
resolution showed multiple ncAA binding modes. The W475 near the USP30
active site was replaced with Trp analogues, and the effect on the
activity as well as the selectivity toward diubiquitin linkage types
was examined. It was found that the Trp analogue with a formyl group
attached to the nitrogen atom of the indole ring led to an improved
activity of USP30 likely due to enhanced polar interactions and that
another Trp analogue, 3-benzothienyl-l-alanine, induced a
unique K6-specificity. Collectively, genetically encoded noncanonical
Trp analogues by evolved PylRS·tRNACUAPyl pair unravel the spatial role of USP30-W475 in
its diubiquitin selectivity.