Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element

The
RiPP precursor recognition element (RRE) is a conserved domain
found in many prokaryotic ribosomally synthesized and post-translationally
modified peptide (RiPP) biosynthetic gene clusters (BGCs). RREs bind
with high specificity and affinity to a recognition sequence within
the N-terminal leader region of RiPP precursor peptides. Lasso peptide
biosynthesis involves an RRE-dependent leader peptidase, which is
discretely encoded or fused to the RRE as a di-domain protein. Here
we leveraged thousands of predicted BGCs to define the RRE:leader
peptidase interaction through evolutionary covariance analysis. Each
interacting domain contributes a three-stranded β-sheet to form
a hydrophobic β-sandwich-like interface. The bioinformatics-guided
predictions were experimentally confirmed using proteins from discrete
and fused lasso peptide BGC architectures. Support for the domain–domain
interface derived from chemical shift perturbation, paramagnetic relaxation
enhancement experiments, and rapid variant activity screening using
cell-free biosynthesis. Further validation of selected variants was
performed with purified proteins. We developed a p-nitroanilide-based leader peptidase assay to illuminate the role
of RRE domains. Our data show that RRE domains play a dual function.
RRE domains deliver the precursor peptide to the leader peptidase,
and the rate is saturable as expected for a substrate. RRE domains
also partially compose the elusive S2 proteolytic pocket that binds
the penultimate threonine of lasso leader peptides. Because the RRE
domain is required to form the active site, leader peptidase activity
is greatly diminished when the RRE domain is supplied at substoichiometric
levels. Full proteolytic activation requires RRE engagement with the
recognition sequence-containing portion of the leader peptide. Together,
our observations define a new mechanism for protease activity regulation.