posted on 2024-01-18, 00:08authored byWen Zhu, Anthony T. Iavarone, Judith P. Klinman
Interactions among
proteins and peptides are essential
for many
biological activities including the tailoring of peptide substrates
to produce natural products. The first step in the production of the
bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide
precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe
the use of hydrogen–deuterium exchange mass spectrometry (HDX-MS)
to characterize the structure and conformational dynamics in the protein–protein
and protein–peptide complexes necessary for PqqE function.
HDX-MS-identified hotspots can be discerned in binary and ternary
complex structures composed of the peptide PqqA, the peptide-binding
chaperone PqqD, and PqqE. Structural conclusions are supported by
size-exclusion chromatography coupled to small-angle X-ray scattering
(SEC-SAXS). HDX-MS further identifies reciprocal changes upon the
binding of substrate peptide and S-adenosylmethionine (SAM)
to the PqqE/PqqD complex: long-range conformational alterations have
been detected upon the formation of a quaternary complex composed
of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA
binding site in PqqD to the PqqE active site Fe4S4. Interactions among the various regions are concluded to arise from
both direct contact and distal communication. The described experimental
approach can be readily applied to the investigation of protein conformational
communication among a large family of peptide-modifying rSAM enzymes.