Conformational Selection Mechanism of Rhomboid-Catalyzed Intramembrane Proteolysis Revealed by Solid-State NMR

Abstract

Intramembrane rhomboid proteases catalyze the hydrolysis of peptide bonds within membrane-embedded substrates, and play a pivotal role in the physiological processes of both prokaryotic and eukaryotic cells. Although multiple structures of rhomboid proteases have been solved, a comprehensive understanding of their catalytic mechanism remains elusive due to the absence of native transmembrane substrates. Here, we investigate the conformational characteristics and dynamic behavior of PsTatA, a native rhomboid protease substrate from Providencia stuartii, in lipid bilayers by solid-state nuclear magnetic resonance (ssNMR). Secondary chemical shifts indicate an extended N-terminus of PsTatA within the lipid bilayer, contradicting the prior assumption of an α-helical conformation. Moreover, we observed multiple N-terminus conformations that are susceptible to modulation by lipid components and the dynamics of the substrate are influenced by the thickness of the membrane. The introduction of rhomboid protease did not induce a direct alteration in the structural configuration of the substrate but instead selectively stabilized a particular conformation by modulating the local membrane environment. This insight sheds new light on the intricate interplay between rhomboid proteases and their substrates within the lipid environment.