Regulated intramembrane proteolysis (RIP) plays important roles in activating cellular signaling pathway in both prokaryotes and eukaryotes. The realization that transmembrane proteins can be cleaved in their membrane spans in the lipid bilayer inspired the extensive studies of the intramembrane proteases responsible for these cleavages. There are four known families of intramembrane proteases: rhomboid, site two protease, presenilin and signal peptide peptidase. Due to the unique feature of RIP occurring in a hydrophobic environment, it is intriguing to investigate the detailed molecular mechanism of the intramembrane proteolytic reactions.

In this dissertation, I will present two three-dimensional protein structures of the transmembrane core domains from two intramembrane protease families: GlpG, the E. coli homolog of rhomboid; and mjS2P, the M. jannaschii homolog of S2P.

Structure analysis helped shed light on how these proteases carry out their functions in intramembrane proteolysis. First, water molecules, which are essential for the scission of peptide bonds, appear to gain access to the active sites of GlpG and mjS2P via a similar mechanism. GlpG contains an open cavity full of water that converges on the active site residue Ser201 whereas mjS2P has a polar channel that allows water entry to the catalytic zinc atom in the closed conformation. More importantly, the two proteases share significant similarity in substrate entry as well, which is proposed to be a gating mechanism regulated by transmembrane helices. This mechanism involves the lateral movements of transmembrane helices within the lipid bilayer to allow substrate access the active site of intramembrane proteases. In GlpG, bending of the C-terminal half of the transmembrane helix a5 was proposed to open the gate for substrate entry. Likewise, in mjS2P, the rotation and translocation of transmembrane domain (TM) 1 as well as the translocation of TM6/TM5 appear to allow substrate entry.