Unwinding of the Substrate Transmembrane Helix in Intramembrane Proteolysis

Intramembrane-cleaving proteases (I-CLiPs) activate pools of single-pass helical membrane protein signaling precursors that are key in the physiology of prokaryotic and eukaryotic cells. Proteases typically cleave peptide bonds within extended or flexible regions of their substrates, and thus the mechanism underlying the ability of I-CLiPs to hydrolyze the presumably a-helical transmembrane domain (TMD) of these membrane proteins is unclear. Using deep-ultraviolet resonance Raman spectroscopy in combination with isotopic labeling, we show that although predominantly in canonical alpha-helical conformation, the TMD of the established I-CLiP substrate Gurken displays 3(10)-helical geometry. As measured by microscale thermophoresis, this substrate binds with high affinity to the I-CLiPs GlpG rhomboid and MCMJR1 presenilin homolog in detergent micelles. Binding results in deep-ultraviolet resonance Raman spectra, indicating conformational changes consistent with unwinding of the 3(10)-helical region of the substrate's TMD. This 3(10)-helical conformation is key for intramembrane proteolysis, as the substitution of a single proline residue in the TMD of Gurken by alanine suppresses 3(10)-helical content in favor of alpha-helical geometry and abolishes cleavage without affecting binding to the I-CLiP. Complemented by molecular dynamics simulations of the TMD of Gurken, our vibrational spectroscopy data provide biophysical evidence in support of a model in which the transmembrane region of cleavable I-CLiP substrates displays local deviations in canonical a-helical conformation characterized by chain flexibility, and binding to the enzyme results in conformational changes that facilitate local unwinding of the transmembrane helix for cleavage.