Dimeric architecture and membrane thinning govern substrate recognition by human signal peptide peptidase.

Signal peptide peptidase (SPP) is an intramembrane aspartyl protease that cleaves transmembrane segments of diverse origin, such as remnant signal peptides of secretory proteins or transmembrane helices of tail-anchored proteins. Consistent with its ability to cleave multiple substrates, SPP is essential in mammals, and its activity is linked to a wide range of processes from immune regulation to protein quality control and cancer. Here we determine cryo-EM structures of human SPP in its apo form and bound to a signal peptide substrate. Together with molecular dynamics simulations and functional assays, our data show that SPP forms a constitutive homodimer that locally curves and thins the membrane, placing the conserved active site within the bilayer. Substrate engagement drives major conformational changes, including movement of a "latch" helix that positions the signal peptide for cleavage. The substrate adopts a tilted helix-unwound-β-strand architecture that defines the cleavage register independently of sequence. Steric exclusion by folded luminal domains or additional transmembrane helices explains selective processing of certain protein termini.