The Sec11 C-terminal short helix promotes productive engagement of internal signal sequences with the signal peptidase complex.

Proteins destined for secretion typically contain N-terminal signal sequences that target nascent chains to the endoplasmic reticulum. Following targeting, these sequences are cleaved by the heterotetrameric signal peptidase complex (SPC). Despite a conserved N-terminal, hydrophobic core, and C-terminal tripartite organization, their sequences are highly variable. How SPC subunits contribute to recognition of these diverse sequences remains poorly understood. Sec11, the catalytic subunit of the SPC, contains an N-terminal transmembrane domain and a C-terminal hydrophobic region. The latter was unresolved in human SPC cryo-EM structures, likely due to intrinsic flexibility, yet AlphaFold predictions of the yeast SPC suggest that this region forms a C-terminal short helix (CTS) strategically positioned within the presumed signal sequence-binding site. This structural arrangement led us to hypothesize its possible role in substrate handling during signal peptide processing and we undertook to investigate its function using biochemical assays combined with molecular dynamics simulations. Topology mapping confirmed that the Sec11 CTS traverses the endoplasmic reticulum membrane and molecular dynamics simulations showed that removal of the Sec11 CTS does not substantially alter overall SPC architecture or the proximal membrane environment. While N-terminal signal sequences of varying hydrophobicity were efficiently cleaved, internal signal sequences with extended N-terminal region were selectively defective in cleavage in the absence of the Sec11 CTS. These data suggest that the Sec11 CTS specifically stabilizes internal signal sequences for productive engagement with SPC.