Structural and energetic insights into human rhomboid proteases reveal a unique lateral gating mechanism for orphan family members

Abstract

Rhomboid proteases play fundamental roles in human biology and disease by cleaving substrate transmembrane domains. However, the absence of structural data and the inability to identify substrates for orphan human rhomboids limit mechanistic understanding. Lateral gating is considered the primary route by which transmembrane substrates access the rhomboid active site, although this has been demonstrated directly only for the bacterial rhomboid GlpG. To address this, we characterised the structures, conformational ensembles, and energy landscapes of human rhomboid proteases using AI-based structural models and molecular dynamics simulations, benchmarked by simulations seeded with GlpG crystal structures and models. We find that while some human rhomboids readily transition to open conformations compatible with transmembrane substrate access, orphan rhomboids possess unusually narrow lateral gates that require substantially higher energy to open. Our results reveal unexpected diversity in substrate engagement mechanisms among human rhomboids, and provide a rationale for the orphan status of recently evolved family members.