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

Rhomboid proteases play fundamental roles in human biology and disease by cleaving substrate transmembrane domains. The absence of structural data and the inability to identify substrates for orphan human rhomboids hinder both a comprehensive understanding of their mechanism of action and the development of strategies to modulate their activity. 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. We show that all human rhomboid proteases possess a conformationally dynamic lateral gate within the membrane which transmembrane substrates can enter. Whereas some rhomboids transition between closed and open conformations with minimal energetic cost, orphan rhomboids exhibit unusually narrow lateral gates that require substantial energy to open. These insights reveal an unexpected degree of heterogeneity in substrate engagement mechanisms among human rhomboids, and provide an explanation for the orphan status for evolutionarily recent family members. In doing so, we establish a framework for interrogating membrane protein function.