Cryo-EM Structures of Methanogenic Schizorhodopsins Reveal Divergent Strategies for Proton Transport and Thermal Adaptation

Schizorhodopsins (SzRs) are light-driven inward proton pumps found in Asgard archaea, contrasting with most microbial rhodopsins that export protons. Some SzRs, including Methanoculleus taiwanensis SzR ( Mt SzR) and Methanoculleus sp. SzR ( M sSzR), exhibit remarkable heat tolerance, suggesting structural adaptations to extreme environments. Here, we report cryo-electron microscopy structures of Mt SzR and M sSzR at 2.4 and 2.7 Å resolutions, respectively, revealing distinct mechanisms for proton transport and thermostability. Both proteins share a cytoplasm-facing proton acceptor, yet Mt SzR employs a hydrophobic gating mechanism, whereas M sSzR lacks this barrier and stabilizes its acceptor through a salt-bridge network. Thermostability also diverges: Mt SzR relies on proline-rich loops and electrostatic interactions, while M sSzR achieves stability through reinforced trimerization and extensive interhelical aromatic packing. Structural and mutational analyses highlight the adaptability of SzRs and provide a framework for engineering robust photoreceptors for optogenetics and synthetic biology.