Structure of Perinereis linea erythrocruorin reveals a compact extracellular globin megacomplex

Many invertebrates lack erythrocytes and instead rely on extracellular hemoglobin assemblies, termed erythrocruorins, for oxygen transport. Here we report a 2.84 Å cryo-electron microscopy (cryo-EM) structure of Perinereis linea erythrocruorin (PlEc). PlEc is a ∼3.3 MDa megacomplex composed of 180 polypeptide chains organized into 12 protomers, forming a hexagonal bilayer with D6 symmetry. Each protomer consists of 12 globin subunits and three linker subunits, adopting a mushroom-like architecture. The cap of the mushroom is formed by a globin dodecamer associated with a heterotrimeric linker head, and the stem consists of a triple-stranded coiled coil derived from the N-terminal helices of three linker subunits. Biochemical assays show that PlEc has thermal stability and auto-oxidation rate comparable to those of other erythrocruorins, but displays relatively lower oxygen-binding affinity. These findings provide mechanistic insights into the quaternary assembly of invertebrate erythrocruorins and lay the groundwork for the potential biomedical applications.