Structural Basis For Receptor Recognition And Pore Formation Of Zebrafish Pore-forming Protein Dln1

Pore forming proteins (PFPs) have been identified in various organisms from bacteria to vertebrates. They are usually synthesized as a water-soluble pre-form protein, which specifically binds to receptors on the target cell. Upon membrane binding, they oligomerize into a ring-like complex that penetrates the target cell membrane, leading to the leakage of intracellular matrix and eventually the death of target cell. The well characterized bacterial PFPs are able to kill host cells and/or other species of bacteria. In contrast to bacterial PFPs, the eukaryotic PFPs exhibit very diversified functions, such as being involved in host defense, signaling for apoptosis and neurodegenerative diseases. Among these PFPs, bacterial PFPs have been well characterized. In contrast to the systematic analysis of prokaryotic aerolysins, which belong to one of three main PFPs families, mechanisms of receptor recognition and/or pore formation of the eukaryotic members remain unknown.Here we present the first crystal (1.86 A) and electron microscopy (20 A) structures of a vertebrate aerolysin-like protein from Danio rerio, termed Dlnl, before and after pore formation. Crystal structure reveals that each subunit of Dlnl antiparallel dimer contains an N-terminal β-prism lectin module and a C-terminal aerolysin module, providing the first structural information of a vertebrate aerolysin. The lectin module specifically recognizes the high-mannose glycans that contain a non-reducing terminus of Manal-2Man or Manal-3Man, whereas the aerolysin module is responsible for pore formation. Specific binding of the lectin module towards high-mannose glycans triggers drastic conformational changes of the aerolysin module in a pH-dependent manner, ultimately resulting in the formation of a membrane-bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore-forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dlnl displays a very high affinity toward yeast mannan and gp120 of HIV, suggesting its potential role of defense in zebrafish immune system. Besides, Dlnl and homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish-specific defense molecule.