A novel insecticidal protein GNIP1Aa demonstrates strong specific toxicity towards the Western corn rootworm (WCR) larvae, Diabrotica virgifera virgifera.
The crystal structure of the protein has been solved at 2.5 Å resolution, revealing two domains: an N-terminal Membrane Attack Complex/Perforin (MACPF) and a novel C-terminal domain. The MACPF domain is structurally similar to the pore-forming CDC (cholesterol-dependent cytolysin) domains. Many CDCs from Gram-positive bacteria, such as PFO and SLO, are toxic to eukaryotic cells, while the best studied MACPFs, such as complement component C6, C8 and C9, are important constituents of the human immune system. Structural resemblance between CDC and MACPF domains explains their similar mechanism of pore-formation, whereas their accessory domains are responsible for protein specificity. All CDCs are supplemented with structurally homologous C-terminal domains with a highly conserved undecapeptide, the ones that bind first to eukaryotic cholesterol-enriched membranes. On contrary, accessory domains in MACPFs are structurally and functionally diverse; in C9 complement component such domain recognizes bacterial cells, while the perforin’s counterpart identifies virus-infected cells.
The role of target selectivity in GNIP1Aa is performed by its novel C-terminal domain, composed of three pseudo-symmetric subdomains. The individual subdomains share structure similarity, a feature that is not obvious from protein sequence examination. Structure-based sequence analysis allowed to identify functionally important amino-acid residues within the domain. Their significance was validated by site-directed mutagenesis. Our studies reveal putative target-recognition sites in GNIP1Aa and provide molecular details of the novel insecticidal mode of action.