Structure–function characterization of an insecticidal protein GNIP1Aa, a member of an MACPF and β-tripod families

Abstract

Significance GNIP1Aa is a protein from Chromobacterium piscinae that demonstrates specific toxicity toward Western corn rootworm, one of the most devastating corn pests in the United States. Our studies provide insight into the GNIP1Aa structure and place this protein into an insecticidal protein class, membrane attack complex/PerForin–β-tripod, different from all insect-control products of modern agricultural technology available on the market. Protein activity and uniqueness make GNIP1Aa an excellent commercial candidate for development into a transgenic product. Such a product might have a high potential to combat crop damage in corn and to delay development of resistance in insects. Our work also contributes to the general understanding of the mechanism of action of pore-forming proteins and their target specificity. The crystal structure of the Gram-negative insecticidal protein, GNIP1Aa, has been solved at 2.5-Å resolution. The protein consists of two structurally distinct domains, a MACPF (membrane attack complex/PerForin) and a previously uncharacterized type of domain. GNIP1Aa is unique in being a prokaryotic MACPF member to have both its structure and function identified. It was isolated from a Chromobacterium piscinae strain and is specifically toxic to Diabrotica virgifera virgifera larvae upon feeding. In members of the MACPF family, the MACPF domain has been shown to be important for protein oligomerization and formation of transmembrane pores, while accompanying domains define the specificity of the target of the toxicity. In GNIP1Aa the accompanying C-terminal domain has a unique fold composed of three pseudosymmetric subdomains with shared sequence similarity, a feature not obvious from the initial sequence examination. Our analysis places this domain into a protein family, named here β-tripod. Using mutagenesis, we identified functionally important regions in the β-tripod domain, which may be involved in target recognition.