Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX

Abstract

The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals—a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi , Aedes albopictus, and Culex tarsalis larvae—substantially increasing the potential use of this binary toxin in mosquito control.

Droplet microfluidics for time-resolved serial crystallography

“…The minimum crystal size is another consideration. Given that diffraction data can be obtained from sub-micron crystals (Gati et al ., 2017, Bücker et al ., 2020, Williamson et al ., 2023), and the 2–3 m long lysozyme and Pdx1 crystals prepared in ∼1 pL droplets, there is scope to further reduce droplet volumes. While it is feasible to prepare monodisperse 5.4-m-diameter droplets with a volume of 82 fL, the effect of greatly reduced seed occupancy and lower crystal formation frequency [Fig.…”

The Perpetual Vector Mosquito Threat and Its Eco-Friendly Nemeses

“…The main structural difference between Tpp1Aa1 and Tpp2Aa1 occurs at the β-trefoil domain, which could be implicated in distinct roles of these proteins during the intoxication process in the midgut epithelia, where Tpp2Aa1 has a less prominent role in carbohydrate binding when compared to Tpp1Aa1. The few structural differences between Tpp1Aa1 and Tpp2Aa1 indicate they can form a heteromeric pore assembly complex with a topology similar to the aerolysin family of pore-forming toxins [ 227 , 228 ]. The crystal structure of other Tpps, such as Tpp49Aa1, Tpp1A2, Tpp80Aa1, and Tpp35Ab1, have been elucidated.…”

“…The crystal structure of other Tpps, such as Tpp49Aa1, Tpp1A2, Tpp80Aa1, and Tpp35Ab1, have been elucidated. Although they have a few differences, they maintain similar structures, i.e., the receptor and pore-forming domains [ 228 ].…”

Factors Related to Bacillus thuringiensis and Gut Physiology. Comment on Rajan, V. An Alkaline Foregut Protects Herbivores from Latex in Forage, but Increases Their Susceptibility to Bt Endotoxin. Life 2023, 13, 2195

“…stephensi and Aedes albopictus but not against Anopheles gambiae or Ae. aegypti [34,35]; Tpp1/Tpp2 kill Aedes atropalpus but have very limited to no effect against Ae. aegypti [36].…”