Helix α-3 inter-molecular salt bridges and conformational changes are essential for toxicity of Bacillus thuringiensis 3D-Cry toxin family

Abstract

Bacillus thuringiensis insecticidal Cry toxins break down larval midgut-cells after forming pores. The 3D-structures of Cry4Ba and Cry5Ba revealed a trimeric-oligomer after cleavage of helices α-1 and α-2a, where helix α-3 is extended and made contacts with adjacent monomers. Molecular dynamic simulations of Cry1Ab-oligomer model based on Cry4Ba-coordinates showed that E101 forms a salt-bridge with R99 from neighbor monomer. An additional salt bridge was identified in the trimeric-Cry5Ba, located at the extended helix α-3 in the region corresponding to the α-2b and α-3 loop. Both salt-bridges were analyzed by site directed mutagenesis. Single-point mutations in the Lepidoptera-specific Cry1Ab and Cry1Fa toxins were affected in toxicity, while reversed double-point mutant partially recovered the phenotype, consistent with a critical role of these salt-bridges. The single-point mutations in the salt-bridge at the extended helix α-3 of the nematicidal Cry5Ba were also non-toxic. The incorporation of this additional salt bridge into the nontoxic Cry1Ab-R99E mutant partially restored oligomerization and toxicity, supporting that the loop between α-2b and α-3 forms part of an extended helix α-3 upon oligomerization of Cry1 toxins. Overall, these results highlight the role in toxicity of salt-bridge formation between helices α-3 of adjacent monomers supporting a conformational change in helix α-3.

Rearrangement of N-Terminal α-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity

“…The final structure of Cry toxin oligomers when inserted into the membrane remains unsolved. It was proposed that the interaction of Cry1Ab with membrane receptors triggers cleavage of helix α-1, facilitating the rearrangement of α-helices at the N-terminal end of domain I and leading to oligomer assembly [ 15 , 19 ]. Evidence based on disulfide cross-linking of various α-helices from domain I among them or between α-helices from domain I with domain II indicated that domain I swings out from domains II and III, resulting in insertion of helix α-5 into the membrane [ 10 ].…”

“…This long helix α-3 is located at the central core of the trimeric conformation presented by both proteins, showing multiple contacts with helices α-3, α-4 and α-6 from the adjacent monomers [ 16 , 17 ]. We previously showed that helix α-3 plays an important role in Cry toxin oligomerization, where intermolecular salt bridges were shown to participate in oligomerization and toxicity [ 18 , 19 , 20 ]. Furthermore, conserved charged residues found in the loop between helices α-2b and α-3 were shown to be involved in a salt bridge with adjacent monomers in several Cry toxins, implying that a conformational change of this loop into an α-helix is required to allow oligomer assembly [ 19 ].…”

“…We previously showed that helix α-3 plays an important role in Cry toxin oligomerization, where intermolecular salt bridges were shown to participate in oligomerization and toxicity [ 18 , 19 , 20 ]. Furthermore, conserved charged residues found in the loop between helices α-2b and α-3 were shown to be involved in a salt bridge with adjacent monomers in several Cry toxins, implying that a conformational change of this loop into an α-helix is required to allow oligomer assembly [ 19 ]. In fact, the residues D129 and K131 of Cry5Ba toxin, located in this loop region, form an intermolecular salt bridge in the trimeric conformation of this protein, and mutations affecting these residues resulted in inactive toxins [ 19 ].…”

“…Furthermore, conserved charged residues found in the loop between helices α-2b and α-3 were shown to be involved in a salt bridge with adjacent monomers in several Cry toxins, implying that a conformational change of this loop into an α-helix is required to allow oligomer assembly [ 19 ]. In fact, the residues D129 and K131 of Cry5Ba toxin, located in this loop region, form an intermolecular salt bridge in the trimeric conformation of this protein, and mutations affecting these residues resulted in inactive toxins [ 19 ]. Similar evidence was observed by in silico modeling data of helix α-3 of domain I of Cry1A toxin [ 21 ].…”

Triple arginines as molecular determinants for pentameric assembly of the intracellular domain of 5-HT3A receptors

“…In this regard, it is intriguing to find that the 5-HT 3A –ICD is structured as pentamers in the absence of both the ECD and the TMD ( Pandhare et al, 2016 ). Multiple studies underscore the importance of salt-bridge formation in the stabilization of a protein oligomeric assembly ( Binter et al, 2009 ; Skinner et al, 2017 ; Pacheco et al, 2018 ). Therefore, we investigated whether some of the aforementioned function-governing salt bridges additionally play a fundamental role in pentameric assembly.…”