Cross-Talk Between Intramolecular and Intermolecular Amino Acid Networks Orchestrates the Assembly of the Cholera Toxin B Pentamer via the Residue His94

Protein assembly is the mechanism of combining two or more protein chains. Living organisms to trigger biological activity often uses this mechanism. The cholera toxin B subunit pentamer (CtxB5), the binding moiety of CtxAB5, a member of the AB5 toxin family, is presented here as a model for studying protein assembly. Experimental results showed the importance of histidine residues in CtxB5 assembly. Nevertheless, the histidines are located outside the pentamer interfaces suggesting an indirect role. Gemini and Spectral-Pro, two network-based models developped in our team, were used in combination with in silico mutations produced with Fold X to investigate this question. All the residues of the pentamer interfaces, so-called hotspots, were identified and some appeared to be chemical neighbors of the His 94, making possible a propagation path of conformational changes from the His 94 to the interface to regulate the pentamer assembly. Mutations of the residues along intra-to-inter amino acid paths produce non additive mutational perturbations of the interface energy, indicating influences of His 94 on its hotspot neighbors to regulate the interface. The role of intra-to-inter amino acid paths in regulating the pentamer interface was confirmed by applying similar approach to the heat labile toxin B pentamer (LTB5), which share 84% sequence identity, structural and functional similarity with CtxB5. Different intra-to-inter amino acid paths were identified for LTB5 consistently with the different assembly mechanisms followed by both toxin pentamers. These results open up avenues for understanding why these two toxins follow different assembly mechanisms.