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 (CtxB 5), the binding moiety of CtxAB 5, a member of the AB 5 toxin family, is presented here as a model for studying protein assembly. Experimental results showed the importance of histidine residues in CtxB 5 assembly. Nevertheless, the histidines are located outside the pentamer interfaces suggesting an indirect role. Gemini and Spectral-Pro, two networkbased 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 intrato-inter amino acid paths in regulating the pentamer interface was confirmed by applying similar approach to the heat labile toxin B pentamer (LTB 5), which share 84% sequence identity, structural and functional similarity with CtxB 5. Different intra-to-inter amino acid paths were identified for LTB 5 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.