Making and Breaking Supramolecular Synthons for Modular Protein Frameworks

Anionic calixarenes are useful mediators of protein assembly. In some cases, protein - calixarene cocrystallization yields multiple polymorphs. Ralstonia solanacearum lectin (RSL) cocrystallizes with p-sulfonato-calix[8]arene (sclx₈) in at least four distinct pH-dependent arrangements. One of these polymorphs, occurring at pH ≤ 4, is a cubic framework in which RSL nodes are connected by sclx₈ dimers. These dimers are supramolecular synthons that occur in distinct crystal structures. Now, we show that the discus-shaped dimer of p-phosphonato-calix[6]arene (pclx₆), can replace the sclx₈ dimer yielding a new assembly of RSL. Remarkably, just one type of RSL - pclx₆ cocrystal was formed, irrespective of pH or crystallization condition. These results with pclx₆ contrast starkly with sclx₈ and suggest that the calixarene type (e.g., phosphonate versus sulfonate) dictates the synthon durability, which in turn exerts control over protein assembly and polymorph selection. Breaking the pclx₆ dimer required a mutant of RSL with an affinity tag for macrocycle binding. This highly accessible, dicationic site resulted in a significantly altered and porous framework with pclx₆ (but not with sclx₈). Experiments with ternary mixtures of RSL, pclx₆, and sclx₈ provide evidence of pH-driven self-sorting. Thus, the "mix-and-match" of protein and supramolecular synthons is a promising approach to protein crystal engineering.