Auto‐regulated Protein Assembly on a Supramolecular Scaffold

Abstract

Controlled protein assembly provides a means to regulate function. Supramolecular building blocks, including rigid macrocycles, are versatile triggers of protein assembly. Now it is shown that sulfonato-calix[8]arene (sclx ) mediates the formation of cytochrome c tetramers in solution. This tetramer spontaneously disassembles at ≥2 equivalents of sclx , providing a remarkable example of auto-regulation. Using X-ray crystallography the sclx binding sites on cytochrome c were characterized. Crystal structures at different protein-ligand ratios reveal varying degrees (up to 35 %) of protein surface coverage by the flexible calixarene and suggest a mechanism for oligomer disassembly. The solution structure of the oligomer was characterized by small-angle X-ray scattering. Overall, the data indicate calixarene-controlled protein assembly and disassembly without the requirement for a competitive inhibitor, and point to protein encapsulation by a flexible macrocycle.

A Thermodynamic Model of Auto‐regulated Protein Assembly by a Supramolecular Scaffold

“…These systems are characterized by bell‐shaped dose‐response curves, where there is a maximum concentration of ligand required to achieve peak response, above which the response is reduced . This effect was reported more than a century ago for antibodies (“prozone effect” or “hook effect”), and has since been observed in DNA transcription (“auto‐regulation”), heparin‐regulated enzyme activity (“template mechanism”),, scaffold proteins (“combinatorial inhibition”), phytate‐mediated protein precipitation,, and supramolecular control of protein assembly ,. Here, we refer to this property as auto‐regulated assembly.…”

“…This mechanism has been referred to as polysteric linkage,, contrasting with allosteric linkage in which protein conformation and dynamics are regulated by ligands. Recently, protein dimerization and higher order assembly has been engineered via synthetic molecules, including bivalent ligands and supramolecular scaffolds . Competitive inhibitors of these molecules can be used to control disassembly ,…”

“…Auto‐regulated assembly was demonstrated by using a variety of techniques including, light scattering, NMR measurements and X‐ray crystallography. It was found that tetramerization of cytochrome c can be triggered by ∼1 equivalent of sclx 8 , while disassembly to the (ligand‐coated) monomer occurs at >2 equivalents . Structural characterization revealed that cytochrome c has at least two sclx 8 binding sites and that sclx 8 can link two cytochrome c molecules (Figure ).…”

Calixarene capture of partially unfolded cytochrome c

“…Here, we have shown that calix[6]arene binding, in combination with imidazole coordination, results in significant structural rearrangement of cyt c . In the cyt c – sclx 6 crystal, the calixarene provides a scaffold that stabilizes the restructured Ω‐loop D mainly through complexation of Lys73 and Lys79. The loss of packing interactions has knock‐on effects for Ω‐loop C, which is disordered.…”

“…Furthermore, highly porous crystalline architectures have been obtained with cyt c and sclx 8 . In one case a protein‐calixarene framework (PDB http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6gd9) is mediated exclusively by the calixarene .…”