Protein Dimerization on a Phosphonated Calix[6]arene Disc

Abstract

Complex formation between cationic cytochrome c and the water-soluble, poly-anionic p-phosphonatocalix[6]arene (pclx ) was investigated. A crystal structure (at 1.8 Å resolution) revealed a remarkable dimeric disc of pclx that acts like glue to mediate a symmetric (C ) protein dimer. The calixarene disc has a diameter of about 1.5 nm and masks about 360 Å of protein surface. The key protein-calixarene contacts occur via two linchpin lysines, with additional contacts provided by a small hydrophobic patch. The protein-calixarene supramolecular assemblies were observed in solution by size-exclusion chromatography with multi-angle light scattering and NMR spectroscopy. Using isothermal titration calorimetry and NMR data, an apparent K in the low micromolar range was determined for the charge-rich protein-calixarene complex. In contrast to p-sulfonatocalix[4]arene, the larger pclx has a single, well-defined binding site that mediates the assembly of cytochrome c in solution.

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

“…Alternatively, the reduced entropy may be rationalized by considering that the first sclx 8 can ‘explore’ a larger protein surface, while the second ligand is restricted. The binding affinities of sclx 8 are comparable to the K d ∼2–8 μM for binding of the related ligand, phosphonato‐calix[6]arene ( pclx 6 ), and are much tighter than the K d ∼30 μM for the smaller sulfonato‐calix[4]arene . Interestingly, pclx 6 triggers dimerization of cytochrome c but not in an auto‐regulated fashion, suggesting a fundamentally different assembly mechanism compared to sclx 8 .…”

“…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 ,…”

“…Recently, we uncovered such an example studying the interaction between a cationic model protein, cytochrome c , and a flexible anionic calixarene, sulfonato‐calix[8]arene ( sclx 8 ) . Cytochrome c is a monomeric electron transport protein, while calixarenes are synthetic macrocycles that have been shown to modulate protein assembly ,,. Auto‐regulated assembly was demonstrated by using a variety of techniques including, light scattering, NMR measurements and X‐ray crystallography.…”

Calixarene capture of partially unfolded cytochrome c

“…The other face of sclx 6 masks a smaller patch (~ 310 Å 2 ) on the second molecule of cyt c . Here, the key residues are Lys4 and Lys100, in a binding mode similar to pclx 6 complexation . The observation of crystal growth at 1 but not 2 eq.…”

“…In the pursuit of understanding how synthetic receptors recognize and bind to lysine we have characterized complex formation between cationic proteins and various anionic calixarenes . Calixarenes are phenol‐based macrocycles, commonly comprising four, six, or eight units.…”