Calixarene-mediated assembly of a small antifungal protein

Abstract

Synthetic macrocycles such as calixarenes and cucurbiturils are increasingly applied as mediators of protein assembly and crystallization. The macrocycle can facilitate assembly by providing a surface on which two or more proteins bind simultaneously. This work explores the capacity of the sulfonato-calix[n]arene (sclx n ) series to effect crystallization of PAF, a small, cationic antifungal protein. Co-crystallization with sclx4, sclx6 or sclx8 led to high-resolution crystal structures. In the absence of sclx n , diffraction-quality crystals of PAF were not obtained. Interestingly, all three sclx n were bound to a similar patch on PAF. The largest and most flexible variant, sclx8, yielded a dimer of PAF. Complex formation was evident in solution via NMR and ITC experiments, showing more pronounced effects with increasing macrocycle size. In agreement with the crystal structure, the ITC data suggested that sclx8 acts as a bidentate ligand. The contributions of calixarene size/conformation to protein recognition and assembly are discussed. Finally, it is suggested that the conserved binding site for anionic calixarenes implicates this region of PAF in membrane binding, which is a prerequisite for antifungal activity.

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

“…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 restructured Ω‐loop D effectively wraps around the calixarene scaffold, with both Lys73 and Lys79 involved directly in sclx 6 complexation. The Lys73 side chain is encapsulated by a calix[2] motif, similar to the Lys27 – s clx 6 interaction in the PAF structure . And the involvement of Pro71 (CH–π interaction with a phenol ring) is also reminiscent of the PAF – s clx 6 complexation (compare Figs and C).…”

“…Detail of the PAF– sclx 6 binding site (PDB 6hah) with key residues shown as sticks . The double partial‐cone conformation of the calixarene is evident.…”

“…Recently, we reported the first crystal structure of a protein–sulfonato‐calix[6]arene ( sclx 6 ) complex . Complexation with the cationic Penicillium antifungal protein (PAF) occurred at the Pro29‐Lys30‐Phe31 motif with additional interactions provided by Lys27 (PDB http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6hah).…”

“…The calixarene adopted a ‘double partial‐cone’ conformation to straddle the protein surface at a pair of neighboring lysines (Fig. ) . Although a variety of cyt c structures have been obtained with sclx 8 and calix[4]arene derivatives , cocrystallization of cyt c with sclx 6 yielded just one crystal form that requires the presence of imidazole.…”