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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.

SUBMITTER: Alex JM

PROVIDER: S-EPMC6400181 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Calixarene-mediated assembly of a small antifungal protein.

Alex Jimi M JM Rennie Martin L ML Engilberge Sylvain S Lehoczki Gábor G Dorottya Hajdu H Fizil Ádám Á Batta Gyula G Crowley Peter B PB

IUCrJ 20190205 Pt 2

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 sclx ...[more]

PMID: 30867921

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Project description:This Account summarizes the progress in protein-calixarene complexation, tracing the developments from binary recognition to the glue activity of calixarenes and beyond to macrocycle-mediated frameworks. During the past 10 years, we have been tackling the question of protein-calixarene complexation in several ways, mainly by cocrystallization and X-ray structure determination as well as by solution state methods, NMR spectroscopy, isothermal titration calorimetry (ITC), and light scattering. Much of this work benefitted from collaboration, highlighted here. Our first breakthrough was the cocrystallization of cationic cytochrome c with sulfonato-calix[4]arene leading to a crystal structure defining three binding sites. Together with NMR studies, a dynamic complexation was deduced in which the calixarene explores the protein surface. Other cationic proteins were similarly amenable to cocrystallization with sulfonato-calix[4]arene, confirming calixarene-arginine/lysine encapsulation and consequent protein assembly. Calixarenes bearing anionic substituents such as sulfonate or phosphonate, but not carboxylate, have proven useful.Studies with larger calix[n]arenes (n = 6, 8) demonstrated the bigger better binder phenomenon with increased affinities and more interesting assemblies, including solution-state oligomerization and porous frameworks. While the calix[4]arene cavity accommodates a single cationic side chain, the larger macrocycles adopt different conformations, molding to the protein surface and accommodating several residues (hydrophobic, polar, and/or charged) in small cavities. In addition to accommodating protein features, the calixarene can bind exogenous components such as polyethylene glycol (PEG), metal ions, buffer, and additives. Ternary cocrystallization of cytochrome c, sulfonato-calix[8]arene, and spermine resulted in altered framework fabrication due to calixarene encapsulation of the tetraamine. Besides host-guest chemistry with exogenous components, the calixarene can also self-assemble, with numerous instances of macrocycle dimers.Calixarene complexation enables protein encapsulation, not merely side chain encapsulation. Cocrystal structures of sulfonato-calix[8]arene with cytochrome c or Ralstonia solanacearum lectin (RSL) provide evidence of encapsulation, with multiple calixarenes masking the same protein. NMR studies of cytochrome c and sulfonato-calix[8]arene are also consistent with multisite binding. In the case of RSL, a C3 symmetric trimer, up to six calixarenes bind the protein yielding a cubic framework mediated by calixarene dimers. Biomolecular calixarene complexation has evolved from molecular recognition to framework construction. This latter development contributes to the challenge in design and preparation of porous molecular materials. Cytochrome c and sulfonato-calix[8]arene form frameworks with >60% solvent in which the degree of porosity depends on the protein:calixarene ratio and the crystallization conditions. Recent developments with RSL led to three frameworks with varying porosity depending on the crystallization conditions, particularly the pH. NMR studies indicate a pH-triggered assembly in which two acidic residues appear to play key roles. The field of supramolecular protein chemistry is growing, and this Account aims to encourage new developments at the interface between biomolecular and synthetic/supramolecular chemistry.

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Calixarene-mediated assembly of a small antifungal protein.

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Calixarene-mediated assembly of a small antifungal protein.

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