Multivalent Calixarene Complexation of a Designed Pentameric Lectin.

We describe complex formation between a designed pentameric ß-propeller and the anionic macrocycle sulfonato-calix[8]arene (sclx8), as characterized by X-ray crystallography and NMR spectroscopy. Two crystal structures and 15N HSQC experiments reveal a single calixarene binding site in the concave pocket of the ß-propeller toroid. Despite the symmetry mismatch between the pentameric protein and the octameric macrocycle, they form a high affinity multivalent complex, with the largest protein-calixarene interface observed to date. This system provides a platform for investigating multivalency.

Cage versus sheet: Probing the Determinants of Protein - Cucurbit[7]uril Crystalline Architectures.

The donut-shaped cucurbit[n]urils (Qn) are a class of rigid macrocyclic receptor with protein recognition capabilities. Qn encapsulation of amino acid side chains can enable protein assembly. Recently, cucurbit[7]uril (Q7) has been applied as a molecular glue for organizing protein building blocks into crystalline architectures. Q7 co-crystallization with dimethylated Ralstonia solanacearum lectin (RSL*) has yielded novel crystalline architectures. Co-crystallization of RSL* and Q7 yields either cage- or sheet-like architectures which may be modulated via protein engineering. However, questions remain as to the factors dictating the formation of one architecture over another (cage versus sheet). Here, we make use of an engineered RSL*-Q7 system which co-crystallizes as the cage or sheet assembly with easily-distinguished crystal morphologies. Using this model system, we probe how the crystallization conditions dictate which crystalline architecture is adopted. Protein-ligand ratios and the sodium concentration were identified as key determinants for the growth of the cage versus sheet assemblies.

Protein-Calixarene Complexation: From Recognition to Assembly.

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.

Where is the Macrocycle? Comment on "Simultaneous Organic and Inorganic Host-Guest Chemistry within Pillararene-Protein Cage Frameworks."

Kostiainen and co-workers claim to have prepared crystals of ferritin by "electrostatically bridging" the protein with a cationic pillar[5]arene. Questions remain regarding the contribution of the pillar[5]arene to protein assembly and the putative role as a molecular glue.

Complex Formation between Cytochrome c and a Tetra-alanino-calix[4]arene.

Owing to their remarkable features, calix[n]arenes are being exploited to study different aspects of molecular recognition, including protein complexation. Different complexation modes have been described, depending on the moieties that complement the aromatic cavity, allowing for function regulation and/or controlled assembly of the protein target. Here, a rigid cone calix[4]arene, bearing four anionic alanine units at the upper rim, was tested as a ligand for cytochrome c. Cocrystallization attempts were unfruitful, preventing a solid-state study of the system. Next, the complex was studied using NMR spectroscopy, which revealed the presence of two binding sites at lysine residues with dissociation constants (Kd) in the millimolar range.

Porous assembly of an antifungal protein mediated by zinc and sulfonato-calix[8]arene.

Controlled protein assembly holds great potential in the fabrication of biohybrid materials. However, the structural diversity and complexity of proteins present an obstacle to controlled assembly. Supramolecular chemistry is a possible solution as it offers tools to mediate self-assembly with molecular precision. This paper deals with the calixarene- and zinc-mediated assembly and crystallization of the histidine-rich Penicillium chrysogenum antifungal protein B (PAFB). We report crystal structures of pure PAFB, PAFB in complex with Zn2+, and the ternary complex of PAFB, Zn2+ and sulfonato-calix[8]arene (sclx8). A comparison of the three crystal structures revealed the structural plasticity of PAFB. While the flexible and highly anionic sclx8 resulted in large molecular weight aggregates of PAFB in solution, diffraction-quality crystals of PAFB-sclx8 were not obtained. We report crystals of PAFB-Zn2+-sclx8 in which a trinuclear zinc cluster occurred adjacent to a calixarene binding site. Interestingly, the combination of sclx8 complexation and zinc coordination resulted in a porous framework with channels of circa 2 nm diameter. Detailed analysis of the crystal structure highlighted novel molecular recognition features. This research enriches the set of supramolecular interactions available to promote protein assembly.

Facile Fabrication of Protein-Macrocycle Frameworks.

Precisely defined protein aggregates, as exemplified by crystals, have applications in functional materials. Consequently, engineered protein assembly is a rapidly growing field. Anionic calix[n]arenes are useful scaffolds that can mold to cationic proteins and induce oligomerization and assembly. Here, we describe protein-calixarene composites obtained via cocrystallization of commercially available sulfonato-calix[8]arene (sclx8) with the symmetric and "neutral" protein RSL. Cocrystallization occurred across a wide range of conditions and protein charge states, from pH 2.2-9.5, resulting in three crystal forms. Cationization of the protein surface at pH ∼ 4 drives calixarene complexation and yielded two types of porous frameworks with pore diameters >3 nm. Both types of framework provide evidence of protein encapsulation by the calixarene. Calixarene-masked proteins act as nodes within the frameworks, displaying octahedral-type coordination in one case. The other framework formed millimeter-scale crystals within hours, without the need for precipitants or specialized equipment. NMR experiments revealed macrocycle-modulated side chain pKa values and suggested a mechanism for pH-triggered assembly. The same low pH framework was generated at high pH with a permanently cationic arginine-enriched RSL variant. Finally, in addition to protein framework fabrication, sclx8 enables de novo structure determination.

Segregated Protein-Cucurbit[7]uril Crystalline Architectures via Modulatory Peptide Tectons.

One approach to protein assembly involves water-soluble supramolecular receptors that act like glues. Bionanoarchitectures directed by these scaffolds are often system-specific, with few studies investigating their customization. Herein, the modulation of cucurbituril-mediated protein assemblies through the inclusion of peptide tectons is described. Three peptides of varying length and structural order were N-terminally appended to RSL, a ß-propeller building block. Each fusion protein was incorporated into crystalline architectures mediated by cucurbit[7]uril (Q7). A trimeric coiled-coil served as a spacer within a Q7-directed sheet assembly of RSL, giving rise to a layered material of varying porosity. Within the spacer layers, the coiled-coils were dynamic. This result prompted consideration of intrinsically disordered peptides (IDPs) as modulatory tectons. Similar to the coiled-coil, a mussel adhesion peptide (Mefp) also acted as a spacer between protein-Q7 sheets. In contrast, the fusion of a nucleoporin peptide (Nup) to RSL did not recapitulate the sheet assembly. Instead, a Q7-directed cage was adopted, within which disordered Nup peptides were partially "captured" by Q7 receptors. IDP capture occurred by macrocycle recognition of an intrapeptide Phe-Gly motif in which the benzyl group was encapsulated by Q7. The modularity of these protein-cucurbituril architectures adds a new dimension to macrocycle-mediated protein assembly. Segregated protein crystals, with alternating layers of high and low porosity, could provide a basis for new types of materials.

Protein recognition by cucurbit[6]uril: high affinity N-terminal complexation.

The donut-shaped cucurbit[n]urils (Qn, n = 6-8) are rigid macrocyclic receptors with widespread use in protein recognition. To date, most applications have centred on the encapsulation of N-terminal aromatic residues by Q7 or Q8. Less attention has been placed on Q6, which can recognize lysine side chains due to its high affinity for alkylamines. In this work, we investigated protein-Q6 complexation by using NMR spectroscopy. Attempts to crystallize protein-Q6 complexes were thwarted by the crystallization of Q6. We studied four proteins that vary in size, net charge, and lysine content. In addition to Q6 interactions with specific Lys or dimethylated Lys residues, we report striking evidence for N-terminal recognition. High affinity (micromolar) binding occurred with the N-terminal Met-Lys motif present in one of the four model proteins. Engineering this feature into another model protein yielded a similar high affinity site. We also present evidence for Q8 binding at this N-terminal feature. These data expand the cucurbituril toolkit for protein sensing.

Probing the determinants of porosity in protein frameworks: co-crystals of cytochrome c and an octa-anionic calix[4]arene.

Sulfonato-calix[n]arenes (sclxn) are promising tools to generate crystalline protein frameworks. We report, for the first time, a lower rim functionalised octa-anionic calix[4]arene (sclx4mc) in complex with proteins. Two crystal structures of sclx4mc bound to yeast or horse heart cytochrome c (cytc) are described. Highly porous honeycomb or tubular assemblies were obtained with yeast or horse cytc, respectively. Related frameworks were obtained previously with sclx8 and sclx6 but not with sclx4, suggesting that the ligand charge is a determining factor.

Measuring the Impact of PEGylation on a Protein-Polysaccharide Interaction.

PEGylation is the most widely used half-life extension strategy for protein therapeutics. While it imparts a range of attractive attributes PEGylation can impede protein binding and reduce efficacy. A model system to probe the effects of PEGylation on protein binding has practical applications. Here, we present a system based on complex formation between a hexavalent lectin (RSL) and the globular polysaccharide Ficoll PM70 (a type of glycocluster). Mutants of the lectin were used to generate conjugates with 3, 6, or 12 PEG (1 kDa) chains. Using NMR spectroscopy we monitored how the degree of PEGylation impacted the lectin-Ficoll interaction. The binding propensity was observed to decrease with increasing polymer density. Apparently, the extended PEG chains sterically impede the lectin-Ficoll binding. This deduction was supported by molecular dynamics simulations of the protein-polymer conjugates. The implications for protein-surface interactions are discussed.

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

Ligand-mediated regulation of protein assembly occurs frequently in different cellular contexts. Auto-regulated assembly, where a ligand acts as its own competitive inhibitor, provides a mechanism for exquisite control of assembly. Unlike simple protein-ligand systems a quantification of the binding thermodynamics is not straightforward. Here, we characterize the interactions of a recently identified model system in which the oligomerization of cytochrome c is controlled by sulfonato-calix[8]arene, an anionic supramolecular scaffold. Isothermal titration calorimetry and thermodynamic modelling, in combination with Bayesian fitting, were used to quantify the ligand binding and assembly equilibria for this system. The approach and variations of this model may prove useful for the analysis of auto-regulated protein assembly in general.

Noncovalent PEGylation via Sulfonatocalix[4]arene-A Crystallographic Proof.

Noncovalent or supramolecular PEGylation, in combination with the site of administration, has great potential to increase the half-life of therapeutic proteins. To date, a variety of noncovalent PEGylation strategies have been devised. However, questions remain concerning the nature of the protein-PEG interaction. Here, we report structural analyses of a model system that comprised the lysine-rich cytochrome c and two PEGylated variants of sulfonatocalix[4]arene. Complex formation was characterized in solution by NMR spectroscopy. It was found that mono- or di-PEGylated sulfonatocalix[4]arene bound the protein similar to the parent calixarene. X-ray crystal structures at <2.7 Å resolution of the PEGylated derivatives in complex with cytochrome c revealed that the PEG chains were mostly disordered or encapsulated within the calixarene cavity. These results suggest that there was minimal interaction between the PEG and the protein surface, providing further evidence in favor of PEG maintaining a random coil conformation.

Protein Recognition by Functionalized Sulfonatocalix[4]arenes.

The interactions of two mono-functionalized sulfonatocalix[4]arenes with cytochrome c were investigated by structural and thermodynamic methods. The replacement of a single sulfonate with either a bromo or a phenyl substituent resulted in altered recognition of cytochrome c as evidenced by X-ray crystallography. The bromo-substituted ligand yielded a new binding mode in which a self-encapsulated calixarene dimer contributed to crystal packing. This ligand also formed a weak halogen bond with the protein. The phenyl-substituted ligand was bound to Lys4 of cytochrome c, in a 1.7 Šresolution crystal structure. A dimeric packing arrangement mediated by ligand-ligand contacts in the crystal suggested a possible assembly mechanism. The different protein recognition properties of these calixarenes are discussed.

Auto-regulated Protein Assembly on a Supramolecular Scaffold.

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 (sclx8 ) mediates the formation of cytochrome c tetramers in solution. This tetramer spontaneously disassembles at ≥2 equivalents of sclx8 , providing a remarkable example of auto-regulation. Using X-ray crystallography the sclx8 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.

Cucurbit[7]uril-Dimethyllysine Recognition in a Model Protein.

Here, we provide the first structural characterization of host-guest complexation between cucurbit[7]uril (Q7) and dimethyllysine (KMe2 ) in a model protein. Binding was dominated by complete encapsulation of the dimethylammonium functional group. While selectivity for the most sterically accessible dimethyllysine was observed both in solution and in the solid state, three different modes of Q7-KMe2 complexation were revealed by X-ray crystallography. The crystal structures revealed also entrapped water molecules that solvated the ammonium group within the Q7 cavity. Remarkable Q7-protein assemblies, including inter-locked octahedral cages that comprise 24 protein trimers, occurred in the solid state. Cucurbituril clusters appear to be responsible for these assemblies, suggesting a strategy to generate controlled protein architectures.

Short Arginine Motifs Drive Protein Stickiness in the Escherichia coli Cytoplasm.

Although essential to numerous biotech applications, knowledge of molecular recognition by arginine-rich motifs in live cells remains limited. 1H,15N HSQC and 19F NMR spectroscopies were used to investigate the effects of C-terminal -GRn (n = 1-5) motifs on GB1 interactions in Escherichia coli cells and cell extracts. While the "biologically inert" GB1 yields high-quality in-cell spectra, the -GRn fusions with n = 4 or 5 were undetectable. This result suggests that a tetra-arginine motif is sufficient to drive interactions between a test protein and macromolecules in the E. coli cytoplasm. The inclusion of a 12 residue flexible linker between GB1 and the -GR5 motif did not improve detection of the "inert" domain. In contrast, all of the constructs were detectable in cell lysates and extracts, suggesting that the arginine-mediated complexes were weak. Together these data reveal the significance of weak interactions between short arginine-rich motifs and the E. coli cytoplasm and demonstrate the potential of such motifs to modify protein interactions in living cells. These interactions must be considered in the design of (in vivo) nanoscale assemblies that rely on arginine-rich sequences.

Protein Dimerization on a Phosphonated Calix[6]arene Disc.

Complex formation between cationic cytochrome c and the water-soluble, poly-anionic p-phosphonatocalix[6]arene (pclx6 ) was investigated. A crystal structure (at 1.8 Šresolution) revealed a remarkable dimeric disc of pclx6 that acts like glue to mediate a symmetric (C2 ) protein dimer. The calixarene disc has a diameter of about 1.5 nm and masks about 360 Å2 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 Kd in the low micromolar range was determined for the charge-rich protein-calixarene complex. In contrast to p-sulfonatocalix[4]arene, the larger pclx6 has a single, well-defined binding site that mediates the assembly of cytochrome c in solution.

Anomer-Specific Recognition and Dynamics in a Fucose-Binding Lectin.

Sugar binding by a cell surface ∼29 kDa lectin (RSL) from the bacterium Ralstonia solanacearum was characterized by NMR spectroscopy. The complexes formed with four monosaccharides and four fucosides were studied. Complete resonance assignments and backbone dynamics were determined for RSL in the sugar-free form and when bound to l-fucose or d-mannose. RSL was found to interact with both the α- and the ß-anomer of l-fucose and the "fucose like" sugars d-arabinose and l-galactose. Peak splitting was observed for some resonances of the binding site residues. The assignment of the split signals to the α- or ß-anomer was confirmed by comparison with the spectra of RSL bound to methyl-α-l-fucoside or methyl-ß-l-fucoside. The backbone dynamics of RSL were sensitive to the presence of ligand, with the protein adopting a more compact structure upon binding to l-fucose. Taking advantage of tryptophan residues in the binding sites, we show that the indole resonance is an excellent reporter on ligand binding. Each sugar resulted in a distinct signature of chemical shift perturbations, suggesting that tryptophan signals are a sufficient probe of sugar binding.

Protein Camouflage: Supramolecular Anion Recognition by Ubiquitin.

Progress in the field of bio-supramolecular chemistry, the bottom-up assembly of protein-ligand systems, relies on a detailed knowledge of molecular recognition. To address this issue, we have characterised complex formation between human ubiquitin (HUb) and four supramolecular anions. The ligands were: pyrenetetrasulfonic acid (4PSA), p-sulfonato-calix[4]arene (SCLX4), bisphosphate tweezers (CLR01) and meso-tetrakis (4-sulfonatophenyl)porphyrin (TPPS), which vary in net charge, size, shape and hydrophobicity. All four ligands induced significant changes in the HSQC spectrum of HUb. Chemical shift perturbations and line-broadening effects were used to identify binding sites and to quantify affinities. Supporting data were obtained from docking simulations. It was found that these weakly interacting ligands bind to extensive surface patches on HUb. A comparison of the data suggests some general indicators for the protein-binding specificity of supramolecular anions. Differences in binding were observed between the cavity-containing and planar ligands. The former had a preference for the arginine-rich, flexible C terminus of HUb.