Probing the dynamical interaction of the para-sulfonato-calix[4]arene with an antifungal protein

Abstract

Calixarenes are hallmark molecules in supramolecular chemistry as guest cages for small ligands. They have also conversely proved their interest as auxiliary ligands toward assisted co-crystallization of proteins. These functionalized macromolecular cages target positively-charges residues, and notably surface-exposed lysines, with a site-selectivity finely characterized experimentally, but that remains to be assessed. Relying on a tailored molecular dynamics simulations protocol, we explore the association of para-sulfonato-calix[4]arenes with an antifungal protein, as a small yet most competitive system with 13 surface-exposed lysines. Our computational approach probe "de novo" the electrostatically-driven interaction, ruled out by a competition with salt bridges, not only corroborating the main binding site probed by X-ray, but also characterizing a second binding site that can act as a transient hub spot. The attach-pull-release (APR) method provides a very good assessment of the overall binding free energy measured experimentally (-6.42+-0.5 vs -5.45 kcal.mol-1 by isothermal titration calorimetry). This work also probes dynamical allosteric modifications upon ligand binding, and our computational protocol could be generalized to situate the supramolecular forces ruling out the calixarene-assisted co-crystallization of proteins.

Situating the Phosphonated Calixarene–Cytochrome C Association by Molecular Dynamics Simulations.

“…In the exo one, K91 and K92's -NH + 3 groups form HBs with the oxygens of two phosphonatomethyl groups (∼2.8 Å). This results in an elongated conformation, as reported previously for sclx 4 55, 56 , favoring with the pmclx 4 phenyl rings, in one case, a cation-π interaction with the -NH + 3 terminal, and in the other a CH• • •π interaction with their alkyl side chains (see Figure 2A-3). In parallel, R13 coordinates a third phosphonate group forming two HBs between its two -NH + 2 groups and two oxygens (∼3.1 and ∼3.2 Å), while a cationπ interaction is established with the phenyl ring (see Figure S9).…”

“…Each structure identifies a different system, for which the total production time amounts to 3 µs (see section "Bound-State Simulations" in ESI). The ligand interaction turns out to be very dynamic along our MD replica: it undergoes local rearrangements and binding site changes (see Figure S5), as previously noticed by MDs 55,56 and experiments 31,32 for psulfonato-calix [4]arene. The most representative structures from cluster analysis are shown in Figure S7, while their relative percentages and the binding site in which the ligand is interacting.…”

“…A remarkable adaptability of the sclx 4 ligand to the protein surface and interaction by both sides was probed experimentally, and influenced the crystal packing of the protein 31,32 . This was also pointed out by MD simulations 55,56 , and we proposed an approach angle descriptor to discriminate between endo and exo configurations. Figure S6 reports the distributions of this angle for pmclx 4 along "Bound State" simulations.…”

“…Our computational protocol, previously used for protein-calixarene associations [54][55][56] , was employed here to identify "De-novo" binding sites at the Cytc surface for pmclx 4 . Initial unbiased MDs were performed where the ligand was allowed to diffuse towards the protein (see "Diffusion Simulations" section in SI), and reach relevant binding sites.…”

“…Such sites, as described also in ref. 55,56 , serve as transient sites ("hub") to more stable ones, such as Site 1 and Site 2.…”