Cucurbit[8]uril Binds Nonterminal Dipeptide Sites with High Affinity and Induces a Type II β-Turn

Abstract

In an effort to target polypeptides at nonterminal sites, we screened the binding of the synthetic receptor cucurbit[8]uril (Q8) to a small library of tetrapeptides, each containing a nonterminal dipeptide binding site. The resulting leads were characterized in detail using a combination of isothermal titration calorimetry, 1 H NMR spectroscopy, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), and X-ray crystallography. The equilibrium dissociation constant values determined for the binding of Q8 to nonterminal dipeptide sites Lys-Phe (KF) and Phe-Lys (FK) were 60 and 86 nm, respectively. These are to the best of our knowledge the highest affinities reported to date for any synthetic receptor targeting a nonterminal site on an unmodified peptide. A 0.79 Å resolution crystal structure was obtained for the complex of Q8 with the peptide Gly-Gly-Leu-Tyr-Gly-Gly-Gly (GGLYGGG) and reveals structural details of the pair-inclusion motif. The molecular basis for recognition is established to be the inclusion of the side chains of Leu and Tyr residues, as well as an extensive network of hydrogen bonds between the peptide backbone, the carbonyl oxygens of Q8, and proximal water molecules. In addition, the crystal structure reveals that Q8 induces a type II β-turn. The sequence-selectivity, high affinity, reversibility, and detailed structural characterization of this system should facilitate the development of applications involving ligand-induced polypeptide folding.

Peptide recognition by a synthetic receptor at subnanomolar concentrations

“…When KF and FK dipeptide sites were located at non-terminal positions, their affinities for Q8 differed by less than 2-fold. 23 …”

“…We recently investigated the binding of Q8 to non-terminal dipeptide sites. 23 Although binding was expected to be weaker at non-terminal sites due to the lack of involvement of the N-terminal ammonium group, we discovered that Q8 binds to non-terminal KF and FK dipeptide sites in the pair-inclusion motif with K d values of 60 and 86 nM, respectively. These affinities were higher than that of non-terminal YL and LY, which were the highest affinity sites observed in previous work.…”

“…22,23 Puzzled by these reports, we revisited the H-Tyr-Leu-Ala-NH 2 and agree that the binding constant is closer to 100 nM than 10 nM. This is important because YL has been the benchmark target dipeptide site in four studies on the pair-inclusion motif, 19,[21][22][23] and similarly we wanted to compare the binding affinities observed for KFGGY and FKGGY to the YL benchmark. Therefore, two additional pentapeptides, H-Tyr-Leu-Gly-Gly-Gly-OH (YLGGG) and H-Leu-Tyr-Gly-Gly-Gly-OH (LYGGG) were obtained commercially and characterized by ITC (Table 1, Fig.…”

“…When KF and FK dipeptide sites were located at nonterminal positions, their affinities for Q8 differed by less than 2-fold. 23 In our rst publication on the pair-inclusion motif, 19 we reported the K d value for Q8 in complex with the tripeptide H-Tyr-Leu-Ala-NH 2 as 8 nM using competitive ITC. Scherman and coworkers also studied the YL dipeptide binding site and reported K d values for Q8 binding to H-Tyr-Leu-Ala-NH 2 (120 nM), H-Tyr-Leu-NH 2 (120 nM), and H-Tyr-Leu-Ala-Ala-NH 2 (140 nM) using ITC by direct titration.…”

“…In order to target the many peptides and proteins that exist in biological systems at or below the nanomolar concentration range, it is necessary for the equilibrium dissociation constants to be at or below the nanomolar range. Although there have been a few reports of K d values in the nanomolar range, 15,[18][19][20][21]23 to the best of our knowledge there have been no reports to date of sub-nanomolar binding of unmodied peptides by synthetic receptors. We recently investigated the binding of Q8 to non-terminal dipeptide sites.…”