Macrocyclic β-Sheet Peptides That Mimic Protein Quaternary Structure through Intermolecular β-Sheet Interactions

Abstract

This paper reports the design, synthesis, and characterization of a family of cyclic peptides that mimic protein quaternary structure through β-sheet interactions. These peptides are 54-membered-ring macrocycles comprising an extended heptapeptide β-strand, two Hao β-strand mimics [JACS 2000, 122, 7654] joined by one additional α-amino acid, and two δ-linked ornithine β-turn mimics [JACS 2003, 125, 876]. Peptide 3a, as the representative of these cyclic peptides, contains a heptapeptide sequence (TSFTYTS) adapted from the dimerization interface of protein NuG2 [PDB ID: 1mio]. 1 H NMR studies of aqueous solutions of peptide 3a show a partially folded monomer in slow exchange with a strongly folded oligomer. NOE studies clearly show that the peptide selfassociates through edge-to-edge β-sheet dimerization. Pulsed-field gradient (PFG) NMR diffusion coefficient measurements and analytical ultracentrifugation (AUC) studies establish that the oligomer is a tetramer. Collectively, these experiments suggest a model in which cyclic peptide 3a oligomerizes to form a dimer of β-sheet dimers. In this tetrameric β-sheet sandwich, the macrocyclic peptide 3a is folded to form a β-sheet, the β-sheet is dimerized through edge-to-edge interactions, and this dimer is further dimerized through hydrophobic face-to-face interactions involving the Phe and Tyr groups. Further studies of peptides 3b-3n, which are homologues of peptide 3a with 1-6 variations in the heptapeptide sequence, elucidate the importance of the heptapeptide sequence in the folding and oligomerization of this family of cyclic peptides. Studies of peptides 3b-3g show that aromatic residues across from Hao improve folding of the peptide, while studies of peptides 3h-3n indicate that hydrophobic residues at positions R 3 and R 5 of the heptapeptide sequence are important in oligomerization.

Out-of-register β-sheets suggest a pathway to toxic amyloid aggregates

“…These proteins form amyloid-like oligomers and fibrils and exhibit cytotoxicity. Our previous studies showed that macrocyclic peptides form oligomers in solution (36) and mimic amyloid oligomers for structural studies (37). Here, we find that, on incubation at 37°C for 0.5 h, BAMs form amyloid-like oligomers that are recognized by A11, the polyclonal amyloid-oligomer-specific conformational antibody (Fig.…”

“…S4A). Diffusion coefficients of BAMs (Aβ [30][31][32][33][34][35][36] ) measured by NMR confirmed that they self-associate in solution at millimolar concentrations, consistent with the formation of BAM oligomers. (Fig.…”

“…Crystal Structure of BAM (Aβ [30][31][32][33][34][35][36] ): An Out-of-Register, Cylindrin-Like Oligomer. In previous work, we determined the crystal structure of BAM (Aβ [30][31][32][33][34][35][36] ) in oligomeric form (34), which we now recognize as having cylindrin-like structure and properties.…”

“…4D). However, instead of repeating in 1D, leading to an out-of-register fibril, the macrocyclic rings of BAM (Aβ [30][31][32][33][34][35][36] ) roll into a β-barrel. A key driving force of this barrel-like structure is the formation of a dry interface; the barrel core is devoid of water, and instead, it is filled with the side chains of methionine and phenylalanine (Fig.…”

Computational design of a symmetric homodimer using β-strand assembly

“…One approach targeted amyloid fibrils by computationally designing a peptide to form a terminating β-strand on a growing fibril (28). Another study took the sequence of the β-strand of a known β-strand mediated homodimer and embedded it in a cyclic peptide (29). A crystal structure of the peptide showed it formed an antiparallel β-strand paired dimer as predicted (30).…”