X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides

Abstract

The self-assembly of peptides and proteins into higher-ordered structures is encoded in the amino acid sequence of each peptide or protein. Understanding the relationship among the amino acid sequence, the assembly dynamics, and the structure of well-defined peptide oligomers expands the synthetic toolbox for these structures. Here, we present the X-ray crystallographic structure and solution behavior of de novo peptides that form antiparallel coiled-coil hexamers (ACC-Hex) by an interaction motif neither found in nature nor predicted by existing peptide design software. The 1.70 Å X-ray crystallographic structure of peptide 1a shows six α-helices associating in an antiparallel arrangement around a central axis comprising hydrophobic and aromatic residues. Size-exclusion chromatography studies suggest that peptides 1 form stable oligomers in solution, and circular dichroism experiments show that peptides 1 are stable to relatively high temperatures. Small-angle X-ray scattering studies of the solution behavior of peptide 1a indicate an equilibrium of dimers, hexamers, and larger aggregates in solution. The structures presented here represent a new motif of biomolecular self-assembly not previously observed for de novo peptides and suggest supramolecular design principles for material scaffolds based on coiled-coil motifs containing aromatic residues.

Maintaining and breaking symmetry in homomeric coiled-coil assemblies

“…( N.B . Consistent with this, a CC-Hex 24 variant with Ile at d and Leu→Phe at a collapses to give a solid core 40 .)

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De Novo-Designed α-Helical Barrels as Receptors for Small Molecules

“…34 There are other examples of coiledcoil oligomers above pentamer but, to our knowledge, these either do not have central channels or their binding/transport properties have not been fully characterised. [35][36][37] Indeed, adding specificity of binding for small molecules has not been addressed formally by any of the above reports. The coiled coil is a parameterizable protein fold; 38 i.e., there are equations that allow coiled-coil structures to be built ab initio making them accessible to computational design.…”

How coiled-coil assemblies accommodate multiple aromatic residues

“…In this study, the three structures contain Phe at some of the a or d positions, which has previously been shown in most cases to form structures with consolidated cores. 25–27 If this is true generally, designing Tyr-containing sequences without Phe might lead to barrel structures.…”

“…15–17 Specific examples of Ar-containing de novo and engineered CCs include: engineered pentameric barrels containing buried Trp or Phe residues (Figure 1C), 20,21 and heterotrimeric CCs with a buried Trp. 22–24 More recent examples include various parallel and antiparallel helical bundles with consolidated cores that feature Phe residues, 25–27 and homomeric and heteromeric helix-loop-helix motifs with isolated Tyr, His and Trp residues that contribute to buried hydrogen-bond networks (Figure 1D & E). 14…”