Navigating the Structural Landscape of De Novo α-Helical Bundles

Abstract

The association of amphipathic a helices in water leads to a-helical-bundle protein structures. However, the driving force for this-the hydrophobic effect-is not specific and does not define the number or the orientation of helices in the associated state. Rather, this is achieved through deeper sequence-to-structure relationships, which are increasingly being discerned. For example, for one structurally extreme but nevertheless ubiquitous class of bundle-the a-helical coiled coils-relationships have been established that discriminate between all-parallel dimers, trimers and tetramers. Association states above this are known, as are antiparallel and mixed arrangements of the helices. However, these alternative states are less-well understood. Here, we describe a synthetic-peptide system that switches between parallel hexamers and various updown-up-down tetramers in response to single-amino-acid changes and solution conditions. The main accessible states of each peptide variant are characterized fully in solution and, in most cases, to high resolution with X-ray crystal structures. Analysis and inspection of these structures helps rationalize the different states formed. This navigation of the structural landscape of a-helical coiled coils above the dimers and trimers that dominate in nature has allowed us to design rationally a well-defined and hyperstable antiparallel coiled-coil tetramer (apCC-Tet). This robust de novo protein provides another scaffold for further structural and functional designs in protein engineering and synthetic biology. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Supporting Figures and Tables (PDF) Crystal-Structure Files The crystal structures are all available from the Protein Data Bank using the following accession codes:

A defined structural unit enables de novo design of small-molecule–binding proteins

“…Two of the three keystone interactions identified by COMBS contribute appreciably to binding affinity. Substitution of His 49 or Gln 14 with alanine individually decreases affinity by approximately 1 kcal/mol (~3-fold) ( Fig. 6D and fig.…”

“…Instead of repurposing natural binding proteins or folds that have been shown to bind a similar ligand, in this work, we use de novo four-helix bundles because they are mathematically parameterized (10,11), designable (12), and share no similarity to the fold of factor Xa. Four-helix bundles generally do not bind small molecules and instead bind metal ions or metalloporphyrins by strong coordinate bonds (10,(13)(14)(15)(16). However, fourhelix bundles are tubular and can be designed to have high thermodynamic stability (11,13) to compensate for the energetically demanding process of building binding cavities replete with buried polar functionality (17).…”

“…S18). Gln 14 was observed in its intended rotamer, whereas His 49 occupied an alternate rotamer that nevertheless maintained the intended position of apixaban's carbonyl relative to the main chain (Fig. 6A).…”

How coiled-coil assemblies accommodate multiple aromatic residues

“…Fraying is observed in structures of designed CC bundles containing polar residues near their termini and in natural CC structures in the PDB (Figure 1B). 52,53 To test this, we replaced the most-terminal Tyr residues with Phe in CC-Type2-(I a Y d ) 4 to give CC-Type2-(I a Y d ) 4 -Y3F & CC-Type2-(I a Y d ) 4 -Y3F-Y24F; and we shifted the register of the sequence (CC-Type2-(I a Y d ) 4 _r) or added an extra half heptad (CC-Type2-(I a Y d ) 4.5 ) to move those Tyr residues more centrally into the sequences.…”

Bent into shape: Folded peptides to mimic protein structure and modulate protein function

“…For larger oligomers (hexamers and larger), a solvent accessible channel is formed in the center of the bundle and is capable of binding small molecules . Different oligomers show varying levels of sensitivity to mutation that is, some designed hexamers formed both parallel and antiparallel tetramers, as well as open and collapsed hexamers, when mutations were incorporated …”