De novo design of orthogonal peptide pairs forming parallel coiled‐coil heterodimers

Abstract

We used the principles governing the selectivity and stability of coiled-coil segments to design and experimentally test a set of four pairs of parallel coiled-coil-forming peptides composed of four heptad repeats. The design was based on maximizing the difference in stability between desired pairs and the most stable unwanted combinations using N-terminal helix initiator residues, favorable combinations of the electrostatic and hydrophobic interaction motifs and negative design motif based on burial of asparagine residues. Experimental analysis of all 36 pair combinations among the eight peptides was performed by circular dichroism (CD). On the basis of CD spectra, each peptide formed a high level of α-helical structure exclusively in combination with its designed peptide partner which demonstrates the orthogonality of the designed peptide pair set.

De novo design of protein homo-oligomers with modular hydrogen-bond network–mediated specificity

“…We therefore turned our attention to coiled-coils, which are abundant in nature{Gruber:2003by, Burkhard:2001wa}, the subject of many protein design studies{Huang:2014jt, Ogihara:1997fr, Reinke:2010bp, Boyle:2011id}, and can be generated parametrically{Grigoryan:2011dr, Harbury:1998ub}, resulting in repeating geometric cross-sections. Coiled coil packing and oligomerization state is largely determined by position-specific identities of nonpolar residues that pack between the helices{Woolfson:1995gl, Harbury:1993ub, Acharya:2006ey}; salt bridge and hydrogen bonding interactions between residues on the periphery can provide additional specificity{Gradisar:2011ef, Kaplan:2014bk, Baker:2015bv}. In natural and designed coiled coils, buried polar interactions can also alter specificity; however, most of these cases involve at most one or two sidechain-sidechain hydrogen bonds with remaining polar atoms satisfied by water or ions{Eckert:1998fa, Akey:2001tr, Lumb:1995uh, Tatko:2006kq, Gonzalez:1996vl} – the relatively small cross-sectional interface area of canonical coiled-coils limits the diversity and location of possible networks.…”

Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments

“…1), which is the topology with the largest number of parallel segments. Although both parallel and antiparallel coiled-coil dimeric arrangements are represented in nature 20 , significantly higher number of isolated parallel coiled-coil dimers have been characterized and designed 15,21,22 . We selected the pairs for the formation of 3 edges of the polyhedron from the orthogonal coiled-coil-forming pairs P3-P4, P5-P6 and P7-P8.…”

“…We selected the pairs for the formation of 3 edges of the polyhedron from the orthogonal coiled-coil-forming pairs P3-P4, P5-P6 and P7-P8. Peptides P3 to P8, composed of 4 heptad repeats (see Online Methods for amino acid sequences), were designed on the basis of known coiled-coil stability and selectivity principles 21 and the orthogonality of the peptide pairs was demonstrated (Supplementary Fig. 6).…”

Self-assembled bionanostructures: proteins following the lead of DNA nanostructures

“…The rules governing coiled-coil formation, their oligomerization state and interaction partner specificity have been considerably established over the last decades [68,69]. On the basis of those rules sets of orthogonal designed coiled-coils as the toolkit for the designed protein assemblies were developed [70-75]. Engineered coiled-coil polypeptides have been used to assemble different nanomaterials: nanofibres [76,77], membranes [78], nanotubes [79], nanostructured films [80], spherical structures [81], responsive hydrogels [82,83], spheres [84] etc.…”