Side-Chain Pairing Preferences in the Parallel Coiled-Coil Dimer Motif: Insight on Ion Pairing between Core and Flanking Sites

Abstract

A new strategy for rapid evaluation of sequence-stability relationships in the parallel coiled-coil motif is described. The experimental design relies upon thiol-thioester exchange equilibria, an approach that is particularly well suited to examination of heterodimeric systems. Our model system has been benchmarked by demonstrating that it can quantitatively reproduce previously reported trends in interhelical a-a' side chain pairing preferences at the coiled-coil interface. This new tool has been used to explore the role of Coulombic interactions between a core position on one helix and a flanking position on the other helix (a-g'). This type of interhelical contact has received relatively little attention to date. Our results indicate that such interactions can influence coiled-coil partner preferences.Proteins collectively display a broad array of tertiary and quaternary structures, with many different modes of packing between neighboring secondary structure elements. Among the possibilities, the α-helical coiled coil is unusual in that it is both common and regular. 1 In the simplest case, two α-helices associate side-by-side, wrapping around one another with a slight left-handed superhelical twist. A characteristic "knobs-into-holes" interdigitation of sidechains is observed at the helix-helix interface, whether the helices are parallel or antiparallel. 2 The relative simplicity of this architecture has led to extensive exploration of sequencestability relationships, 3 motivated by the prospects of predicting coiled-coil structure from sequence information alone, refining computational tools, and using coiled coils as building blocks in rational protein design and synthetic biology. 4 Although some principles that govern coiled-coil stability have been elucidated, our understanding remains incomplete.Here we introduce a heterodimeric parallel coiled-coil model system designed to provide new insights on the origins of stability and helix-pairing preferences. Our system employs relatively short peptide segments (20 or 21 residues), which facilitates broad exploration of sequence variations. Parallel, two-helix assembly is promoted by a thioester linkage between the Cterminus of one segment and the side-chain of a C-terminal Cys residue on the other; this design enables us to monitor coiled-coil stability under native conditions via thiol-thioester exchange equilibration. 5 Our experimental design (Figure 1) is based on well-known characteristics of sequences that form coiled coils. The segments intended to adopt α-helical conformations feature a heptad sequence repeat pattern (abcdefg), in which side-chains at a and d dominate the helix-helix contacts. Two-helix stoichiometry (rather than alternate three-or four-helix assemblies) is directed by placing Leu at the d sites, Ile at the N-terminal a positions, and Asn at the a sites closest to the covalent connection.6 ,7 The remaining (central) a positions of each segment (designated X and Ψ) are "guest" sites for substitutions that allow us to probe ...

Helix Propensities of Amino Acid Residues via Thioester Exchange

“…5 Specifically, we have used the thioester exchange strategy to evaluate thermodynamic contributions of side chains at the α-helical interface to coiled-coil stability. 6 Here we show that varying residues at sites that are distal to the helix-helix interface provides an accurate measure of α-helical propensity for residues bearing side chains that are not charged.…”

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

“…[39][40][41] In a comprehensive study, Vinson and colleagues determined the thermodynamic stabilities of 100 heterodimeric coiled-coil mutants varying a-a' residue pairs. They state: "the most extreme example of two amino acids regulating dimer preference has to do with Ile and Asn".…”

“…[35][36][37][38] Studies have also been performed to quantify the contribution of these Asn-Asn pairs to dimer stability at least. [39][40][41] To summarize a large body of literature, N@a is destabilising, but it specifies parallel dimer over alternative states such as antiparallel dimer and parallel trimer. Asn inclusions do occur in trimers, though less frequently than in dimers.…”

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

“…4 De novo peptides that fold into coiled coils have proven to be a robust scaffold with interesting properties. [5][6][7][8][9][10] De novo coiled coils can be designed to associate into diverse supramolecular assemblies including dimers, [11][12][13][14] trimers, 15,16 tetramers, [17][18][19] pentamers, [20][21][22] hexamers, 23 and larger assemblies. 3,24 De novo coiled coils have also been designed to form fibers, 12,13,25,26 act as enzymes, 27 and mimic membrane transport proteins.…”