Design of a heterotetrameric coiled coil

Abstract

We have successfully designed a simple peptide sequence that forms highly stable coiled-coil heterotetramers. Our model system is based on the GCN4-pLI parallel coiled-coil tetramer, first described by Kim and coworkers (Harbury et al., Science 1993;262:1401-1407.We introduced glutamates at all of the e and c heptad positions of one sequence (ecE) and lysines at the same positions in a second sequence (ecK). Based on a modeling study, these sidechains are close enough in space to form structure-stabilizing salt bridges. We show that ecE and ecK are highly unstable by themselves but form very stable parallel helical tetramers when mixed, as judged by circular dichroism, analytical ultracentrifugation, and disulfide crosslinking studies. The origin of the difference in stabilities between the homomeric structures and the heteromeric structures comes from a combination of the relief of electrostatic repulsions with concomitant formation of electrostatic attractive interactions based on pH and NaCl screening experiments. We quantify the stability of the heterotetrameric coiled coil from a thermodynamic analysis and compare the finding to other similar coiled-coil systems.

Computational Design and Elaboration of a de Novo Heterotetrameric α-Helical Protein That Selectively Binds an Emissive Abiological (Porphinato)zinc Chromophore

“…In contrast, the symmetry in six-coordinate Fe 3+ ligation can be leveraged in the design of proteins that encapsulate iron porphyrins 10–12. Such preferred local asymmetry in the metal coordination of PZn suggests use of helical bundles having reduced symmetry, e.g ., heterotetrameric bundles 46,47. Along these lines, heterotetramers have been designed that present a dinuclear metal ion site48,49 including an A 2 B 2 protein 50.…”

Polyglutamine fibrils are formed using a simple designed β‐hairpin model

“…In contrast, a 1.0 m M sample of tCQ8 dissolved in 12 m M sodium borate, pH 8.5, in which the lysines are now significantly deprotonated, showed conversion to a β‐sheet spectrum within seconds, as judged by CD, with a well‐defined minimum at 218 nm. Peptides with similar lysine composition can show p K a shifts in their ϵ‐amino groups as low as 9.0, providing greater than 10% deprotonation at this higher pH 36…”

Evaluation of the protein interfaces that form an intermolecular four-helix bundle as studied by computer simulation

“…If the binding interfaces form the ideal four-helix bundle, [10,11] the distance between charged amino acids shown in Table 3 should be , 8 Å . Therefore, the charged amino acid residues on the N-terminal regions of the helices contribute to weak interactions by intermolecular Coulomb force.…”

“…[3,20] In this method, by means of using the four-helix bundle that is stabilised by hydrophobic contacts and ionic interactions, we can switch-ON (bind) and -OFF (disassociation) by modulating the environment of solution such as salt concentration, pH, temperature and the presence of ligands. [2,10,21] For example, if ion concentration is increased, electrostatic interaction will become weak, thus inducing switch-OFF. In the future, we may be able to facilitate protein-protein binding between any proteins with a-helix on their surface.…”

“…In a helix bundle motif, an a-helix contains heptad repeats where every seventh amino acid is located at an equivalent position with respect to the helix axis. The seven positions are referred to as a-g. Root et al [10] introduced charged amino acids into the 'b' and 'c' positions of the heptad repeat and stabilised the helix bundle structure. [11] Komatsu et al [12] performed molecular dynamics (MD) simulations with a coarsegrained model using Lac repressor four-helix protein (LARFH).…”