title: Structural basis for the oligomerization-state switch from a dimer to a trimer of an engineered cortexillin-1 coiled-coil variant.
abstract: Coiled coils are well suited to drive subunit oligomerization and are widely used in applications ranging from basic research to medicine. The optimization of these applications requires a detailed understanding of the molecular determinants that control of coiled-coil formation. Although many of these determinants have been identified and characterized in great detail, a puzzling observation is that their presence does not necessarily correlate with the oligomerization state of a given coiled-coil structure. Thus, other determinants must play a key role. To address this issue, we recently investigated the unrelated coiled-coil domains from GCN4, ATF1 and cortexillin-1 as model systems. We found that well-known trimer-specific oligomerization-state determinants, such as the distribution of isoleucine residues at heptad-repeat core positions or the trimerization motif Arg-h-x-x-h-Glu (where h = hydrophobic amino acid; x = any amino acid), switch the peptide's topology from a dimer to a trimer only when inserted into the trigger sequence, a site indispensable for coiled-coil formation. Because high-resolution structural information could not be obtained for the full-length, three-stranded cortexillin-1 coiled coil, we here report the detailed biophysical and structural characterization of a shorter variant spanning the trigger sequence using circular dichroism, anatytical ultracentrifugation and x-ray crystallography. We show that the peptide forms a stable α-helical trimer in solution. We further determined the crystal structure of an optimised variant at a resolution of 1.65 Å, revealing that the peptide folds into a parallel, three-stranded coiled coil. The two complemented R-IxxIE trimerization motifs and the additional hydrophobic core isoleucine residue adopt the conformations seen in other extensively characterized parallel, three-stranded coiled coils. These findings not only confirm the structural basis for the switch in oligomerization state from a dimer to a trimer observed for the full-length cortexillin-1 coiled-coil domain, but also provide further evidence for a general link between oligomerization-state specificity and trigger-sequence function.
authors.affiliation: Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland. sasa.bjelic@case.edu
authors.forename: Saša
authors.initials: S
authors.lastname: Bjelić
authors.forename: Mara
authors.initials: M
authors.lastname: Wieser
authors.forename: Daniel
authors.initials: D
authors.lastname: Frey
authors.forename: Christian U
authors.initials: CU
authors.lastname: Stirnimann
authors.forename: Mark R
authors.initials: MR
authors.lastname: Chance
authors.forename: Rolf
authors.initials: R
authors.lastname: Jaussi
authors.forename: Michel O
authors.initials: MO
authors.lastname: Steinmetz
authors.forename: Richard A
authors.initials: RA
authors.lastname: Kammerer
mesh_terms: D000595:Amino Acid Sequence; D008840:Microfilament Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
chemical_list: D008840:Microfilament Proteins