title: A distinct 14 residue site triggers coiled-coil formation in cortexillin I.
abstract: We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two-stranded, parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311-Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled-coil formation. Moreover, deletion of the six N-terminal heptad repeats of Ir led to the formation of a four- rather than a two-helix structure, suggesting that the full-length cortexillin I coiled-coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled-coil 'trigger' site Arg311-Asp324 yielded approximately 30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptide's helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311-Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled-coil formation of cortexillin I.
authors.affiliation: M.E.Müller Institute for Microscopy, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
authors.forename: M O
authors.initials: MO
authors.lastname: Steinmetz
authors.forename: A
authors.initials: A
authors.lastname: Stock
authors.forename: T
authors.initials: T
authors.lastname: Schulthess
authors.forename: R
authors.initials: R
authors.lastname: Landwehr
authors.forename: A
authors.initials: A
authors.lastname: Lustig
authors.forename: J
authors.initials: J
authors.lastname: Faix
authors.forename: G
authors.initials: G
authors.lastname: Gerisch
authors.forename: U
authors.initials: U
authors.lastname: Aebi
authors.forename: R A
authors.initials: RA
authors.lastname: Kammerer
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002942:Circular Dichroism; D004023:Dictyostelium / Q000737:chemistry; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017384:Sequence Deletion; D012965:Sodium Chloride / Q000494:pharmacology; D014461:Ultracentrifugation
chemical_list: D000596:Amino Acids; D008840:Microfilament Proteins; D009842:Oligopeptides; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C101109:ctxA protein, Dictyostelium discoideum; D012965:Sodium Chloride