title: Malleability of folding intermediates in the homeodomain superfamily.
abstract: Members of the homeodomain superfamily are three-helix bundle proteins whose second and third helices form a helix-turn-helix motif (HTH). Their folding mechanism slides from the ultrafast, three-state framework mechanism for the engrailed homeodomain (EnHD), in which the HTH motif is independently stable, to an apparent two-state nucleation-condensation model for family members with an unstable HTH motif. The folding intermediate of EnHD has nearly native HTH structure, but it is not docked with helix1. The determinant of whether two- or three-state folding was hypothesized to be the stability of the HTH substructure. Here, we describe a detailed Φ-value analysis of the folding of the Pit1 homeodomain, which has similar ultrafast kinetics to that of EnHD. Formation of helix1 was strongly coupled with formation of HTH, which was initially surprising because they are uncoupled in the EnHD folding intermediate. However, we found a key difference between Pit1 and EnHD: The isolated peptide corresponding to the HTH motif in Pit1 was not folded in the absence of H1. Independent molecular dynamics simulations of Pit1 unfolding found an intermediate with H1 misfolded onto the HTH motif. The Pit1 folding pathway is the connection between that of EnHD and the slower folding homeodomains and provides a link in the transition of mechanisms from two- to three-state folding in this superfamily. The malleability of folding intermediates can lead to unstable substructures being stabilized by a variety of nonnative interactions, adding to the continuum of folding mechanisms.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom.
authors.forename: Wiktor
authors.initials: W
authors.lastname: Banachewicz
authors.forename: Tomasz L
authors.initials: TL
authors.lastname: Religa
authors.forename: R D
authors.initials: RD
authors.lastname: Schaeffer
authors.forename: Valerie
authors.initials: V
authors.lastname: Daggett
authors.forename: Alan R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D019077:Helix-Turn-Helix Motifs / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics* / Q000502:physiology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D017510:Protein Folding; D050817:Transcription Factor Pit-1 / Q000378:metabolism*
chemical_list: D018398:Homeodomain Proteins; C494187:POU1F1 protein, human; D050817:Transcription Factor Pit-1