Malleability of folding intermediates in the homeodomain superfamily

Wiktor Banachewicz, Tomasz L. Religa, R. D. Schaeffer, Valerie Daggett, Alan R. Fersht

Research output: Contribution to journalArticlepeer-review

20 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)5596-5601
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number14
StatePublished - Apr 5 2011


  • Diffusion collision
  • Protein folding
  • Temperature jump
  • Transition state

ASJC Scopus subject areas

  • General


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