Structural insights into an equilibrium folding intermediate of an archaeal ankyrin repeat protein

doi:10.1073/pnas.0710657105

Structural insights into an equilibrium folding intermediate of an archaeal ankyrin repeat protein

*Institut für Physik, Biophysik,
^†Institut für Biochemie und Biotechnologie, Physikalische Biotechnologie,
^§Institut für Biochemie/Biotechnologie, Biotechnologie, and
^¶Mitteldeutsches Zentrum für Struktur und Dynamik der Proteine (MZP), Martin-Luther-Universität Halle-Wittenberg, D-06120 Halle (Saale), Germany; and
^‡Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, SE-10691 Stockholm, Sweden

Edited by Robert T. Sauer, Massachusetts Institute of Technology, Cambridge, MA, and approved January 9, 2008 (received for review November 9, 2007)

Abstract

Repeat proteins are widespread in nature, with many of them functioning as binding molecules in protein–protein recognition. Their simple structural architecture is used in biotechnology for generating proteins with high affinities to target proteins. Recent folding studies of ankyrin repeat (AR) proteins revealed a new mechanism of protein folding. The formation of an intermediate state is rate limiting in the folding reaction, suggesting a scaffold function of this transient state for intrinsically less stable ARs. To investigate a possible common mechanism of AR folding, we studied the structure and folding of a new thermophilic AR protein (tANK) identified in the archaeon Thermoplasma volcanium. The x-ray structure of the evolutionary much older tANK revealed high homology to the human CDK inhibitor p19^INK4d, whose sequence was used for homology search. As for p19^INK4d, equilibrium and kinetic folding analyses classify tANK to the family of sequential three-state folding proteins, with an unusual fast equilibrium between native and intermediate state. Under equilibrium conditions, the intermediate can be populated to >90%, allowing characterization on a residue-by-residue level using NMR spectroscopy. These data clearly show that the three C-terminal ARs are natively folded in the intermediate state, whereas native cross-peaks for the rest of the molecule are missing. Therefore, the formation of a stable folding unit consisting of three ARs is the necessary rate-limiting step before AR 1 and 2 can assemble to form the native state.

Footnotes

^‖To whom correspondence should be addressed. E-mail: jochen.balbach{at}physik.uni-halle.de
Author contributions: C.L. and J.B. designed research; C.L., U.W., P.N., and H.L. performed research; C.L., U.W., P.N., M.K., and H.L. analyzed data; and C.L., M.T.S., and J.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2RFM).
This article contains supporting information online at www.pnas.org/cgi/content/full/0710657105/DC1.