PDBsum entry 2rfm

Protein binding

PDB id: 2rfm

Contents

Protein chains

183 a.a.

Ligands

SO4 ×8

144

BU2 ×4

GOL ×6

Metals

_CL ×3

Waters ×479

References listed in PDB file

Key reference

Title

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

Authors

C.Löw, U.Weininger, P.Neumann, M.Klepsch, H.Lilie, M.T.Stubbs, J.Balbach.

Ref.

Proc Natl Acad Sci U S A, 2008, 105, 3779-3784. [DOI no: 10.1073/pnas.0710657105]

PubMed id

18305166

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.

Figure 1.
Schematic representation of the structure of the thermophilic ankyrin repeat protein tANK (Protein Data Base ID code 2RFM). Five ARs (AR1–AR5), each comprising a loop, a β-turn, and two sequential α-helices form the elongated structure, extended by an α-helical N terminus are shown. Side chains of the wild-type fluorescence probes Trp-71 and Trp-104 are indicated as sticks. The figure was created by using MOLMOL (34).

Figure 6.
Simplified folding model of tANK. The protein folds via an on-pathway intermediate in which the two N-terminal repeats are unfolded and the three C-terminal repeats are natively folded. Additional NMR cross-peaks of the intermediate state, which do not show a random coil chemical shift, and CD data suggest that there is some residual secondary structure in AR 1 and 2.