Designed to be stable: Crystal structure of a consensus ankyrin repeat protein

Abstract

Ankyrin repeat (AR) proteins mediate innumerable protein-protein interactions in virtually all phyla. This finding suggested the use of AR proteins as designed binding molecules. Based on sequence and structural analyses, we designed a consensus AR with fixed framework and randomized interacting residues. We generated several combinatorial libraries of AR proteins consisting of defined numbers of this repeat. Randomly chosen library members are expressed in soluble form in the cytoplasm of Escherichia coli constituting up to 30% of total cellular protein and show high thermodynamic stability. We determined the crystal structure of one of those library members to 2.0-Å resolution, providing insight into the consensus AR fold. Besides the highly complementary hydrophobic repeat-repeat interfaces and the absence of structural irregularities in the consensus AR protein, the regular and extended hydrogen bond networks in the ␤-turn and loop regions are noteworthy. Furthermore, all residues found in the turn region of the Ramachandran plot are glycines. Many of these features also occur in natural AR proteins, but not in this rigorous and standardized fashion. We conclude that the AR domain fold is an intrinsically very stable and well-expressed scaffold, able to display randomized interacting residues. This scaffold represents an excellent basis for the design of novel binding molecules.

Ankyrin Repeat: A Unique Motif Mediating Protein−Protein Interactions

“…Hence, one might think that AR proteins differ from globular proteins in folding and stability and each AR in a protein could unfold and refold individually. However, folding studies on INK4 proteins (12,17,23,32,33), Notch AR domain (34,35), myotrophin (22), and some engineered AR proteins (14,15,36,37) have demonstrated that AR proteins exhibit an equilibrium two-state transition between native and unfolded proteins by spectroscopic and calorimetric criteria, similar to most globular proteins. Furthermore, no partially folded intermediates have been found in these equilibrium folding-unfolding reactions, except that a NMR study of p19 in the presence of moderate concentrations of urea revealed the possible existence of an equilibrium intermediate with poor chemical shift dispersions, suggesting that it is largely unfolded (32).…”

“…In the third approach, consensus design was combined with a combinatorial library to generate AR proteins containing a common structural framework and novel binding specificities (15,36,(80)(81)(82) (Figures 1a and 6c). For binding scaffolds such as AR proteins, its diversity in binding specificity reflects its diversity in function.…”

Repeat-protein folding: New insights into origins of cooperativity, stability, and topology

“…High resolution crystal structures revealed that proteins containing up to five consensus ankyrin repeats [25,41] and up to eight consensus TPR repeats [42] adopt folds that have root-mean-square-deviations of around 0.9 and 1.6Å, respectively, from analogous structures of naturally-occurring repeat proteins. Thus, structurally speaking, this simple consensus method for repeat-protein design is highly successful.…”

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

“…DARPins were produced in the E. coli XL10 Gold strain with the pQE30 expression plasmid with isopropyl ␤-D-1-thiogalactopyranoside (IPTG) induction and purified using Ninitrilotriacetic acid (NTA) affinity chromatography as described previously (45). The DARPin proteins obtained were checked by size exclusion chromatography coupled to multiangle light scattering (SEC-MALS) for the oligomerization state.…”