Designed Armadillo Repeat Proteins: Library Generation, Characterization and Selection of Peptide Binders with High Specificity

Abstract

Designed Armadillo repeat proteins (ArmRPs) are a novel class of binding proteins intended for general modular peptide binding and have very favorable expression and stability properties. Using a combination of sequence and structural consensus analyses, we generated a 42-amino-acid designed Armadillo repeat module with six randomized positions, having a theoretical diversity of 9.9×10(6) per repeat. Structural considerations were used to replace cysteine residues, to define less conserved positions and to decide where to introduce randomized amino acid residues for potential interactions with the target peptide. Based on these concepts, combinatorial libraries of designed ArmRPs were assembled. The most stable version of designed ArmRP in library format was the N5C format, with three randomized library repeat modules flanked by full consensus repeat modules on either side and, in turn, flanked by N- and C-terminal capping repeats. Unselected members of this library were well expressed in the Escherichia coli cytoplasm, monomeric and showed the expected CD spectra and cooperative unfolding. N5C libraries were used in ribosome display selections against the peptide neurotensin. Highly specific peptide binders were enriched after four rounds of selections using ribosome display. Four peptide side chains were shown to contribute most of the interaction energy, and single alanine mutants could be discriminated. Thus, designed ArmRP libraries can become valuable sources for peptide binding molecules because of their favorable biophysical properties and with a potential for application in general modular peptide recognition.

A Combined NMR and Computational Approach to Investigate Peptide Binding to a Designed Armadillo Repeat Protein

“…The simulations agree with previous experimental findings [23] and the PRE and CSP data. Concerning the latter data, the ring of Pro7 NT packs against the aromatic ring of Trp77, while its Cα atom remains in close proximity to Ser80 (Table S4 and Figure 6) whose amide and Cβ resonances are both strongly perturbed (Δδ CB ~ 6 ppm) in the presence of NT.…”

“…These studies confirmed that the N-terminal part of the protein contributes more towards peptide binding (Figure 2 and 3 Figure 10B). Moreover, the non-localized distribution of mostly weak attenuations induced by a spin label at position 1 ("NT-Q1C") confirms that the N-terminal hexapeptide is not involved in a specific binding mechanism, consistent with previous binding data [23] (see Supp. Mat.…”

Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

“…From an initial version of a consensus ArmRP library, a binder to neurotensin was selected with a K d of 7 µM [29], but the binding mode was different from the intended canonical binding [30]. More recently, picomolar affinities for the interaction between the designed ArmRP Y III M 6 A II and the (KR) 4 -peptide have been measured [31].…”

“…The target peptides (expressed as pD-fusion [5,29] or chemically synthesized (JPT) (Supplementary Table ST4)) were immobilized via their biotin residues on NeutrAvidin (100 µl, 200 nM in PBS-TB).…”

Combined expression of ALEX1 and HNF-1beta could improve the ability of differentiation between squamous cell carcinoma and adenocarcinoma of the uterine cervix

“…cell adhesions, signal transductions, development and carcinogenesis by interactions of their armadillo-domains with many binding-molecules [19]. Iseki et al [8] found that overexpression of ALEX-1 plays a role in carcinogenesis suppression.…”