Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

Abstract

Armadillo repeat proteins (ArmRP) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. They may thus form the basis for building a modular system, in which each repeat is complementary to a piece of the target peptide. Accordingly, preselected repeats could be assembled into specific binding proteins on demand and thereby avoid the traditional generation of every new binding molecule by an independent selection from a library.Stacked armadillo repeats, each consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed curvature variations in natural ArmRPs, and identified a repeat pair from yeast importin-α as having the optimal curvature geometry to be complementary to a peptide over its whole length. We employed a symmetric in silico design to obtain a uniform sequence for a stackable repeat while maintaining the desired curvature geometry.Computationally designed armadillo repeat proteins (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics (MD) simulations in explicit solvent. Using an N-capping repeat from the consensus-design approach, two different crystal structures of dArmRP were determined. Although the experimental structures of dArmRP deviated from the designed curvature, the insertion of the most conserved binding pockets of natural ArmRPs onto the surface of dArmRPs resulted in binders against the expected peptide with low nanomolar affinities, similar to the binders from the consensus-design series.

Designing repeat proteins: a modular approach to protein design

“…As de novo design allows complete control of the structure, idealized units of native repeat families can also be directly created by incorporating native features as sequence and structure constraints. This approach readily created idealized ANK, TPR, LRR, WD40, HEAT and ARM repeat proteins [35,36]. With the exception of LRRs, capping repeats could be directly designed from the structures.…”

Peptide‐Guided Assembly of Repeat Protein Fragments

“…[8][9][10][11] Each internal module Mc ontains three tightly packed a-helices H1, H2, and H3 ( Figure 1A). [8][9][10][11] Each internal module Mc ontains three tightly packed a-helices H1, H2, and H3 ( Figure 1A).…”

“…[13] We recently discovered that the complementary dArmRP fragments YM 2 and MA assemble with a K d of 126 nm into the YM 2 :MA complex that structurally resembles the full-length YM 3 Ap rotein. [8] Based on these observations,w es et out to investigate whether mixtures of complementary dArmRP fragments enrich those combinations that constitute the best binder towards agiven target peptide.The principle is demonstrated for the protein YM 4 Au sing mixtures of ap articular N-terminal fragment with an umber of complementary C-terminal fragments that display different affinities towards atarget peptide ( Figure 2). [8] Based on these observations,w es et out to investigate whether mixtures of complementary dArmRP fragments enrich those combinations that constitute the best binder towards agiven target peptide.The principle is demonstrated for the protein YM 4 Au sing mixtures of ap articular N-terminal fragment with an umber of complementary C-terminal fragments that display different affinities towards atarget peptide ( Figure 2).…”

“…Designed armadillo repeat proteins (dArmRPs) form elongated, rod-like molecules that consist of multiple,tightly packed internal modules M, and are terminated at the N-and C-terminal ends by capping modules,Y iii and A ii ,r espectively. [8][9][10][11] Each internal module Mc ontains three tightly packed a-helices H1, H2, and H3 ( Figure 1A). They propagate ar ight-handed triangular spiral ( Figure 1B), which exposes as upercoiled binding surface consisting of helix H3 of each repeat.…”

“…[14] Importantly,t he assembled fragments of adArmRP binder for the peptide ligand neurotensin retained the ability to bind the peptide. [8] Based on these observations,w es et out to investigate whether mixtures of complementary dArmRP fragments enrich those combinations that constitute the best binder towards agiven target peptide.The principle is demonstrated for the protein YM 4 Au sing mixtures of ap articular N-terminal fragment with an umber of complementary C-terminal fragments that display different affinities towards atarget peptide ( Figure 2).…”