TY - JOUR
T1 - Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition
AU - Reichen, Christian
AU - Hansen, Simon
AU - Forzani, Cristina
AU - Honegger, Annemarie
AU - Fleishman, Sarel J
AU - Zhou, Ting
AU - Parmeggiani, Fabio
AU - Ernst, Patrick
AU - Madhurantakam, Chaithanya
AU - Ewald, Christina
AU - Mittl, Peer R E
AU - Zerbe, Oliver
AU - Baker, David
AU - Caflisch, Amedeo
AU - Plückthun, Andreas
N1 - Copyright © 2016 Elsevier Ltd. All rights reserved.
PY - 2016/11/6
Y1 - 2016/11/6
N2 - Armadillo repeat proteins (ArmRPs) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. Thus, they may 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 aa arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed the curvature variations in natural ArmRPs and identified a repeat pair from yeast importin-α as having the optimal curvature geometry that is 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 ArmRPs (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics 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.
AB - Armadillo repeat proteins (ArmRPs) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. Thus, they may 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 aa arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed the curvature variations in natural ArmRPs and identified a repeat pair from yeast importin-α as having the optimal curvature geometry that is 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 ArmRPs (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics 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.
KW - armadillo repeat protein
KW - computational protein design
KW - Rosetta
KW - peptide binding
KW - molecular dynamics
KW - synthetic biology
U2 - 10.1016/j.jmb.2016.09.012
DO - 10.1016/j.jmb.2016.09.012
M3 - Article (Academic Journal)
C2 - 27664438
VL - 428
SP - 4467
EP - 4489
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 22
ER -