title: Structure and Energetic Contributions of a Designed Modular Peptide-Binding Protein with Picomolar Affinity.
abstract: Natural armadillo repeat proteins (nArmRP) like importin-α or β-catenin bind their target peptides such that each repeat interacts with a dipeptide unit within the stretched target peptide. However, this modularity is imperfect and also restricted to short peptide stretches of usually four to six consecutive amino acids. Here we report the development and characterization of a regularized and truly modular peptide-specific binding protein, based on designed armadillo repeat proteins (dArmRP), binding to peptides of alternating lysine and arginine residues (KR)n. dArmRP were obtained from nArmRP through cycles of extensive protein engineering, which rendered them more uniform. This regularity is reflected in the consistent binding of dArmRP to (KR)-peptides, where affinities depend on the lengths of target peptides and the number of internal repeats in a very systematic manner, thus confirming the modularity of the interaction. This exponential dependency between affinity and recognition length suggests that each module adds a constant increment of binding energy to sequence-specific recognition. This relationship was confirmed by comprehensive mutagenesis studies that also reveal the importance of individual peptide side chains. The 1.83 Å resolution crystal structure of a dArmRP with five identical internal repeats in complex with the cognate (KR)5 peptide proves a modular binding mode, where each dipeptide is recognized by one internal repeat. The confirmation of this true modularity over longer peptide stretches lays the ground for the design of binders with different specificities and tailored affinities by the assembly of dipeptide-specific modules based on armadillo repeats.
authors.affiliation: Department of Biochemistry, University of Zürich , Winterthurerstrasse 190, 8057 Zürich, Switzerland.
authors.forename: Simon
authors.initials: S
authors.lastname: Hansen
authors.affiliation: Department of Biochemistry, University of Zürich , Winterthurerstrasse 190, 8057 Zürich, Switzerland.
authors.forename: Dirk
authors.initials: D
authors.lastname: Tremmel
authors.affiliation: Department of Biochemistry, University of Zürich , Winterthurerstrasse 190, 8057 Zürich, Switzerland.
authors.forename: Chaithanya
authors.initials: C
authors.lastname: Madhurantakam
authors.affiliation: Department of Biochemistry, University of Zürich , Winterthurerstrasse 190, 8057 Zürich, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Reichen
authors.affiliation: Department of Biochemistry, University of Zürich , Winterthurerstrasse 190, 8057 Zürich, Switzerland.
authors.forename: Peer R E
authors.initials: PR
authors.lastname: Mittl
authors.affiliation: Department of Biochemistry, University of Zürich , Winterthurerstrasse 190, 8057 Zürich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D051186:Armadillo Domain Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D040761:Biomimetic Materials / Q000737:chemistry / Q000378:metabolism; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism; D028884:Karyopherins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D020449:Repetitive Sequences, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D051186:Armadillo Domain Proteins; D004151:Dipeptides; D028884:Karyopherins; D010455:Peptides; D001120:Arginine; D008239:Lysine