title: DARPin-Based Crystallization Chaperones Exploit Molecular Geometry as a Screening Dimension in Protein Crystallography.
abstract: DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18 kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 β-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept.
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Alexander
authors.initials: A
authors.lastname: Batyuk
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Yufan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Annemarie
authors.initials: A
authors.lastname: Honegger
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Heberling
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Electronic address: plueckthun@bioc.uzh.ch.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D017089:Ankyrin Repeat; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D001618:beta-Lactamases / Q000737:chemistry
chemical_list: D058619:Maltose-Binding Proteins; D018832:Molecular Chaperones; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1
keywords: X-ray crystallography; designed ankyrin repeat proteins; protein design; protein engineering; rigid domain fusions