title: Designed ankyrin repeat protein binders for the crystallization of AcrB: plasticity of the dominant interface.
abstract: The formation of well-diffracting crystals is a major bottleneck in structural analysis of membrane proteins by X-ray crystallography. One approach to improve crystal quality is the use of DARPins as crystallization chaperones. Here, we present a detailed analysis of the interaction between DARPins and the integral membrane protein AcrB. We find that binders selected in vitro by ribosome display share a common epitope. The comparative analysis of three crystal structures of AcrB-DARPin complexes allowed us to study the plasticity of the interaction with this dominant binding site. Seemingly redundant AcrB-DARPin crystals show substantially different diffraction quality as a result of subtle differences in the binding geometry. This work exemplifies the importance to screen a number of crystallization chaperones to obtain optimal diffraction data. Crystallographic analysis is complemented by biophysical characterization of nine AcrB binders. We observe that small variations in the interface can lead to differing behavior of the DARPins with regards to affinity, stoichiometry of the complexes and specificity for their target.
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. nmonroe@bioc.uzh.ch
authors.forename: Nicole
authors.initials: N
authors.lastname: Monroe
authors.forename: Gaby
authors.initials: G
authors.lastname: Sennhauser
authors.forename: Markus A
authors.initials: MA
authors.lastname: Seeger
authors.forename: Christophe
authors.initials: C
authors.lastname: Briand
authors.forename: Markus G
authors.initials: MG
authors.lastname: Grütter
mesh_terms: D000595:Amino Acid Sequence; D017089:Ankyrin Repeat; D001667:Binding, Competitive; D003460:Crystallization; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D046911:Macromolecular Substances / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry*; D016415:Sequence Alignment
chemical_list: D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011994:Recombinant Proteins; C083496:AcrB protein, E coli