title: Affinity maturation of a computationally designed binding protein affords a functional but disordered polypeptide.
abstract: Computational methods have been recently applied to the design of protein-protein interfaces. Using this approach, a 61 amino acid long protein called Spider Roll was engineered to recognize the kinase domain of the human p21-activated kinase 1 (PAK1) with good specificity but modest affinity (KD=100μM). Here we show that this artificial protein can be optimized by yeast surface display and fluorescence-activated cell sorting. After three rounds of mutagenesis and screening, a diverse set of tighter binding variants was obtained. A representative binder, MSR7, has a >10(2)-fold higher affinity for PAK1 when displayed on yeast and a 6 to 11-fold advantage when produced free in solution. In contrast to the starting Spider Roll protein, however, MSR7 unexpectedly exhibits characteristics typical of partially disordered proteins, including lower α-helical content, non-cooperative thermal denaturation, and NMR data showing peak broadening and poor signal dispersion. Although conformational disorder is increasingly recognized as an important property of proteins involved in cellular signaling and regulation, it is poorly modeled by current computational methods. Explicit consideration of structural flexibility may improve future protein designs and provide deeper insight into molecular events at protein-protein interfaces.
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland. Electronic address: mb879@cam.ac.uk.
authors.forename: Maren
authors.initials: M
authors.lastname: Butz
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland. Electronic address: kast@org.chem.ethz.ch.
authors.forename: Peter
authors.initials: P
authors.lastname: Kast
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland. Electronic address: hilvert@org.chem.ethz.ch.
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
mesh_terms: D000595:Amino Acid Sequence; D003198:Computer Simulation; D019020:Directed Molecular Evolution; D005434:Flow Cytometry; D005454:Fluorescence Polarization; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae; D054462:p21-Activated Kinases / Q000737:chemistry*
chemical_list: D064267:Intrinsically Disordered Proteins; D011993:Recombinant Fusion Proteins; C515903:PAK1 protein, human; D054462:p21-Activated Kinases
keywords: Computational design; Directed evolution; Intrinsically disordered proteins; Molten globule; Protein–protein interactions; Protein–receptor interface; Yeast surface display