Identification of novel functional mini-receptors by combinatorial screening of split-WW domains

• Verfasst von: Neitz, Hermann [VerfasserIn]
• Verfasst von: Paul, Niels Benjamin [VerfasserIn]
• Verfasst von: Häge, Florian [VerfasserIn]
• Verfasst von: Lindner, Christina [VerfasserIn]
• Verfasst von: Graebner, Roman [VerfasserIn]
• Verfasst von: Kovermann, Michael [VerfasserIn]
• Verfasst von: Thomas, Franziska [VerfasserIn]
• Titel: Identification of novel functional mini-receptors by combinatorial screening of split-WW domains
• Verf.angabe: Hermann Neitz, Niels Benjamin Paul, Florian R. Häge, Christina Lindner, Roman Graebner, Michael Kovermann and Franziska Thomas
• E-Jahr: 2022
• Jahr: 14 Jul 2022
• Umfang: 12 S.
• Fussnoten: Gesehen am 13.02.2023
• Titel Quelle: Enthalten in: Chemical science
• Ort Quelle: Cambridge : RSC, 2010
• Jahr Quelle: 2022
• Band/Heft Quelle: 13(2022), 31, Seite 9079-9090
• ISSN Quelle: 2041-6539
• DOI: doi:10.1039/D2SC01078J
• Datenträger: Online-Ressource
• Sprache: eng
• Bibliogr. Hinweis: Forschungsdaten: Thomas, Franziska, 1980 - : Identification of novel functional mini-receptors by combinatorial screening of split-WW domains [research data]
• K10plus-PPN: 1834593573

Abstract

β-Sheet motifs such as the WW domain are increasingly being explored as building blocks for synthetic biological applications. Since the sequence-structure relationships of β-sheet motifs are generally complex compared to the well-studied α-helical coiled coil (CC), other approaches such as combinatorial screening should be included to vary the function of the peptide. In this study, we present a combinatorial approach to identify novel functional mini-proteins based on the WW-domain scaffold, which takes advantage of the successful reconstitution of the fragmented WW domain of hPin1 (hPin1WW) by CC association. Fragmentation of hPin1WW was performed in both loop 1 (CC-hPin1WW-L1) and loop 2 (CC-hPin1WW-L2), and the respective fragments were linked to the strands of an antiparallel heterodimeric CC. Structural analysis by CD and NMR spectroscopy revealed structural reconstitution of the WW-domain scaffold only in CC-hPin1WW-L1, but not in CC-hPin1WW-L2. Furthermore, by using 1H-15N HSQC NMR, fluorescence and CD spectroscopy, we demonstrated that binding properties of fragmented hPin1WW in CC-hPin1WW-L1 were fully restored by CC association. To demonstrate the power of this approach as a combinatorial screening platform, we synthesized a four-by-six library of N- and C-terminal hPin1WW-CC peptide fragments that was screened for a WW domain that preferentially binds to ATP over cAMP, phophocholine, or IP6. Using this screening platform, we identified one WW domain, which specifically binds ATP, and a phosphorylcholine-specific WW-based mini-receptor, both having binding dissociation constants in the lower micromolar range.