Identification of novel functional mini-receptors by combinatorial screening of split-WW domains. Issue 31 (21st July 2022)

• Record Type: Journal Article
• Authors: Neitz, Hermann; Paul, Niels Benjamin; Häge, Florian R.; Lindner, Christina; Graebner, Roman; Kovermann, Michael; Thomas, Franziska
• Is Part Of: Chemical science. Volume 13:Issue 31(2022)
• Page Start: 9079
• Page End: 9090
• Publication Date: 2022-07-21
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/d2sc01078j
• Languages: English
• ISSNs: 2041-6520

Abstract

Abstract : A combinatorial approach toward novel functional WW domains based on coiled-coil-mediated reconstitution of split WW domains is presented. As such, an ATP-binding WW domain was found from a 4-by-6 library of N- and C-terminal WW domain fragments. 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 1 H– 15 N 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.