Identification of novel functional mini-receptors by combinatorial screening of split-WW domains [Research Data]

DOI

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.

Identifier
DOI https://doi.org/10.11588/data/SGN2CN
Related Identifier https://doi.org/10.1039/D2SC01078J
Metadata Access https://heidata.uni-heidelberg.de/oai?verb=GetRecord&metadataPrefix=oai_datacite&identifier=doi:10.11588/data/SGN2CN
Provenance
Creator Thomas, Franziska ORCID logo; Neitz, Hermann ORCID logo; Paul, Niels Benjamin ORCID logo; Häge, Florian ORCID logo; Lindner, Christina ORCID logo; Graebner, Roman ORCID logo; Kovermann, Michael ORCID logo
Publisher heiDATA
Contributor Thomas, Franziska; Kovermann, Michael
Publication Year 2023
Funding Reference Deutsche Forschungsgemeinschaft (DFG) 414261058, 2082/1–390761711 (3DMM2O) ; Young Scholar Fund (M.K) ; University of Konstanz (M.K) ; Carl Zeiss Foundation (F.H) ; Dr Sophie-Bernthsen-Stiftung
Rights CC BY 4.0; info:eu-repo/semantics/openAccess; http://creativecommons.org/licenses/by/4.0
OpenAccess true
Contact Thomas, Franziska (Heidelberg University, Institute of Organic Chemistry); Kovermann, Michael (University of Konstanz, Department of Chemistry)
Representation
Resource Type Dataset
Format application/zip; application/vnd.openxmlformats-officedocument.wordprocessingml.document
Size 1335881; 25903068; 258524; 54876945; 494704; 58029
Version 1.1
Discipline Chemistry; Construction Engineering and Architecture; Engineering; Engineering Sciences; Natural Sciences