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

beta-Sheet motifs such as the WW domain are increasingly being explored as building blocks for synthetic biological applications. Since the sequence-structure relationships of beta-sheet motifs are generally complex compared to the well-studied alpha-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 (hPin1(WW)) by CC association. Fragmentation of hPin1(WW) was performed in both loop 1 (CC-hPin1(WW)-L1) and loop 2 (CC-hPin1(WW)-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-hPin1(WW)-L1, but not in CC-hPin1(WW)-L2. Furthermore, by using H-1-N-15 HSQC NMR, fluorescence and CD spectroscopy, we demonstrated that binding properties of fragmented hPin1(WW) in CC-hPin1(WW)-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 hPin1(WW)-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.

Automated summary

The study presents a combinatorial approach to identify novel functional mini-proteins based on the WW-domain scaffold. The WW domain fragments were reconstituted through coiled coil association, and the binding properties were fully restored. Using this approach, specific binding domains for ATP and phosphorylcholine were identified.