The WW-domain scaffold as a model system for the de novo design of miniaturized phosphate receptors, phosphatases and sulfatases

• Applicant: Professorin Dr. Franziska Thomas
• Subject Area: Biological and Biomimetic Chemistry
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 414261058

Enzymes catalyze most of the biochemical processes in living systems, and this with astonishing substrate specificity, stereoselectivity and efficiency. Hence, it is no surprise that efforts are made to use enzymes for chemical synthesis. Next of using native enzymes for chemical reactions, engineering of catalytic properties on protein scaffolds or designing enzymes completely from scratch is of increasing scientific interest. Most strategies in enzyme design comprise redesign of natural protein scaffolds. The advantage resides in the huge number of available protein structures. However, the sequence-to-structure relationships of most protein scaffolds are not yet understood, and modifications might lead to protein misfolding. In small, well-characterized protein folding motifs the sequence positions, which allow modification, are known and these structures are accessible by chemical synthesis. On the downside, few protein folds are thoroughly studied and only the coiled-coil motif is well-understood. Consequently, mostly α-helical bundles were previously used for the de novo design of miniaturized enzymes.This research concept is centered on the design of miniaturized enzymes. The WW domain, a small, three-stranded β-sheet protein folding motif, will be investigated as potential alternative scaffold in the design of mini enzymes. In preliminary studies, sequence alignments and alanine scans of the ligand binding site were used to identify amino acid residues, which are relevant for structure and/or function. Based on this, designed sequences are proposed, which encode for a basic WW-domain scaffold. This scaffold is the starting point for the design of WW domains with different binding properties or even catalytic activity. As a proof-of-concept, the de novo design of representatives of the main WW-domain groups is planned. Furthermore, WW domains showing binding to phosphorylated peptides and phosphorylated organic molecules will be created. This in turn is a starting point for the de novo design of WW-domain based phosphatases and sulfatases. To identify active WW-domain scaffolds, a combinatorial approach based on the reconstitution of split WW domains by coiled-coil association is anticipated. For this purpose, the WW-domain fragments are linked to the strands of an antiparallel coiled coil, respectively. Previously, this concept was successfully tested with the split WW domain of PIN1.The presented research project is understood as initiator for the design of further ß-sheet-based protein folding motifs with catalytic activity. In future studies catalysis of ligation reactions, phophorylations and transfer reactions will be of interest.

• DFG Programme: Research Grants