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Abstract

The structural diversity of proteins may appear endless, nevertheless even large protein complexes can be decomposed into protein domains and smaller sub-domain sized fragments. Only recently, we could identify such fragments employing sequence-based comparisons of different folds, as the TIM-barrel and the flavodoxin-like fold (Farias-Rico et al., 2014). As an extension of this work, we compared all a/b proteins and identified several fragments shared by different folds illustrating how nature may have achieved structural and functional diversity from a reduced set of building blocks. Inspired by this combinatorial concept, we searched for homologous fragments bearing active sites to engineer a func- tional fold-chimera. We extracted the vitamin-B12 binding part from methylmalonyl CoA mutase, which belongs to the flavodoxin-like fold (FL) and used it to replace the corresponding fragment in uropor- phyrinogen III synthase, which belongs to the hemD-like fold (HDL). The new hybrid resulted in a stable and well-folded protein whose structure was determined by X-ray crystallography. Moreover, cobalamin-binding function was successfully transferred to the new protein from the FL parent, which shows the advantage of using this approach for the design of new functional proteins. In addition, pro- file alignments revealed sequence and structural evidence that suggested an evolutionary path for HDL from FL by gene duplication. To test this hypothesis, we expressed a modified C-terminal half of uro- porphyrinogen III synthase and solved its structure by NMR spectroscopy, thereby confirming the pre- dicted FL architecture. Altogether, our approach facilitates the detection of common ancestry among different folds contributing to our understanding of protein development. Furthermore, our results show how new complex proteins can be designed using fragments of existing proteins that serve as building blocks in a Lego-like manner. We believe that combining fragments containing existing proper- ties will provide a successful method for the design of novel functionalities in the future.