Playing Lego: A functional protein designed from fragments of two different folds

The complex and highly functional proteins that we observe today evolved from simpler and less specialized subunits. In this process domains as well as subdomains are recruited and the existing functionalities of the recruited units are subsequently adapted. Here we show that this concept of combinatorial assembly and recruitment can also be applied to protein design.

In particular, we combined fragments of two different folds, the flavodoxin-like and the (βα)₈-barrel fold, to build new βα-barrel proteins. The crystal structure of one of these proteins revealed the molecular interactions of the fragments and showed that the pieces retain their structures within the new protein context [1]. It further uncovered the presence of an additional strand in the inner barrel, which is formed by residues of the C-terminus. This new and unexpected structural element relieves stress at the interface. The interface was then optimized by computational design with the Rosetta program, thus creating a stable protein whose structure could be verified by NMR spectroscopy. The intrinsic properties of the fragments were further used to improve functionality: an intact phosphate-binding site provided by one parent protein supplemented by two amino acid mutations enabled us to quickly establish ligand-binding affinities comparable to that of wild-type proteins. The approach illustrates the advantages of using subdomain-sized fragments that contribute interesting properties for the design of new proteins.

Altogether this modular assembly enables new combinations of functional properties encoded in fold fragments. It further demonstrates how new proteins can quickly develop and be competitive in today’s protein world.