Data collection and refinement statistics.

<p>*Highest resolution shell is shown in parenthesis.</p><p>Data collection and refinement statistics.</p

The thalidomide binding domain of mouse cereblon exhibits flexibility comparable to MsCI4.

<p>The mouse domain in complex with (blue, sand) and without thalidomide (yellow, shades of brown) is compared to MsCI4•thalidomide (transparent white). (A) Two thalidomide-bound domains from 4TZC arranged as an intertwined dimer. (B) Superposition of one monomer to MsCI4•thalidomide, illustrating the unfolded nature of the first flexible region. (C) Same as (B) but from another perspective, in stereo. (D) Apo mouse domains in 3WX2 are found in two conformations, “yellow” and “brown”, forming an endless array of interactions in the crystal lattice. (E) Superposition of both apo conformations onto MsCI4•thalidomide, showing the first flexible region in different conformations. (F) Same as (E) but from another perspective, in stereo. Note that the conformation of the tryptophans on the left is reminiscent of the flipped tryptophan in the intertwined MsCI4 dimer.</p

Crystallization conditions and cryo protection / washing procedure.

<p>Crystallization conditions and cryo protection / washing procedure.</p

Partial unfolding of MsCI4 upon loss of thalidomide.

<p>Crystals of the orthorhombic crystal from two crystallization conditions were washed for 40 h in a solution without thalidomide. For the initial structure (grey) and both experiments, “Wash I” (brown) and “Wash II” (black), the monomer that had the ligand washed out (chain C) is depicted in two perspectives. Loss of ligand was accompanied by the unfolding of three regions. The structures after washing are overlaid with the initial structure in transparent. On the bottom, the sequence alignment of MsCI4 with human cereblon details the unfolded regions and indicates secondary structure elements. The unfolded regions are shaded. In “Wash I”, the unfolding of the first region is incomplete, so the hairpin formed by the 3^rd and 4^th β-strand is still folded, albeit shifted in position. The C-terminal segment deleted in the R419X mutant of human cereblon and the corresponding segment in MsCI4 are underlined. Key-residues are highlighted red.</p

The flexible regions and mental retardation mutation in the context of human full length cereblon.

<p>At the top, interaction partner DDB1 is represented by a sphere. The thalidomide binding domain is in green (rigid core), yellow (flexible regions) and red (deleted C-terminal part in the MsCI4^WWK/FFX mutant). The exact boundaries of the segments are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128342#pone.0128342.g002" target="_blank">Fig 2</a>. The N-terminal extension of cereblon is in brown, the remainder of the protein grey.</p

Potential of Fragment Recombination for Rational Design of Proteins

It is hypothesized that protein domains evolved from smaller intrinsically stable subunits via combinatorial assembly. Illegitimate recombination of fragments that encode protein subunits could have quickly led to diversification of protein folds and their functionality. This evolutionary concept presents an attractive strategy to protein engineering, e.g., to create new scaffolds for enzyme design. We previously combined structurally similar parts from two ancient protein folds, the (βα)₈-barrel and the flavodoxin-like fold. The resulting “hopeful monster” differed significantly from the intended (βα)₈-barrel fold by an extra β-strand in the core. In this study, we ask what modifications are necessary to form the intended structure and what potential this approach has for the rational design of functional proteins. Guided by computational design, we optimized the interface between the fragments with five targeted mutations yielding a stable, monomeric protein whose predicted structure was verified experimentally. We further tested binding of a phosphorylated compound and detected that some affinity was already present due to an intact phosphate-binding site provided by one fragment. The affinity could be improved quickly to the level of natural proteins by introducing two additional mutations. The study illustrates the potential of recombining protein fragments with unique properties to design new and functional proteins, offering both a possible pathway of protein evolution and a protocol to rapidly engineer proteins for new applications