Structural plasticity of a designer protein sheds light on β‐propeller protein evolution

Abstract

b-propeller proteins are common in nature, where they are observed to adopt 4-to 10-fold internal rotational pseudo-symmetry. This size diversity can be explained by the evolutionary process of gene duplication and fusion. In this study, we investigated a distorted b-propeller protein, an apparent intermediate between two symmetries. From this template, we created a perfectly symmetric 9-bladed b-propeller named Cake, using computational design and ancestral sequence reconstruction. The designed repeat sequence was found to be capable of generating both 8-fold and 9fold propellers which are highly stable. Cake variants with 2-10 identical copies of the repeat sequence were characterised by X-ray crystallography and in solution. They were found to be highly stable, and to self-assemble into 8-or 9-fold symmetrical propellers. These findings show that the bpropeller fold allows sufficient structural plasticity to permit a given blade to assemble different forms, a transition from even to odd changes in blade number, and provide a potential explanation for the wide diversity of repeat numbers observed in natural propeller proteins.

Influence of circular permutations on the structure and stability of a six‐fold circular symmetric designer protein

“…Alternatively, they may have just retained the closure present in the ancestral motif prior to duplication and fusion. Future experiments using different recently designed proteins, such as Tako8 and Cake9, belonging to different propeller families may shed further light on the matter 29,30 . Nevertheless, the propeller fold may be highly successful because of an intrinsic adaptability that allows individual proteins to adopt different permutations to regulate their flexibility.…”

Structure and Stability of the designer protein WRAP-T and its permutants

“…Some motifs permit multiple symmetries. The Cake sequence for example can both fold into an eight-and nine-bladed propeller depending on the number of repeats expressed [Mylemans et al, 2020a], and the WRAP sequence has also been shown to possess this property [Afanasieva et al, 2019]. Most sequences however will only properly fold in a specific symmetry; Pizza will only adopt a six-bladed propeller, even when seven repeats are tandemly placed in a single polypeptide Voet et al [2014].…”

“…[Voet et al, 2015, Shang andNienhaus, 2015] Other Pizza derivatives exhibited catalytic activity [Clarke et al, 2019], bound inorganic metaloxoclusters [Vandebroek et al, 2020], or were joined with α-helices to create protein cages [Vrancken et al, 2020]. As symmetric proteins are highly interesting for the creation of symmetric macromolecular assemblies, the same computational design strategy was subsequently used to make the eight-bladed propeller, Tako8 [Noguchi et al, 2019] and the ninebladed propeller Cake9 [Mylemans et al, 2020a]. The latter can also fold as an eight-bladed propeller when only eight repeats are expressed.…”

Development and applications of artificial symmetrical proteins

“…Mylemans et al attempted to recreate a symmetric -propeller based on a naturally occurring incomplete propeller domain. By following the RE 3 volutionary protein design approach with a 9-fold symmetric backbone, an idealized sequence was obtained, named Cake [66] . Surprisingly, truncated variants were found to fold as -propellers with either eight or nine blades ( Fig.…”