Protein design by fusion: implications for protein structure prediction and evolution

Abstract

Domain fusion is a useful tool in protein design. Here, the structure of a fusion of the heterodimeric flagella-assembly proteins FliS and FliC is reported. Although the ability of the fusion protein to maintain the structure of the heterodimer may be apparent, threading-based structural predictions do not properly fuse the heterodimer. Additional examples of naturally occurring heterodimers that are homologous to full-length proteins were identified. These examples highlight that the designed protein was engineered by the same tools as used in the natural evolution of proteins and that heterodimeric structures contain a wealth of information, currently unused, that can improve structural predictions.

Protein rethreading: A novel approach to protein design

“…These loop residues can be expected to be flexible, thus they can adjust to a new conformation bridging the distance. We have observed this to be true in a previous heterodimeric fusion 25 . Although, the termini of the flagellar assembly proteins (FliS and FliC) are ~14 Å apart, it was possible to fuse the termini with just a two amino acids linker with minimal changes to the structure of the heterodimeric complex.…”

“…Although the rethreading process performed here is analogous to MLP, rethreading is not limited to swapping loops. Rethreading can be more expansive than MLP in the following ways: 1- breaks within secondary structural elements can be introduced, 2- a number of residues may be removed as shown with rDHFR-1 which is 12 residues shorter than wtDHFR, 3- the original N and C termini can be used as connection points to a newly introduced terminus, and 4-rethreading can be used on multi-domain proteins or to fuse protein complexes 25 . Therefore, the potential number of protein architectures that can be engineered using rethreading is likely to be vast.…”

“…These 16 residues are more than sufficient to bridge the 4 Å distance between the two incision points ( Figs 1 and 2 ). The loop residues are expected to be flexible and able to adopt the required changes 25 . The number of loop residues varies depending on which model is used for this analysis.…”

FliS/flagellin/FliW heterotrimer couples type III secretion and flagellin homeostasis

“…While our knowledge on the functional roles of FliS, FliW and CsrA is steadily increasing, structural information is lagging behind. We present the high-resolution structure of FliS bound to full-length flagellin showing the FliS/Flagellin interaction is more complex than indicated by previous partial structures 21 , 22 . Moreover, we show that FliW and FliS bind to opposing interfaces located at the N- and C-termini of flagellin, respectively.…”

“…We first wanted to determine a structure elucidating the complex of the FliS chaperone in complex with its client flagellin. Thus far, the only structural information that was available was of FliS bound to a C-terminal fragment of flagellin (residues 478–518; pdb: 1ORY, 21 , 4IWB, 22 ). To obtain the complete structure, the flagellin/FliS complex from B. subtilis was co-expressed in E .…”