Protein rethreading: A novel approach to protein design

Abstract

Protein engineering is an important tool for the design of proteins with novel and desirable features. Templates from the protein databank (PDB) are often used as initial models that can be modified to introduce new properties. We examine whether it is possible to reconnect a protein in a manner that generates a new topology yet preserves its structural integrity. Here, we describe the rethreading of dihydrofolate reductase (DHFR) from E. coli (wtDHFR). The rethreading process involved the removal of three native loops, and the introduction of three new loops with alternate connections. The structure of the rethreaded DHFR (rDHFR-1) was determined to 1.6 Å, demonstrating the success of the rethreading process. Both wtDHFR and rDHFR-1 exhibited similar affinities towards methotrexate. However, rDHFR-1 showed no reducing activity towards dihydrofolate, and exhibited about ~6-fold lower affinity towards NADPH than wtDHFR. This work demonstrates that protein rethreading can be a powerful tool for the design of a large array of proteins with novel structures and topologies, and that by careful rearrangement of a protein sequence, the sequence to structure relationship can be expanded substantially.

A Single-domain Protein Catenane of Dihydrofolate Reductase

“…Notably, the previously reported rethreaded DHFR shows no reducing activity at all. 40 In fact, even circular permutation alone could cause the decrease of catalytic activity to varying degrees. 45,47 The reconstituted physical complex of two DHFR fragments (split between α-helix-C and β-sheet-5) retained only 15% of the enzymatic activity of the full-length DHFR.…”

“…A single protein domain usually has one closely packed hydrophobic core and consists of rigid secondary motifs connected by flexible loops. The highly conserved hydrophobic core allows extensive engineering of the structure, such as circular permutation, [33][34][35][36] domain swapping, [37][38][39] loop rethreading, 40 and split-reconstitution. 41,42 The mutual complementarity among segments preserves the structural integrity despite distinct connectivity.…”

“…41,42 The mutual complementarity among segments preserves the structural integrity despite distinct connectivity. Previously, both green fluorescent protein 43 and dihydrofolate reductase 40 (DHFR) have been shown to tolerate rethreading, giving a linear variant with a swapped sequence that still folds into an overall similar structure with more or less preserved function. Rewiring has also been performed on the SpyTag-SpyCatcher complex, leading to the development of active template for protein heterocatenane synthesis.…”

Step‐by‐step design of proteins for small molecule interaction: A review on recent milestones

“…Lin et al have recently developed and summarized a set of rules for precise loop design 103 . Similarly, Agah et al focused on rethreading only the loop regions of dihydrofolate reductase from E. coli , where the loops were removed and reconnected to different secondary structures on the same original structure, originating a novel fold, albeit devoid of functionality 29 …”

A Single-domain Protein Catenane of Dihydrofolate Reductase

“…A single protein domain usually has one closely packed hydrophobic core and consists of rigid secondary motifs connected by flexible loops. The highly conserved hydrophobic core allows extensive engineering of the structure, such as circular permutation, [33][34][35][36] domain swapping, [37][38][39] loop rethreading, 40 and split-reconstitution. 41,42 The mutual complementarity among segments preserves the structural integrity despite distinct connectivity.…”