Biology subjects

Kosugi, T.

Publications and source records attributed to Kosugi, T..

6 recordsLinked to original sources

bioRxiv2024

De novo design of ATPase based on the blueprint optimized for harboring the P-loop motif

De novo design of proteins has seen remarkable recent progress and has provided understanding of folding and functional expression. However, rationally creating enzymes with high activity comparable to most naturally occurring enzymes remains challenging. Here, we attempted to design an ATPase de novo, through the exploration of an optimal backbone blueprint to incorporate a conserved phosphate binding motif, the P-loop, into designed structures. The designed protein, based on the identified blueprint, was found to be a monomer with high thermal stability, and exhibited the ATPase ability. The crystal structure was closely matched to the design model, both at the overall structure level and within the P-loop motif. Interestingly, AlphaFold was not able to predict the designed structure accurately, indicating the difficulties of predicting folded structures for novel protein sequences. Remarkably, the designed protein exhibited ATPase ability even at temperatures around 100 {degrees}C, with significantly increased activity. However, the ATPase activity was still not comparable to those of naturally occurring enzymes. This suggests that the P-loop motif alone is insufficient to achieve the high ATPase activity seen in naturally occurring enzymes, indicating that other structural components are necessary to reach such activity levels.

biophysics↗

bioRxiv2023

Design of Cyclic Peptides Targeting Protein-Protein Interactions using AlphaFold

More than 930,000 protein-protein interactions (PPIs) have been identified in recent years, but their physicochemical properties differ from conventional drug targets, complicating the use of conventional small molecules as modalities. Cyclic peptides are a promising modality for targeting protein-protein interactions (PPIs), but it is difficult to predict the structure of a target protein-cyclic peptide complex or to design a cyclic peptide sequence that binds to the target protein using computational methods. Recently, AlphaFold with a cyclic offset has enabled predicting the structure of cyclic peptides, thereby enabling de novo cyclic peptide designs. We developed a cyclic peptide complex offset to enable the structural prediction of target proteins and cyclic peptide complexes and found AlphaFold2 with a cyclic peptide complex offset can predict structures with high accuracy. We also applied the cyclic peptide complex offset to the binder hallucination protocol of AfDesign, a de novo protein design method using AlphaFold, and we could design a high predicted local-distance difference test and lower separated binding energy per unit interface area than the native MDM2/p53 structure. Furthermore, the method was applied to 12 other protein-peptide complexes and one protein-protein complex. Our approach shows that it is possible to design putative cyclic peptide sequences targeting PPI.

bioinformatics↗

bioRxiv2023

Structure-based engineering of Tor complexes uncovers different roles of two types of yeast TORC1s

Certain proteins assemble into diverse complex states, each having a distinctive and unique function in the cell. The target of rapamycin complex 1 (TORC1) plays a central role in signaling pathways for cells to respond to their environment, such as nutritional status. TORC1 is widely recognised for its association with various diseases. The budding yeast Saccharomyces cerevisiae has two types of TORC1s comprising different constituent proteins, Tor1- and Tor2-containing TORC1s but are considered to have the same function. Here, we rationally redesigned the complex states by structure-based engineering and constructed a Tor2 mutant to form TORC2 but not TORC1. Functional analysis of the mutant revealed that the two types of TORC1s induced different phenotypes-rapamycin, caffeine and pH dependences of cell growth and replicative and chronological lifespans. These findings are expected to provide further insights into various fields such as molecular evolution and lifespan.

molecular biology↗

bioRxiv2022

Solubility-aware protein binding peptide design using AlphaFold

New protein-protein interactions (PPIs) are being identified, but PPIs have different physicochemical properties compared with conventional targets, making it difficult to use small molecules. Peptides offer a new modality to target PPIs, but designing appropriate peptide sequences by computation is challenging. Recently, AlphaFold and RosettaFold have made it possible to predict protein structures from amino acid sequences with ultra-high accuracy, enabling de novo protein design. We designed peptides likely to have PPI as the target protein using the "binder hallucination" protocol of AfDesign, a de novo protein design method using AlphaFold. However, the solubility of the peptides tended to be low. Therefore, we designed a solubility loss function using solubility indices for amino acids and developed a solubility-aware AfDesign binder hallucination protocol. The peptide solubility in sequences designed using the new protocol increased with the weight of the solubility loss function; moreover, they captured the characteristics of the solubility indices. Moreover, the new protocol sequences tended to have higher affinity than random or single residue substitution sequences when evaluated by docking binding affinity. Our approach shows that it is possible to design peptide sequences that can bind to the interface of PPI while controlling solubility.

bioinformatics↗

bioRxiv2021

Design of complicated all-α protein structures

A wide range of de novo protein structure designs have been achieved, but the complexity of naturally occurring protein structures is still far beyond these designs. To expand the diversity and complexity of de novo designed protein structures, we sought to develop a method for designing "difficult-to-describe"-helical protein structures composed of irregularly aligned -helices like globins. Backbone structure libraries consisting of a myriad of -helical structures with 5- or 6-helices were generated by combining 18 helix-loop-helix motifs and canonical -helices, and five distinct topologies were selected for de novo design. The designs were found to be monomeric with high thermal stability in solution and fold into the target topologies with atomic accuracy. This study demonstrated that complicated -helical proteins are created using typical building blocks. The method we developed would enable us to explore the universe of protein structures for designing novel functional proteins.

biophysics↗

bioRxiv2020

De Novo Design of Allosteric Control into Rotary Motor V1-ATPase by Restoring Lost Function

Protein complexes exert various functions through allosterically controlled cooperative work. De novo design of allosteric control into protein complexes provides understanding of their working principles and potential tools for synthetic biology. Here, we hypothesized that an allosteric control can be created by restoring lost functions of pseudo-enzymes contained as subunits in protein complexes. This was demonstrated by computationally de novo designing ATP binding ability of the pseudo-enzyme subunits in a rotary molecular motor, V1-ATPase. Single molecule experiments with solved crystal structures revealed that the designed V1 is allosterically accelerated than the wild-type by the ATP binding to the created allosteric site and the rate is tunable by modulating the binding affinity. This work opened up an avenue for programming allosteric control into proteins exhibiting concerted functions.

biophysics↗