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Updated: Aug 6, 2025
Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane SSM-Based Electrophysiology
Published on: May 3, 2021
Targeting Ras-binding domain of ELMO1 by computational nanobody design
Chunlai Tam1,2, Mutsuko Kukimoto-Niino3, Yukako Miyata-Yabuki4
1Laboratory for Structural Bioinformatics, Center for Biosystems Dynamics Research, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan.
Abstract:
The control of cell movement through manipulation of cytoskeletal structure has therapeutic prospects notably in the development of novel anti-metastatic drugs. In this study, we determine the structure of Ras-binding domain (RBD) of ELMO1, a protein involved in cytoskeletal regulation, both alone and in complex with the activator RhoG and verify its targetability through computational nanobody design. Using our dock-and-design approach optimized with native-like initial pose selection, we obtain Nb01, a detectable binder from scratch in the first-round design. An affinity maturation step guided by structure-activity relationship at the interface generates 23 Nb01 sequence variants and 17 of them show enhanced binding to ELMO1-RBD and are modeled to form major spatial overlaps with RhoG. The best binder, Nb29, inhibited ELMO1-RBD/RhoG interaction. Molecular dynamics simulation of the flexibility of CDR2 and CDR3 of Nb29 reveal the design of stabilizing mutations at the CDR-framework junctions potentially confers the affinity enhancement.
Insights
Researchers designed a novel nanobody, Nb29, targeting the ELMO1 protein
Area of Science:
- Biochemistry
- Structural Biology
- Drug Discovery
Background:
- Cell movement regulation is crucial for cancer metastasis.
- ELMO1 protein is involved in cytoskeletal regulation and cancer cell migration.
- Targeting protein-protein interactions offers therapeutic strategies for anti-metastasis.
Purpose of the Study:
- To determine the structure of ELMO1's Ras-binding domain (RBD) alone and with RhoG.
- To computationally design nanobodies targeting ELMO1-RBD.
- To validate nanobody binding and inhibitory potential against ELMO1-RBD/RhoG interaction.
Main Methods:
- X-ray crystallography for structural determination.
- Computational nanobody design using a dock-and-design approach.
- Affinity maturation and molecular dynamics simulations for optimization and validation.
Main Results:
- Successfully determined the structure of ELMO1-RBD in complex with RhoG.
- Designed Nb01, a novel nanobody binder, from scratch in the first design round.
- Generated 17 enhanced binding variants, with Nb29 inhibiting ELMO1-RBD/RhoG interaction.
Conclusions:
- Computational nanobody design is effective for targeting protein-protein interactions.
- Nb29 demonstrates therapeutic potential by inhibiting ELMO1-RBD/RhoG interaction.
- Stabilizing mutations in nanobody CDRs contribute to enhanced binding affinity.
