9H1F

Cofilin-1 in complex with high-affinity Sybody B12

Summary for 9H1F

• Entry DOI: 10.2210/pdb9h1f/pdb
• Descriptor: Cofilin-1, SybodyB12 (3 entities in total)
• Functional Keywords: cofilin-1, cfl1, adf/cofilin, g-actin, f-actin, actin, sybody, nanobody, synthetic nanobody, actin binding protein, abp, protein binding
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 2
• Total formula weight: 31787.21

Authors

Paraschiakos, T.,Windhorst, S.,Pogenberg, V. (deposition date: 2024-10-09, release date: 2025-03-19, Last modification date: 2025-03-26)

Primary citation

Paraschiakos, T.,Li, J.,Scholz, J.,Han, S.J.,Deckers, M.,Pogenberg, V.,Faix, J.,Windhorst, S.
A high affinity Sybody blocks Cofilin-1 binding to F-actin in vitro and in cancer cells.
Biochem Pharmacol, 236:116866-116866, 2025

PubMed Abstract: Upregulation of the actin-severing protein Cofilin-1 is implicated in enhancing malignancy of various cancer types by promoting actin turnover and increasing cellular motility. Despite the importance of targeting Cofilin-1, currently there is a lack of inhibitors specifically targeting its actin-severing activity. To address this issue, we generated synthetic anti-Cofilin-1 nanobodies (Sybodies) that interfere with human Cofilin-1 binding to filamentous actin. We identified four high affinity Sybodies against human Cofilin-1 with dissociation constants (K) in the nanomolar range that inhibited G-actin sequestration, and actin-severing activity of Cofilin-1 in vitro. Notably, Sybody B12, with the lowest K of approximately 27 nM, competitively blocked actin binding to Cofilin-1, and also inhibited G-actin sequestration of murine Cofilin-1. The crystal structure of the Sybody-B12-Cofilin-1 complex, resolved at 1.8 Å, revealed that Sybody B12 binds to the G-actin binding site of Cofilin-1, showing that Sybody B12 engages the same binding site on Cofilin-1 as actin. Consistently, transient expression of mPlum-tagged Sybody B12 in human H1299 lung cancer cells inhibited the formation of enhanced green fluorescent protein (EGFP)-Cofilin-actin rods. Notably, stable expression of Sybody B12 did not affect viability of H1299 cells, and no compensatory up-regulation of Cofilin-2 or actin-depolymerization factor (ADF) mRNA were detectable in Sybody B12 expressing H1299 cells. Together, these findings suggest that Sybody B12 exhibits a strong potential as tool for inhibiting the interaction of Cofilin-1 with actin. In addition, it could serve as a promising lead structure for designing Cofilin-1 inhibitors in silico.

PubMed: 40064451
DOI: 10.1016/j.bcp.2025.116866

Experimental method

X-RAY DIFFRACTION (1.8 Å)