INTERACTION STUDY BETWEEN ACAP3 AND SEPT6-SEPT7 HETERODIMERIC COILED COIL

Septins are filamentous proteins involved in cytokinesis, cell polarization,cytoskeletal stabilization and many other cellular processes. Septins possess a C-terminal domain that usually includes α-helical coiled coil (CC) sequences. In addition to mediating selective septin interactions, septin CCs have been implicated in binding to membranes and other protein partners. The crystallographic structure of a heterodimeric septin coiled coil (PDB 8SJJ) shows imperfections in the CC helical packing, which seems important for the binding of partners. A well-characterized example is BORG3, whose BD3 motif binds to the heterodimeric coiled coil formed by SEPT6 and SEPT7 (67CC), enhancing septin polymerization.[1] Aside from the BORGs, no other septin partner [2] has had its interaction structurally characterized. An affinity capture-mass spectrometry high-throughput experiment have identified ACAP3 (ArfGAP with coiled-coil, ankyrin repeat and PH domain containing protein 3) as a septin interacting protein.[3] Deep-learning structural modeling of ACAP3, SEPT6 and SEPT7 suggests a possible interaction between the C-terminal region of ACAP3 (dubbed here as ACAP3-c) and 67CC. The objective of this work is to express, purify and characterize the interaction between ACAP3-c and 67CC. Recombinant ACAP3-c was expressed in E. coli Rosetta(DE3) and purified under both native and denaturing conditions. The protocol purification included washing steps with 2 M urea and 0.1% Triton X-100 to remove contaminants. Protein stabilization was assisted by adding 5-10% glycerol and 1 mM DTT to the buffer. Differential scanning fluorimetry (DSF) was used to verify the stability of different ACAP3-c preparations. The protein obtained from the soluble fraction was the most thermally stable, a fact also confirmed by the low presence of aggregates in dynamic light scattering (DLS) experiments (hydrodynamic diameter, D_H = 8.4 ± 2.3 nm). Pull-down experiments confirmed the interaction of His-tagged ACAP3-c with 67CC. This study will contribute to expand our understanding of septin-binding partners and may offer insights into how ACAP3 modulate the human cytoskeleton.
References:
[1] CASTRO, D. K. S. V. et al. Dissecting the binding interface of the septin polymerization enhancer Borg BD3. Journal of Molecular Biology, v. 435, n. 13, p. 168132, 2023.
[2] NEUBAUER, K. & ZIEGER, B. et al. The Mammaliam Septin Interactome. Frontiers in Cell and Developmental Biology, v. 5, p. 3, 2017.
[3] HUTTLIN, E. L. et al. Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell, v. 184, n. 11, p. 3022-3040. e28, 2021.

This work was supported by Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES-PROEX scholarship). We thank Prof. Maria Cristina Nonato for providing wet laboratory infrastructure and the Structural Biology of Septin group for providing cloned plasmids.
Keywords: septin, coiled coil, protein-protein interaction.