Fig. 4: SPIRE interactions with FMN-subgroup formins are conserved across distant.

a Domain organisation of human (Hs Homo sapiens) and Monosiga brevicollis (Mb) SPIRE proteins and formin (FMN) subfamily formins. SPIRE proteins share KIND (kinase non-catalytic C-lobe domain), WH2 (Wiskott-Aldrich syndrome protein homology 2) domains, GTBM (globular tail domain binding motif) and FYVE_2 domain (after Fab1/YOTB/Vac1/EEA1). FMN proteins share the formin homology domains (FH1 and FH2), the C-terminal formin-SPIRE interaction sequence (FSI) and as-yet uncharacterised large N-terminus. Protein structures are drawn to scale and numbers indicate amino acid residues. b WebLogo^108,109 showing amino acid conservation within vertebrate FMN-FSI protein sequences. Residues forming hydrogen bonds (#) and salt bridges (+) with the SPIRE-KIND are labeled respectively¹⁷. Below the corresponding C-terminal sequence part of Mb-FMN is depicted, adjusted to the WebLogo and conserved residues are labeled (*). c Multiple sequence alignment of vertebrate (Hs Homo sapiens, Mm Mus musculus) and choanoflagellate (Mb Monosiga brevicollis, Sr Salpingoeca rosetta) SPIRE proteins. The acidic cluster of the SPIRE KIND domain is shown spanning α-helices α3 and α4 as well as the β-sheet β4¹⁷. Coloring indicates amino acid conservation at specific positions ranging from high (dark blue) to low (light blue) conservation. Asterisks indicate contact sites between vertebrate SPIRE1-KIND and FMN2-FSI sequences¹⁷. Contact sites conserved in Monosiga brevicollis are labelled accordingly. Numbering indicates amino acid residues. d Protein structure alignment of the ESMFold⁵⁸ predicted Mb-SPIRE KIND domain (grey) and the crystal structure of the vertebrate SPIRE1-KIND:FMN2-FSI complex (PDB-ID: 2YLE, blue¹⁷). SPIRE1-KIND amino acid residues forming contact sites with FMN2-FSI are colored in red. Contact sites which are conserved in Monosiga brevicollis are labelled accordingly (compare with (c)). The α-helices α3 and α4 as wells as β-sheet β4 are indicated. The FSI peptide structure is hidden for clarity. e GST-pulldown assay with purified GST-Mb-FMN-eFSI and lysates from HEK 293 cells transiently expressing N-terminal (KIND and WH2) AcGFP1-tagged Mb-SPIRE (GFP-Mb-SPIRE-KW, input). GST and AcGFP1 (GFP) were used as controls and Ponceau S staining shows equal amounts of GST and GST-tagged proteins. N = 2 experimental repeats. f Localisation of transiently co-expressed tagged full-length Mb-SPIRE and Mb-SPIRE-ΔKW (AcGFP1; GFP-Mb-SPIRE, GFP-Mb-SPIRE-ΔKW; green) and Mb-FMN-FH2-FSI (mRuby3; Ruby3-Mb-FMN-FH2-FSI; red) in human HeLa cells was analysed by fluorescence microscopy. AcGFP1 (GFP) and mRuby3 (Ruby3) expressions were used as controls. Deconvoluted images indicate the localisation of Mb-SPIRE and Mb-SPIRE-ΔKW proteins on vesicular structures. Mb-FMN-FH2-FSI colocalises with Mb-SPIRE, but not with Mb-SPIRE-ΔKW. Scale bars represent 5 µm. At least six cells from two distinct experiments were imaged for each condition and the cytoplasmic region of one representative cell is presented here.