Affinity to cellulose is a shared property among coiled-coil domains of intermediate filaments and prokaryotic intermediate filament-like proteins

Abstract

Coiled-coil domains of intermediate filaments (IF) and prokaryotic IF-like proteins enable oligomerisation and filamentation, and no additional function is ascribed to these coiled-coil domains. However, an IF-like protein from Streptomyces reticuli was reported to display cellulose affinity. We demonstrate that cellulose affinity is an intrinsic property of the IF-like proteins FilP and Scy and the coiled-coil protein DivIVA from the genus Streptomyces. Furthermore, IF-like proteins and DivIVA from other prokaryotic species and metazoan IF display cellulose affinity despite having little sequence homology. Cellulose affinity-based purification is utilised to isolate native FilP protein from the whole cell lysate of S. coelicolor. Moreover, cellulose affinity allowed for the isolation of IF and IF-like protein from the whole cell lysate of C. crescentus and a mouse macrophage cell line. The binding to cellulose is mediated by certain combinations of coiled-coil domains, as demornstrated for FilP and lamin. Fusions of target proteins to cellulose-binding coiled-coil domains allowed for cellulose-based protein purification. The data presented show that cellulose affinity is a novel function of certain coiled-coil domains of IF and IF-like proteins from evolutionary diverse species.

Ultrastructure of Exospore Formation in Streptomyces Revealed by Cryo-Electron Tomography

“…We predict that the layer represents a putative polarisome and thus, the observed structure likely represents a complex of DivIVA and Scy (Holmes et al, 2013). While purified FilP formed striated bundles in vitro when imaged with negative staining EM (Soderholm et al, 2018;Javadi et al, 2019), we did not observe such structures in our tomograms, suggesting that FilP may adopt alternate conformations under native conditions or in association with other proteins. Additional filaments were occasionally observed in the cytoplasm, along the length of the vegetative hyphae and near hyphal tips (Supplementary Figure S1 and Supplementary Movie S2).…”

Assembly mechanisms of the bacterial cytoskeletal protein FilP

“…Next, the cellophane was washed with PBS buffer before covering with permeabilization buffer (20 mM Tris–HCl, pH 8, 50 mM glucose, 10 mM EDTA, and 2 mg/ml lysozyme) for 1 min. After washing the cellophane with PBS buffer, it was covered with blocking solution (1× PBS, 2% BSA) for 5 min, washed with PBS, and incubated with primary polyclonal rabbit anti-FilP antibody (Söderholm et al, 2018) diluted 1:1,000 in blocking buffer. Afterwards, the cellophane was washed with PBS and incubated with goat anti-rabbit, Alexa Fluor 647 (Life technologies) secondary antibody diluted 1:1,000 in blocking buffer for 1 h. The cellophane samples were washed with PBS and mounted onto agar pads for imaging.…”

“…Proteins were transferred to PVDF membranes (Bio-Rad) based on the manufacturer’s protocol. Polyclonal rabbit anti-FilP (Söderholm et al, 2018) was used as a primary antibody, and horseradish peroxidase-conjugated antirabbit antibodies were used for detection. Blots were visualized using ECL reagents and scanned with a C-DiGiT blot scanner (LI-COR).…”

“…IF-like proteins from other bacterial species have also been reported, such as CfpA (Izard, 2006), Scc (Mazouni et al, 2006), Scy (Walshaw et al, 2010), and FilP (Bagchi et al, 2008). FilP is an IF-like protein from Streptomyces coelicolor (Bagchi et al, 2008; Fuchino et al, 2013; Söderholm et al, 2018). Streptomyces are Gram-positive bacteria with characteristics such as polar growth, multicellularity, differentiation, and sporulation (Flärdh & Buttner, 2009; Jakimowicz & van Wezel, 2012; McCormick & Flärdh, 2012).…”

Molecular characterization of the N-terminal half of TasA during functional amyloid assembly and its contribution toBacillus subtilisbiofilm formation

“…These fimbriae are maintained attached to the cell surface via their interaction with cellulose, which seems to serve as a template for the polymerization of the fimbriae 40 . Moreover, a more recent work has proposed that coiled-coil domains in intermediate filament-like proteins have an intrinsic affinity for cellulose in different bacterial species 41 , demonstrating a connection between specific domains in filamentous proteins and interactions with polysaccharides.…”