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Page 2 of 15 Alekseeva et al. Microbiome Res Rep 2023;2:10 https://dx.doi.org/10.20517/mrr.2023.06

Results: We experimentally showed that neither 2D FN3 nor CD FN3 alone can bind to TNFα. Prediction of the 3D
structures of ΔFN3.1, 2D FN3, and CD FN3 suggested that only ΔFN3.1 can form a pocket allowing binding with TNF
α to occur. Polymorphism analysis of amino acid sequences of ΔFN3.1 proteins in B. longum strains uncovered
substitutions that can alter the conformation of the spatial structure of the ΔFN3.1 protein. We also analyzed
human gut-derived bacterial proteins harboring FN3 domains which allowed us to differentiate between those
containing motifs of cytokine receptors (MCRs) in their FN3 domains and those lacking them.
Conclusion: Only the complete FN3.1 protein can selectively bind to TNFα. Analysis of 3D models of the 2D FN3,
CD FN3, and ΔFN3.1 proteins showed that only the ΔFN3.1 protein is potentially capable of forming a pocket
allowing TNFα binding to occur. Only FN3 domains containing MCRs exhibited sequence homology with FN3
domains of human proteins.

Keywords: Fibronectin domain type III (FN3), bifidobacteria, TNFα, host-bacteria interaction

INTRODUCTION
[1-3]
Type III fibronectin domains (FNIII, FN3) were first identified in eukaryotic fibronectin , where they
[4]
function as structural spacers or mediate protein-protein interactions . The FN3 domain contains 90-100
amino acid residues, which form a conserved beta (β)-sandwich fold. The fold of the FN3 module contains
seven strands, forming two antiparallel β-sheets of three and four strands, which are stacked on top of each
other, forming a hydrophobic core that does not require stabilization by intrachain disulfide bonds .
[1]

The FN3 domain is widespread among proteins of both eukaryotic organisms such as humans, animals,
plants and fungi and prokaryotic organisms such as bacteria, archaea and viruses. FN3 domains have been
found in various eukaryotic extracellular and intracellular protein families, including extracellular matrix
molecules, enzymes, muscle tissue proteins, and cell surface receptors [including motifs of cytokine
[5,6]
receptors (MCRs)] . FN3 domains often contain the consensus motif WSXWS (WS motif; MCR; random
amino acid in the middle of the motif) . This motif plays a role in receptor folding, ligand (cytokine)
[7,8]
binding, and signal transduction [9,10] . Mutations in this motif alter the receptor’s ability to bind cytokines .
[8]
In bacteria, FN3 domains are found in proteins with different enzymatic functions: cellulases [11,12] ,
hydrolases [1,13] , chitinases [14,15] , and proteases . Presumably, FN3 domains play a structural, stabilizing role
[16]
in ensuring the normal functioning of the enzyme . FN3 domains have been shown to be evolutionarily
[12]
conserved and involved in a wide range of cellular functions: cell adhesion, migration, growth and
[21]
differentiation [17,18] , the transmission of nerve impulses [19,20] and biofilm formation . Researchers based at
the Laboratory of Microbial Genetics of the Vavilov Institute of General Genetics were the first to discover
the unique and species-specific PFNA operon and to shed light on its role in communication with the
human immune system via interaction with cytokines [22,23] . The PFNA operon was found in the genomes of
77 out of 99 species of Bifidobacterium, including 11 species that are commonly found in humans. The
PFNA operon consists of five to eight genes depending on the concrete species of Bifidobacterium . The
[23]
main genes are pkb2, fn3, aaa-atp, duf58 and tgm. The structure of the PFNA operon of Bifidobacterium (B.)
longum subsp. longum GT15, which consists of eight genes, is illustrated in Figure 1.

The serine-threonine protein kinase Pkb2, encoded by the pkb2 gene, is a signal transduction protein
capable of binding to an external signal; the ligand is currently not known. The fn3 gene encodes the FN3
protein, which contains two fibronectin domains that have MCRs [Figure 1]. This protein has a signal
peptide and a transmembrane domain. It is found only in Bifidobacterium. We demonstrated for the first
time that a fragment of the FN3 protein, which consists of two FN3 domains and a C-terminal region
(∆FN3.1; Figure 1), can selectively bind to the tumor necrosis factor TNFα . The studied AAA+ ATPase of
[24]