Teneurins are evolutionarily ancient cell surface receptors which have emerged as important regulators of many neurobiological processes in both vertebrates and invertebrates, including synaptic partner matching (Hong et al., 2012), synaptic organization (Mosca et al., 2012), neuronal migration (Drabikowski et al., 2005) and axonal guidance and targeting (Leamey et al., 2007; Berns et al., 2018). This variety of functions has been attributed to both homophilic interactions between teneurins expressed on adjacent cells and also heterophilic interactions with other cell surface receptors, such as the synaptic adhesion G-protein-coupled receptor, latrophilin, which is essential for hippocampal synapse formation in mice (Silva et al., 2011; Boucard et al., 2014; Anderson et al., 2017). The relative importance of heterophilic vs. homophilic interactions of teneurins is not fully understood.

Consistent with their evolutionarily ancient origins, bioinformatic and phylogenetic analyses have revealed that eukaryotic teneurins arose via a horizontal gene transfer event from bacteria early during metazoan evolution (Tucker et al., 2012). However, their relationship to these bacterial proteins has remained unclear due to a lack of structural data for the teneurins. The structural analysis of teneurins has long been hampered by difficulties with recombinant expression and purification of these large, glycosylated and intricately folded type II transmembrane protein receptors. The advent of advanced eukaryotic expression systems, especially those using mammalian HEK293 cells (Seiradake et al., 2015), and insect cells in suspension (Berger and Poterszman, 2015) have made the production of high quality samples tractable and led to the recent publications of the first teneurin structures (Jackson et al., 2018; Li et al., 2018). These recently published structures agree with each other overall, but there are some discrepancies that cannot be easily explained by differences in the sequences used, and which may arise from the methods employed to determine the structures. Where there are differences, we focus on the results obtained with the most completely modeled and highest resolution structure (solved by X-ray crystallography, up to 2.4 Å resolution), which is that of chicken Teneurin 2 (Figure 1A,B; Jackson et al., 2018). This structure is in good agreement with the structure of murine Teneurin 3, solved by cryo-electron microscopy by the same authors (up to 3.8 Å resolution), and with the structure of human Teneurin 2 solved by Li et al. (2018), also using cryo-electron microscopy (up to 3.1 Å resolution). The structure of chicken Teneurin 2 (residues 955-2802) obtained by X-ray crystallography is >99% complete, although less structural rigidity is observed in its peripheral domains. This is reflected by increased thermal motion (B-factors) in these regions (Figure 1E). The two models derived from cryo-electron microscopy reconstructions are both less complete compared to the X-ray structure, in particular in those regions corresponding to those with the highest B-factors in the crystal structure (Figure 1F). This suggests that these areas of the protein may be inherently flexible, but what causes the differences in magnitude of the domain flexibility in the different structures is currently not clear. Given the availability of the higher resolution and more complete X-ray structure, we will only discuss the lower resolution cryo-EM structures where the data is most reliable.

Alongside their cryoEM structure of human Teneurin2, Li et al. (2018) also present compelling mutagenesis and functional data. To avoid overlap with other chapters of this review series we focus our discussion on the structural features of teneurins with respect to their evolution from the bacterial protein ancestors.

Teneurins are typically ∼2800 amino acids long with about half of this sequence encoding a structurally conserved extracellular scaffold (Figure 1B). Over half of this scaffold region contains a sequence repeat motif known as a tyrosine-aspartate (YD)-repeat [or rearrangement hotspot (Rhs)-repeat] (Zhang et al., 2012). The YD-repeat motif is widespread amongst prokaryotic proteins, and has been structurally characterized in the context of the heterodimeric TcB-TcC subcomplex in bacterial ABC toxin complexes (Busby et al., 2013; Gatsogiannis et al., 2013; Meusch et al., 2014). In both teneurins and TcB-TcC complexes, each YD-repeat forms a pair of β-strands joined by a β-hairpin. Together, multiple repeats form an extended spiraling β-sheet resulting in a large, hollow shell-like structure (the YD-shell) that is sealed at the C-terminal end by spiraling inward to form an “Rhs-associated core domain”-like structure. As well as acting as a central scaffold, the teneurin YD-shell is thought to bind to negatively charged heparin glycans (Minet et al., 1999). A mutation in the YD-shell of human Ten1, P1610L, is linked to congenital anosmia (Alkelai et al., 2016).

Two smaller domains are found N-terminal to the YD-shell and complete the teneurin scaffold. The most N-terminal of these domains is a distinctive fibronectin (FN) type-III domain termed the FN-plug domain (Figure 1A,D). Compared to a canonical FN type-III domain, the FN-plug contains an insertion (44 amino acids in chicken Ten2) between β strands 1 and 2 and an extension of its C-terminus (38 amino acids in chicken Ten2). This insertion and extension form a separate subdomain designated the “plug,” as it resides inside the YD-shell cavity, forming numerous hydrophobic and hydrogen bonding interactions with the shell interior. The YD-shell and FN-plug combination encloses a space of ∼130 nm³.

Between the FN-plug and YD-shell is a six-bladed β-propeller domain (Figure 1A,B,D), the NHL domain. The NHL domain has been shown to determine the specificity of teneurin homophilic interactions (Beckmann et al., 2013), which are thought to underlie initial synaptic partner matching in the murine hippocampus (Berns et al., 2018), the vertebrate visual system (Dharmaratne et al., 2012; Antinucci et al., 2013) and in the Drosophila olfactory map (Hong et al., 2012) and neuromuscular junction (Mosca et al., 2012). The NHL domain is positioned perpendicular to the YD-shell and is held in place by the FN-plug domain. The top face (Chen et al., 2011) of the NHL domain is decorated with extended loops, stabilized by five highly conserved disulphide bonds. One of these loops undergoes alternative splicing. Recent in vitro data indicates that inclusion of this splice site in Ten3 enables homophilic interactions between teneurins expressed on adjacent cells “in trans” (Berns et al., 2018). Consistent with these residues being important for homophilic teneurin interactions in trans, teneurin isoforms including this alternatively spliced region have shown cell–cell adhesive properties (e.g., for Ten2 Beckmann et al., 2013), whilst an isoform lacking these residues did not show any adhesion (Silva et al., 2011; Boucard et al., 2014). Recent reports have also indicated that inclusion of this splice site also abrogates the human teneurin-latrophilin interaction (Li et al., 2018), however, the alternatively spliced chicken Ten2 retains its binding capabilities (Jackson et al., 2018). Thus this area requires further investigation to provide meaningful mechanistic insights across species.

Approximately 500 amino acids link the vertebrate teneurin scaffold region to its single-span transmembrane helix. This region comprises a membrane-proximal region of ∼180 amino acids, eight epidermal growth factor-like (EGF) repeats, a short cysteine-rich motif and a recently identified transthyretin-like (TTR) domain, which is flexibly linked to the central scaffold and packs against the FN-plug domain in the high resolution crystal structure of chicken Ten2, where it is stabilized in a discrete conformation by crystal lattice contacts (Jackson et al., 2018). The TTR domain is not resolved, presumably due to its flexibility, in any of the available structures determined by cryo-electron microscopy (Jackson et al., 2018; Li et al., 2018). The EGF region has not yet been structurally resolved, but bears sequence similarity to the extracellular matrix protein tenascin (Baumgartner et al., 1994; Levine et al., 1994), and has received particular attention due to its ability to dimerise teneurins (Feng et al., 2002). Two of these eight EGF repeats contain only five cysteine residues, compared to the six which would be typical in an EGF repeat. This results in the formation of intermolecular disulphide bridges that presumably form in cis i.e., between teneurin molecules expressed on the same cell. Early rotary shadowing electron microscopy data revealed the overall architecture of these dimers, showing pairs of globular domains (now thought to represent the teneurin scaffold and peripheral TTR, ABD and Tox-GHH), connected by thin elongated rods, presumed to be the EGF repeats (Feng et al., 2002).

Like the NHL domain, the teneurin EGF region also contains an alternatively spliced exon, which lies between the seventh and eighth EGF repeat. Sequencing of mRNA from hippocampal neurons (Berns et al., 2018) revealed three splice variants at this site. Similar to the alternatively spliced site in the NHL domain, inclusion of the alternatively spliced residues of the EGF region enables teneurin homophilic interactions in trans in an in vitro cell aggregation assay (Berns et al., 2018). Future structures of full teneurin ectodomain dimers will likely be valuable in understanding the molecular basis underlying this splicing-dependent homophilic adhesion.

Downstream of the EGF region lies a highly conserved cysteine-rich motif with the consensus sequence ExxCx(D/N)xxDx(D/E)xDxxxDCxxx(D/E)CCxxxxCxxxxxC. The teneurin cysteine-rich motif has not yet been functionally characterized, although bioinformatic analysis has revealed sequence similarity to putative metal-binding motifs in bacterial and algal proteins (Tucker et al., 2012).

As predicted from previously solved structures of YD-repeat-containing proteins (Busby et al., 2013; Meusch et al., 2014), the residues immediately downstream of the Rhs-associated core (residues 2467–2592 in chicken Ten2 and 2382–2507 in murine Ten3) reside inside the shell cavity. These residues form extended loops and short helices, which pack against the Rhs-associated core domain, FN-plug and shell interior, sealing small gaps in the YD-shell. This region has been designated the “internal linker” and has no detectable structural homologs (Figure 3A). The teneurin internal linker is significantly smaller than the proteolytically cleaved C-terminal regions of TcB-TcC complex C-proteins (Figure 3B). In contrast to the teneurin internal linker, the cytotoxic C-terminal regions of bacterial C-proteins are thought to be unfolded when encapsulated within the YD-shell, and fold only upon injection into their insect cell targets (Busby et al., 2013; Meusch et al., 2014;Gatsogiannis et al., 2018).

Strikingly, unlike structures of TcB-TcC toxin complexes, where the entire C-terminal toxic domain is enclosed within the YD-shell, the teneurin C-terminal domain leaves the shell interior via a gap in the shell wall (Figure 3C). These exposed C-terminal residues (∼200 amino acids) form an intricate fold comprising two subdomains, which pack against the shell exterior (Figure 3D). This external C-terminal domain is only fully resolved in the crystal structure of chicken Ten2 and is either completely or partially disordered in both of the teneurin structures solved by single-particle electron microscopy, presumably due to conformational flexibility in this region.

Immediately downstream of the shell exit site, the crystal structure of chicken Ten2 reveals a 120-residue domain (the antibiotic-binding-like domain, ABD), which bears structural homology to a class of bacterial proteins which bind the small-molecule antibiotics bleomycin and zorbamycin (Maruyama et al., 2001; Rudolf et al., 2015). These bacterial proteins confer resistance to the antibiotics by sequestering them, preventing their activation by oxygen (Rudolf et al., 2015). The bleomycin-binding surface of the bacterial protein has been identified crystallographically (Maruyama et al., 2001). The equivalent surface in chicken Ten2 is solvent-exposed, but there are currently no known small molecule ligands for the teneurin ABD.

The ABD wraps around a long helix that leads into the most C-terminal domain of teneurin – the ToxGHH domain. The ToxGHH domain was originally identified bioinformatically and is prevalent amongst bacterial toxins, where it encodes a putative nuclease activity mediated by a HNH catalytic triad (Zhang et al., 2012). The closest structural homolog of the teneurin Tox-GHH domain (residues 2720–2795 in chicken teneurin 2) is the catalytic nuclease domain of a class of bacteriocins known as group A colicins (Figure 3E; Kleanthous et al., 1999). Structural comparison and sequence alignments of teneurin Tox-GHH domains and colicin nuclease domains reveals that teneurins lack both catalytically important histidine residues, and retain only the structurally important asparagine residue (N2790) (Figure 3F; Kleanthous et al., 1999). Recent reports have indicated that, when expressed in isolation, the teneurin C-terminal domain (chicken Ten 1 S2346-R2705 or chicken Ten2 F2407-R2802) is capable of cleaving mitochondrial circular nucleic acids in vitro and inducing apoptosis in vivo. However, in the context of an intact teneurin ectodomain, no nuclease activity was observed (Ferralli et al., 2018). Given the newly available structural data indicating that the teneurin C-terminal domain is solvent-exposed, rather than encapsulated within the YD-shell, future experiments will be necessary to determine how any potential nuclease activity is regulated.

It has previously been shown that the final ∼40 residues of the teneurin Tox-GHH domain bear sequence similarity to pre-pro-hormones and neuropeptide precursors (Qian et al., 2004). This region corresponds to the teneurin C-terminal associated peptide (TCAP), which is thought to function independently of intact teneurins to stimulate neurite outgrowth, regulate dendritic morphology and modulate anxiety behaviors in rats (Qian et al., 2004; Wang et al., 2005; Al Chawaf et al., 2007; Tan et al., 2008). As well as being transcribed independently (Chand et al., 2013), TCAPs may be generated via proteolytic cleavage from full-length teneurins (Lovejoy et al., 2006). The predicted cleavage site (based on alignment with known neuropeptides) (Lovejoy et al., 2006) is located within the second helix of the Tox-GHH domain, suggesting that the protein may unfold in this region to become accessible to proteases (Figure 3E). Recent evidence also suggests that the C-terminus of teneurin is required for binding to the adhesion-G-protein-coupled receptor latrophilin (Lphn) (Silva et al., 2011; Li et al., 2018) as removal of 390 residues from the C-terminus of human Ten2 abolished Lphn binding (Li et al., 2018). The precise nature and stoichiometry of the Lphn-Ten interaction is currently unknown.

A bioinfomatic approach using a wide range of fully sequenced genomes identified teneurins in chordate genomes (e.g., mouse, chicken and zebrafish), and also in the protochordates Ciona intestinalis and Branchiostoma floridae (Tucker et al., 2012). Teneurin genes were also identified in molluscs, annelids, trematodes, nematodes and arthropods. No teneurin sequences could be identified in cnidarians, ctenophores or sponges, indicating either that they are not present in these deeper-branching eukaryotic clades, or that they have highly divergent sequences (Tucker et al., 2012). However, a single teneurin gene in the genome of the choanoflagellate Monosiga brevicollis was discovered and shown to have only three introns. This gene shows strong sequence similarity to some bacterial teneurin-like proteins (with strongest similarity to the teneurin-like protein from B. subtilis; 69% coverage, 30% identity), albeit having a divergent intracellular domain, providing evidence that the gene could have been acquired by M. brevicollis from its prokaryotic prey via a horizontal gene transfer event (Tucker et al., 2012), as observed for other proteins (Foerstner et al., 2008). As choanoflagellates are considered to be the closest living relatives of metazoans, the authors suggest that teneurins may have played a key role in the evolution of multicellular metazoa from their unicellular choanoflagellate ancestors. Further testing of this hypothesis awaits a careful examination of recently sequenced genomes in the light of the recently determined structures described above.

Recent breakthroughs in understanding the first structures of teneurin protein domains have revealed unexpected new insights into this enigmatic family of proteins. First, the structures show that the C-terminal Tox-GHH/TCAP domains are solvent-exposed and therefore will be accessible to external ligands, proteases and other extracellular factors. Second, teneurin family proteins contain a previously unknown type of fibronectin domain (FN-plug) that specifically acts to seal off the YD-shell. Third, understanding the fold of the central teneurin scaffold led to the discovery of a whole family of teneurin-like proteins in prokaryotes, suggesting evolution from an ancient and widespread fold. Future research on these bacterial proteins, comparing their structure-function relationship with teneurins, will likely reveal more unexpected aspects of teneurin biology. Last but not least, the elaborate structure of the teneurin extracellular C-terminus is unlike that of any other cell adhesion receptor, most of which consist of smaller adhesion domain repeats. The intricate nature of this fold, and the presumably large energetic cost of synthesizing and correctly folding these large proteins, suggests that teneurins may encode functions beyond the role of a canonical cell adhesion receptor that are yet to be explored.

All authors contributed to the conception and writing of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.