Introduction

Cellular signaling via proteins that possess calcium-binding domains is fundamental for multiple processes, such as endocytosis, exocytosis, vesicular trafficking, and phospholipase activities (Corbalan-Garcia and Gómez-Fernández 2014). The C2 domain is present in many proteins involved in neurotransmission, such as synaptotagmins (Bagur and Hajnóczky 2017; Evans et al. 2016; Nalefski and Falke 1996; Shin et al. 2005). This domain was initially described as the second of four calcium-binding domains of the calcium-dependent protein kinase C (PKC) (Nalefski and Falke 1996; Nishizuka 1988). Molecular and functional studies have elucidated the role of proteins that possess C2 domains in different signaling cascades; however, questions remain about their structure and role in cell physiology. C2 proteins involved in membrane trafficking, such as synaptotagmins and ferlins, contain a transmembrane region and at least two C2 domains whose affinity for Ca2+ increases upon binding phospholipids (Rizo and Sudhof 1998).

A novel family of C2 proteins known as MCTPs was discovered by in silico genomic sequence analyses (Shin et al. 2005). They contain three C2 domains (C2A, C2B, and C2C) oriented towards the cytoplasm: one to two transmembrane regions that anchor the protein to intracellular membranes and one short C-terminal sequence. Mammals have two MCTP coding genes (MCTP1 and MCTP2), while invertebrates, such as Drosophila melanogaster and Caenorhabditis elegans, have only one MCTP gene (Shin et al. 2005). The cellular distribution of MCTPs has been reported in endosomes and the endoplasmic reticulum, which enables them to sense changes in Ca2+ concentration (Shin et al. 2005; Genç et al. 2017; Espino-Saldaña et al. 2020) and promote lipid droplet biogenesis (Joshi et al. 2018, 2021). Assessment with selective antibodies for each isoform has demonstrated that MCTPs are widely expressed in different tissues, especially in striated muscle and heart (Qiu et al. 2015; Shin et al. 2005).

MCTPs seem to be implicated in heart and brain disorders. For example, a deletion of 2.2 Mb in the 15q26.2 chromosome, where the MCTP2 gene localizes, causes coarctation of the aorta and hypoplastic heart syndrome (Lalani et al. 2013). Furthermore, the importance of the MCTP2 gene for proper cardiogenesis in frogs was tested by knocking down its gene expression with morpholinos, which yielded embryos with a heart condition resembling coarctation of the aorta (Lalani et al. 2013). In other studies, single-nucleotide polymorphisms in the MCTP2 gene were found to be associated with schizophrenia and bipolar disorders; however, their role in these psychiatric diseases remains elusive (Djurovic et al. 2009).

Studies of MCTP function have also been carried out in invertebrates. Only one gene has been identified in the genome of D. melanogaster, and a gene-trap study showed that it is expressed in the accessory cells of the olfactory organ. A P-transposon insertion at the 3'-end of the non-coding region of the MCTP gene is extremely lethal to the fly (Tunstall et al. 2012), while other allelic mutants of the MCTP gene are viable but defective in the presynaptic homeostatic plasticity in motor neurons (Genç et al. 2017). In C. elegans, mctp-1 is regulated by two alternative promoters in the nervous system and spermatheca and gives rise to four alternatively spliced isoforms (Téllez-Arreola et al. 2020). In addition, mctp-1 mutants exhibited impaired sensory function, locomotion, and hyposensitivity to an acetylcholinesterase blocker, suggesting that MCTP-1 is required for neurotransmitter release in the worm (Téllez-Arreola et al. 2020).

While it has been proposed that MCTPs can modulate the neuronal activity in invertebrates and that mutations in the mctp genes are involved in human disease, evidence of such roles has only recently emerged (Genç, et al. 2017, Téllez-Arreola et al. 2020, Liu et al. 2021). To shed some light on the molecular structure and phylogenetic distribution of this protein family, in this study, we present a phylogenetic analysis and a computational structural modeling and docking analysis of the C2 domains of the C. elegans MCTP-1 protein, in an effort to understand the structure of this emerging family of calcium sensors.

Materials and Methods

Mapping MCTP Relatives and Their Orthologs

All sequences for MCTP proteins were found using SMARTBLAST and Orthofinder (https://github.com/davidemms/OrthoFinder) (Emms and Kelly 2019; Schultz et al. 1998). To map the MCTP relatives, we employed the https://shoot.bio/ platform, and all domains in life databases were selected; then, after identifying the close relatives, we added two nodes. Finally, we used the C. elegans MCTP protein sequence (NP_491908.2) as a template in all cases.

Multiple Sequence Alignment and Phylogenetic Analysis

A circular phylogenetic tree connecting MCTPs with other C2 domain functional protein families was constructed using the https://shoot.bio/ platform, then imported into Newick format and parsed to Rstudio using the ggtree package (Yu 2020). The C2 domain phylogenetic tree was generated using the http://www.phylogeny.fr/index.cgi platform. “A la carte” option was used. “MUSCLE” for alignments, Gblocks default settings for curation, and maximum likelihood method with bootstrapping (100 replicates) were selected (Dereeper et al. 2008). Jalview was used to color sequence alignments derived from MUSCLE results (Waterhouse et al. 2009).

MCTP Computational Secondary Structure Prediction, Structural Modeling, and Docking Analysis

We used PSIPRED software for secondary structure prediction and RaptorX Structure Prediction software to model the tertiary structure of MCTP from C. elegans (NP_491908.2) using the default parameters of the server (Jones 1999; Källberg et al. 2012). The automatically generated PDB model was manually cropped in Molsoft ICM-Pro v3.8–3, keeping all the predicted domains in a standard configuration. Three Ca2+ ions were loaded from ChemSpider into the ICM project (CSID: 4,573,905) and fitted into the MCTP C2A domain, using the structure of the C2A domain of PKC as a comparative guide (PDB: 1DSY, Verdaguer et al. 1999) and employing the Pocket Finder function of ICM (Dey and Chen 2011). The final model was submitted to the Yasara energy minimization server (Krieger et al. 2009), and images were generated in PyMOL v2.2.2 Schrodinger LLC (Schrödinger, San Diego, CA).

Results

MCTPs Were Found in Metazoan But Not in Unicellular Eukaryotes

The emergence of MCTP proteins is poorly understood; thus, we first used the C. elegans (NP_491908.2) protein sequence as a query to search for putative MCTPs and analyzed their evolutionary relationship with other C2 domain proteins (Fig. 1). We found that MCTPs belong to a group of PKC-C2 domain families, the closest relatives of which are PCKs, Need4s, Ferlins, and Copines, all considered Ca2+ sensors (Zhang and Aravind 2010) (Fig. 1). We also found that MCTPs are widespread in metazoans, from cnidarians to humans. We did not detect MCTPs in prokaryotic organisms or unicellular eukaryotes. The dendrogram analysis shows the MCTP protein family's divergence into three major classes: MCTP1 and MCTP2 were present in chordates, whereas MCTPi was found only in invertebrates (Fig. 1; see representative species in Table 1). Taking advantage of the 2021 ensemble.org database, we searched for the gene tree of MCTPs. We found gene duplication events in vertebrates for both the MCTP1 and MCTP2 genes (Fig. S1A). MCTP1 is duplicated in perching birds and fish, whereas MCTP2 is duplicated only in fish (Fig. S1A-B). We did not find duplication events for MCTPi (Fig. S1C).

Fig. 1
figure 1

Phylogenetic relationship between MCTPs and other C2 domain functional protein families. The platform https://shoot.bio/ was employed to identify MCTP orthologs and generate an unrooted Maximum likelihood tree. The orange group represents MCTPi, the blue group represents MCTP1, and the green group represents MCTP2. As stated earlier, MCTPi is specific to invertebrates, while MCTP1 and MCTP2 are found in vertebrates. Other members of the PCK-C2 family group are PKC, cPLA, RIM, Need4, Copine, Ras, Doc2, and Ferlins. Abbreviations: PKC-C2, Protein kinase C family; Slps, synaptotagmin-Like Protein; RIM, Rab3-interacting molecule; DOC2, double C2-like domain-containing protein; cPLA, cytosolic phospholipase; Need, neural precursor cell expressed developmentally down-regulated protein 4. UniProt reference proteomes 2020 and https://ensembl.org databases were used for the analysis. See supporting material for bootstrapping values. (Color figure online)

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Table 1 Representative MCTPs orthologs found in https://shoot.bio/
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Calcium-Binding Residues Are Conserved in C2A and C2C Domains But Not in C2B

The classical C2 domain calcium sensors contain five aspartic acid residues that bind Ca2+. As previously described, MCTP proteins possess three cytosolic C2 domains in tandem (Fig. 2A). First, taking advantage of C2 domain similarity across species, we analyzed the amino acid sequences corresponding to individual C2 domains (C2A, C2B, and C2C) from the different classes of MCTPs from animal models to determine their degree of conservation, including the calcium-binding pocket (Fig. 2B). A comparison of the C2 domains revealed a high degree of conservation in the primary sequence: the C2C domain is the most conserved, followed by C2A and, finally, C2B. The calcium-binding sites are more conserved in C2A and C2C than C2B (Fig. 2B). C2C calcium-binding sites have not been previously reported in other proteins; thus, it may have a specialized function in MCTPs. Calcium-binding residues in the top loop region of C2 domains interconnect β-sheets, leading these domains to interact with Ca2+ and membranes. The aspartate residues of C2 domains in MCTPs may bind Ca2+ (Shin et al. 2005). We found that five of these residues are partially conserved in the three C2 domains of MCTPs. Aspartate residues 1 through 4 are conserved in the C2C domain, whereas the fifth presents the substitution D → E in invertebrates (Fig. 2B). C2B has several amino acid substitutions, including D → A, E, H, N, or T (Fig. 2B). Other MCTPi and MCTP2 C2Bs from C. elegans, Haemonchus contortus, Xenopus laevis, Xenopus tropicalis, and some isoforms from humans and mice contain only one or two aspartate residues, suggesting that these C2Bs may not bind Ca2+ (Fig. 2B). The position of amino acid residues predicted correctly that calcium-binding sites are well conserved in C2A and C2C in all MCTPs analyzed, but remarkably, such is not the case for C2B.

Fig. 2
figure 2

Multiple sequence alignment of MCTP C2 domains from different biological model organisms reveals the presence of calcium- and lipid-binding motifs. A General diagram of MCTPs using the consensus sequence generated from sequence alignment of the model species. Three C2 domains followed by two transmembrane propellers at the C-terminus anchor the protein to the membrane. The putative calcium-binding sites are located in C2A and C2C, and the lipid pocket (polybasic motif) is shown in C2A and C2B. B MCTP C2 domain multiple sequence alignment of model organisms. The alignment was colored in JAlVIEW. Secondary structure prediction was performed by PSIPRED. Abbreviations: Cele., C. elegans; Dm, D. melanogaster; HC, Haemonchus contortus; Mm, Mus musculus; Dr, Danio rerio; Xl, Xenopus laevis; Xt, Xenopus tropicalis; Hs, Homo sapiens; PKCα, protein kinase C- alpha

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Sequence Analysis Reveals a Conserved Lipid-Binding Motif in MCTP C2A and C2B Domains

The lipid-binding selectivity of C2 domains is dependent on the electrostatic charges formed by the calcium-binding region (Corbalán-García et al. 2003). The ability to bind lipids independently of the calcium-binding sites has been described in C2 domains of PKCs (Zhang and Aravind 2010). Hence, we performed a systematic conservation sequence analysis of lipid-binding sites of MCTP C2 domains.

We found that the C2A and C2B domains of MCTPs possess a lysine-rich motif, termed the polybasic cluster, between β-sheets three and four (Corbalan-Garcia and Gómez-Fernández 2014; Di Paolo and De Camilli 2006). This polybasic motif is formed by positively charged lysines (K, K, K) flanked by aromatic residues (Y, W) (Figs. 2B, 3) (Guerrero-Valero et al. 2009). The polybasic cluster can bind phosphatidylserine and phosphatidylinositol 4,5-bisphosphate (PIP2) to calcium-binding domains, such as the C2B domain of synaptotagmin proteins (Honigmann et al. 2013). In MCTPs, the lysine-rich motif is found in the C2A and C2B domains. This creates room for a new working hypothesis that phospholipids may be necessary for binding Ca2+, which contrasts with the initial observation that MCTP function is independent of lipid interactions (Shin et al. 2005). Altogether, we found at least two putative PIP2-binding sites that resemble other C2 domains known to penetrate the plasma membrane; these PIP2-binding sites coordinate with the aspartates in C2A, C2B, and C2C and may stabilize Ca2+ ions (Fig. 3).

Fig. 3
figure 3

Model for Ca2+ and PIP2 docking of the MCTP C2 domains. Docking of three C.elegans MCTP1 C2 domains: C2A (brown), C2B (green), and C2C (blue) with Ca2+ and PIP2. The closeup images at the top show the aspartate region (red lines) binding Ca2+ (gray spheres). The closeup images at the bottom show the predicted polybasic motifs (yellow) and their interaction with PIP2 (red). Dashed lines represent hydrogen bridges formed by interactions with the phosphate groups of PIP2 (Color figure online)

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MCTPs Have Two Putative Transmembrane Regions at the C-Terminus

One criterion to establish whether a protein belongs to MCTPs is the presence of two stretches of hydrophobic residues that form the transmembrane regions (TMR), which allows the protein to anchor to cell vesicles (Shin et al. 2005). The presence of transmembrane regions in MCTPs is still debated. Some studies in mammals suggest that this is the only family of calcium sensors to contain two TMRs (Shin et al. 2005), while others report that the C-terminus of MCTP2 is a reticulon domain (ER-shaping RHE) (Joshi et al. 2018). To provide new insights, we analyzed the C-termini of MCTPs from different species. Based on Kyte and Doolittle (not shown) and secondary structure prediction analysis, we found that most MCTPs have two predicted segments, with the exception of the human protein MCTP2–2, which lacks the first TMR due to an alternative splicing event (Fig. 4). It is unknown whether a lack of one TMR misdirects the protein; a single TMR seems to suffice to anchor the protein to intracellular vesicles (Shin et al. 2005). The amino acid sequence of MCTP TMRs is not related to other known C2 proteins involved in membrane trafficking, such as synaptotagmins. In MCTPs, the second TMR is more conserved (70%) in different species, whereas the first TMR sequence is less conserved (10%). Our results are in line with the observation that MCTPs have two TMRs (Shin et al. 2005); however, we do not discard the possibility that the isoforms lacking the TMR1 might be functionally distinct.

Fig. 4
figure 4

Multiple sequence alignment of the putative transmembrane helix. Sequence alignment of putative transmembrane regions. Green rectangles at the top represent the transmembrane 1 and 2 helices. The degree of conservation of the sequence and the consensus of each segment are indicated. The secondary structure was modeled using PSIPRED. Cele., C. elegans; Dm, D. melanogaster; HC, Haemonchus contortus; Mm, Mus musculus; Dr, Danio rerio; Xl, Xenopus laevis; Xt, Xenopus tropicalis; Hs, Homo sapiens; SYT-1, synaptotagmin 1 (Color figure online)

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The Polybasic Motif Is a Fingerprint Among MCTP C2 Domains

C2 domains are detectors which integrate responses in the presence of Ca2+ and lipids (Corbalan-Garcia and Gómez-Fernández 2014; Téllez-Arreola et al. 2020). A wide array of proteins has been identified to contain functional domains that bind Ca2+ and lipids; some participate in membrane trafficking, others in signal transduction (Corbalan-Garcia and Gómez-Fernández 2014; Min et al. 2007). However, the well-conserved amino acid sequences that bind Ca2+ and lipids seem to be crucial elements among MCTP C2 domains. Thus, we investigated whether these sequences are essential to define the C2 domain class. First, we determined whether the amino acid sequence of C2 domains of MCTPs varies among species. These domains span approximately 125 residues with predicted secondary structures suggesting they fold into eight β-strands. This is a characteristic of the C2 domain conformation Class 2 (type II topology) in which the N- and C-termini are near the bottom (Corbalan-Garcia and Gómez-Fernández 2014). Next, amino acid sequences of all three C2 domains from representative species of each clade were used to generate maximum likelihood trees. We observed two general clades: the first clade includes the C2C domain from all species (C. elegans, H. contortus, C. intestinalis, X. laevis, X. tropicalis, D. rerio, M. musculus, and H. sapiens). The second clade includes C2A and C2B, which suggests they may have similar functions as indicated by their primary amino acid sequence and computational analysis. It is worth noting that both C2As and C2Bs possess the lipid-binding residues included within the lysine-rich domain identified by our analysis; in sharp contrast, C2Cs possess calcium-binding sites instead of the lysine-rich cluster, a structural feature which suggests different Ca2+ sensitivities and membrane bridging (Fig. 5).

Fig. 5
figure 5

Maximum likelihood tree of MCTP C2 domain sequences from different biological models. 100 replicates were used for bootstrapping. Bootstrapping values along branches are denoted in red; domains with putative calcium-binding abilities are colored in green and blue. The scale bar represents the tree branch length (Color figure online)

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Discussion

In the present study, we provide new insights into the structure and molecular function of the MCTP proteins. MCTPs are composed of a variable N-terminal region, three C2 domains, one or two TMRs, and one short C-terminal sequence (Shin et al. 2005). We used this approach to determine peculiarities in the amino acid sequence of MCTPs to formulate a working hypothesis to understand their functional role. MCTPs belong to the PCK-C2 family of proteins; all members within this family are considered calcium sensors (Zhang and Aravind 2010). Calcium sensor proteins are central molecular components of cell biology; they appeared early in the evolution of eukaryotes, and there is evidence of their presence in more primitive placozoans (Barber et al. 2009; Lek et al. 2012; Washington and Ward 2006). Our results indicate that MCTPs were not present in early eukaryotes distributed widely across this phylum; thus, it may be inferred that MCTPs emerged as an adaptation in metazoans. We identified three subgroups of MCTPs: MCTP1 and MCTP2 are present in vertebrates, whereas MCTPi is observed only in invertebrates. While MCTPs have been documented in plants, there are remarkable structural differences between MCTPs belonging to plants and animals, such as the number of C2 domains. In both cases, however, these MCTPs belong to the KG009 group according to the eggNOG database (see supporting material).

The number of C2 domains in a protein may indicate a wide range of functions, from vesicular transport to signal transduction. Proteins with multiple C2 domains in tandem act primarily as membrane trafficking effectors, while proteins with only one C2 domain are involved in signal transduction. Such is the case for synaptotagmins and PKCs, two of the most well-known proteins to contain C2 domains (Corbalan-Garcia and Gómez-Fernández 2014).

The sensitivity of C2 domains for binding Ca2+ and lipids and/or sensing protein interactions is influenced by several factors: (a) the orientation of the C2 domains, (b) the degree of conservation of residues involved in Ca2+ binding, and (c) the presence of a polybasic cluster. In some proteins, such as synaptotagmin 1, C2A and C2B bind two or three molecules of Ca2+ with low affinity, an interaction which is strengthened in the presence of phospholipids (Bai et al. 2002; Evans et al. 2016; Honigmann et al. 2013; Kojima 1995; Rizo and Sudhof 1998; Sutton et al. 1995). In other cases, such as with synaptotagmin 4, substitutions within the C2A calcium-binding sites impact calcium affinity, whereas substitutions within the C2B domain affect its conformation such that the domain is unable to properly form calcium pockets. However, the latter is not the case for the homologous protein syt4 in D. melanogaster, whose function relies on binding calcium (Barber et al. 2009; Dai et al. 2004).

The diversity of C2 domains found in MCTPs suggests that they play a role in both calcium- and lipid-binding responses in different scenarios. The secondary structure of the three MCTP C2 domains is highly conserved in all selected organisms. Our results are consistent with those reported by Shin et al. (2005), who showed that several organisms lack the calcium-binding aspartates in the C2B domain. Interestingly, we also found that C. elegans, H. contortus, X. laevis, X. tropicalis, M. musculus, and H. sapiens lack calcium-binding sites in the C2B domain, suggesting that this domain does not bind Ca2+ in the species listed above. However, we do not discard the possibility of a lower binding affinity to Ca2+ as displayed by the C2A synaptotagmin 4 from rats, which despite lacking aspartate residues, demonstrates a lower affinity for Ca2+ (Barber et al. 2009; Dai et al. 2004). Another possibility is that the MCTP C2B domain resembles the RIM C2B domain, whose function relies significantly on PIP2 binding and protein interactions in lieu of binding Ca2+ (Betz et al. 2001; de Jong et al. 2018; Guan et al. 2007; Hu et al. 2013; Wang et al. 1997). Our results indicate that the C2A and C2B domains of MCTP contain a well-conserved polybasic cluster, and that these motifs may function similarly to the synaptotagmin 1 C2 domains, which extend to the plasma membrane by interacting with PIP2. This orientation increases the sensitivity of C2A to Ca2+ while facilitating the role of C2B in protein interactions located within PIP-enriched zones. We also do not discard other possible molecular mechanisms, such as one resembling the C2A and C2B C2 domains of Rabphilin, both of which exhibit Ca2+-binding activity facilitated by the interaction of PIP2; however, they interact differently with other phosphoinositides (Guillen et al. 2013).

Recently, it was discovered that MCTP2 binds in vitro to the charged lipids PI4P; PI4,5P2; PI3,4,5P3; and cardiolipin (Joshi et al. 2021), which supports our observations of lipid-binding in silico; however, the molecular affinity for lipids and responses of each MCTP C2 domain in different biological scenarios are still poorly understood, and further experiments are needed to determine their precise role. The presence of three C2 domains (A-C) opens the door to a variety of scenarios in which MCTP could play a role in membrane biology. For example, the C2C domain of MCTP is similar to the C2C domain in E-Syts and Munc13-1 in that it is an essential component of calcium-independent membrane tethering (Min et al. 2007; Quade et al. 2019). In E-Syts, C2C plays a critical role in ER-PM tethering in E-Syts, while in Munc13-1, it is essential for liposome fusion (Quade et al. 2019). Our findings indicate that the C2C domain of MCTP could be a singular case of calcium-binding pockets, since calcium-binding residues are highly conserved across diverse species. This suggests that the C2C domain is more sensitive to Ca2+ in the context of membrane binding. Even though we did not find a polybasic cluster within the structure of the C2C domain, we do not discard a possible lipid association resembling that of the C2C domain in otoferlins (Padmanarayana et al. 2014).

E-Syts and ferlins are two other families of proteins which, like MCTPs, are calcium sensors and have been found to contain more than three C2 domains in tandem. Otoferlin plays an essential role in the exocytosis of neurotransmitters at the ribbon synapse, whereas E-syts function as tethers in ER-plasma membrane contact sites (Giordano et al. 2013; Herdman and Moss 2016; Saheki et al. 2016). In invertebrates such as C. elegans and D. melanogaster, it has been demonstrated that mctp-null mutants display defective neurotransmitter release (Genç et al. 2017; Téllez-Arreola et al. 2020), whereas in plants, MCTPs play a role as tethers in ER-PM contact sites (Brault et al. 2019). The functional differences between MCTP subgroups are not well defined, although they have similar calcium-binding properties and may play similar or complementary roles to isoforms with two TMRs. It is not known which differences are essential for distinguishing between various classes of MCTPs. The N terminus might be related to the specialized function of the C2 domains of each isoform, as is the case for ferlins and E-syts (Barber et al. 2009; Washington and Ward 2006). It is already known that MCTPs are calcium sensors which contain three C2 domains in tandem with two transmembrane regions. We propose additionally that MCTPs are evolutionarily conserved across multicellular organisms but not in prokaryotic or unicellular eukaryotic cells, and that MCTPs may be sensitive to calcium- and/or lipid-binding events due to the functional properties exhibited by their three C2 domains. For example, the MCTP C2C domain may bind Ca2+ and membrane lipids in response to changes in Ca2+ concentration at the synapse, thus conferring MCTP proteins a unique calcium- and lipid-binding property. Considering the wide variety of scenarios in which MCTPs can bind Ca2+, lipids, or membranes, further experimentation is required to assess the selectivity of the C2 domains and to test whether MCTPs rely on specific membrane interactions to play essential roles in endocytic and exocytic pathways.