The secondary structure conformations adopted by the calcitonin peptide naturally influence its biomolecular interactions, such as receptor binding and self-aggregation. As an amphiphilic and conformationally flexible molecule, with a zero net charge at neutral pH and a hydrophilic central segment flanked by hydrophobic C- and N-terminal regions (Fig. 1), monomeric calcitonin often adopts a random coil structure in aqueous solution (Wüthrich, 1976; Arvinte et al., 1993; Du et al., 2002). The N-terminal region may however sometimes also adopt a β-sheet conformation, or, at acidic pH, an α-helical structure stabilized bv the N-terminal S-S bridge (Kamihira et al., 2000). The C-terminal region may adopt a β-sheet conformation at low pH, instead of the random coil structure typically observed at neutral pH (Kamihira et al., 2000). Human calcitonin monomers can furthermore adopt α-helical structures in non-aqueous solutions containing e.g. trifluoroethanol (TFE) or methanol (Arvinte and Drake, 1993; Siligardi et al., 1994). Salmon calcitonin, which differs from human calcitonin in 16 out of 32 amino acids, has a less hydrophobic C-terminal region, a higher propensity for α-helical secondary structure, and is less prone to aggregate (Motta et al., 1991a; Arvinte and Drake, 1993; Du et al., 2002). In 90% dimethyl sulfoxide (DMSO) with 10% H2O, monomers of both human and salmon calcitonin were reported to adopt an intramolecular β-sheet hairpin structure (Motta et al., 1989; Motta et al., 1991b), similar to the conformation identified as important for aggregation of Aβ peptides (Abelein et al., 2014).
Given the capacity of calcitonin to form cell-toxic amyloid aggregates (Belfiore et al., 2019), for example when over-produced as a result of e.g. thyroid cancer (Gambardella et al., 2019), the aggregation properties of the peptide are of particular importance (Renawala et al., 2021). In general, clarifying the structure and toxic mechanisms of oligomers of various peptides and proteins remains an important research task (Yang et al., 2021). While most polypeptides adopt β-sheet structure during amyloid aggregation (Abelein et al., 2014), sometimes amyloid can form via aggregation of α-helical structures (Jayaraman et al., 2012; Mitra and Paul, 2023).
Earlier work has shown that calcitonin can adopt different structures when aggregating. For example, aggregates with a combination of both α-helix and β-sheet structure have been observed (Arvinte et al., 1993; Du et al., 2002). An aggregation mechanism was suggested where the C-terminal 23–32 residues first form intermolecular β-sheets, which then act as a template for aggregation of α-helical rods comprising residues 8–22 (Arvinte et al., 1993). Another study found that human calcitonin aggregated into a mixture of antiparallel and parallel β-sheets at pH 3.3, but into a mixture of antiparallel β-sheets and random coil structure at pH 7.5 (Kamihira et al., 2000). Furthermore, an extended version of human calcitonin with an additional N-terminal Leu residue and a C-terminal Gly residue was found to mainly aggregate into β-sheet conformation (Du et al., 2002).
Here, we report that human calcitonin forms β-sheet structure when aggregating in phosphate buffer at pH 7.3 (Figs. 3C and 4). Earlier studies have shown that calcitonin aggregation is promoted by various factors, such as increased peptide concentration and temperature (Arvinte et al., 1993), and also by reducing the N-terminal S-S bridge which otherwise stabilizes the N-terminal region (Kamihira et al., 2000; Renawala et al., 2021). Our current results confirm that calcitonin aggregates faster at higher temperatures (Fig. 4), They also demonstrate the importance of agitation for the aggregation process, as aggregation was only observed in the samples that were magnetically stirred (Fig. 3).
In the samples that were not magnetically stirred, gradually increasing the temperature from 5 oC to 55 oC induced a structural conversion involving loss of PPII-helix and likely formation of β-sheet conformation (Figs. 3A and 3D). This structural change was found to be completely reversible when the temperature was slowly returned to 5 oC (Fig. 3B). The presence of PPII-helix in the monomer structure is noteworthy. Human calcitonin has previously been shown to adopt a PPII-helix structure at -85°C, in the cryogenic solvent ethanediol/water (2:l ratio), while salmon calcitonin under those conditions adopted an α-helical structure (Arvinte and Drake, 1993). Here, we observe a linear dependence of the amount of PPII-helix structure with temperature, and demonstrate that almost 50% PPII-helix component is present in human calcitonin at physiological temperatures (Fig. 3D). This is in line with earlier studies showing that short peptides often display PPII-helix conformations (Adzhubei et al., 2013; Narwani et al., 2017). For example, the full-length Aβ(1–40) was found to adopt around 45% PPII-helix at 0°C in 10 mM sodium phosphate buffer at pH 7.4, while the shorter Aβ(1–9) fragment displayed almost 60% PPI-helix under the same conditions (Danielsson et al., 2005). The presence of PPII-helix in calcitonin could be relevant for calcitonin aggregation, as the PPII-helix appears to be less prone to form amyloid fibrils (Danielsson et al., 2005). But the PPII structure might also be relevant for calcitonin receptor interactions.
Interestingly, there appears to exist different variants of calcitonin receptors, with different affinities for helical and nonhelical calcitonin conformations (Nakamuta et al., 1990; Nakamuta et al., 1991; Siligardi et al., 1994; Wu et al., 2002). Experiments on rats showed that calcitonin analogs that do not easily adopt α-helical structure selectively recognized calcitonin receptors in bone tissue, whereas α-helical calcitonin analogs appeared to display a preference for receptors in kidney (Nakamuta et al., 1990). As the calcitonin receptor is a membrane protein, it becomes relevant to study calcitonin in membrane-like environments (Motta et al., 1991a; Arvinte and Drake, 1993; Siligardi et al., 1994; Belfiore et al., 2019). Membrane interactions, including membrane disruption, could also be involved in the toxic mechanisms of oligomeric aggregates of calcitonin and other amyloidic proteins and peptides (Belfiore et al., 2019; Wärmländer et al., 2019).
Micelle-forming surfactants are simple membrane models that can be used to investigate basic properties of membrane-peptide interactions, including structure induction (Rocha et al., 2012; Österlund et al., 2018; Österlund et al., 2019). In this study, we show that human calcitonin in phosphate buffer adopts a combination of α-helix and β-sheet secondary structure in the presence of micelles of anionic SDS (Fig. 5A) and zwitterionic SB3-14 (Fig. 5C) surfactants. It has previously been shown that salmon calcitonin in SDS micelles displays overall α-helix confirmation (Motta et al., 1991a; Siligardi et al., 1994). For human calcitonin in SDS, one study has reported generally similar CD spectra as those presented in Fig. 5A, which then were interpreted as reflecting only small amounts (< 15%) of α-helix structure (Siligardi et al., 1994). This is compatible with the current interpretation of the spectrum as a combination of α-helix and β-sheet structure (Fig. 5A). The current results therefore confirm previous reports that human calcitonin is less prone to adopt α-helical structure than salmon calcitonin (Motta et al., 1991a; Siligardi et al., 1994). Here, pure α-helical structure is only observed for human calcitonin monomers in the presence of micelles of the cationic CTAB surfactant (Fig. 5B).
The CD spectra for calcitonin in CTAB are somewhat different in oxidizing and reducing conditions (Figs. 5B and 6B), which confirms earlier reports that the calcitonin S-S bridge (Fig. 1) is important for N-terminal α-helix structure, i.e. by stabilizing it (Kamihira et al., 2000; Renawala et al., 2021). In the presence of non-charged DDM micelles, only minor structural changes are observed, presumably towards more random coil structure. Taken together, these results show that headgroup charge is an important parameter for surfactant/calcitonin interactions. As biological membranes consist of a multitude of lipids with different properties, including differences in charge (Vorobyov and Allen, 2011; Kitamata et al., 2020; Wang and Majd, 2023), the results presented here are likely important for understanding biological calcitonin-membrane interactions.