Mobility capillary electrophoresis–native mass spectrometry reveals the dynamic conformational equilibrium of calmodulin and its complexes

Abstract

Benefitting from the rapid evolution of artificial intelligence and structural biology, an expanding collection of high-resolution protein structures has greatly improved our understanding of protein functions. Yet, proteins are inherently flexible, and these static structures can only offer limited snapshots of their true dynamic nature. The conformational and functional changes of calmodulin (CaM) induced by Ca²⁺ binding have always been a focus of research. In this study, the conformational dynamics of CaM and its complexes were investigated using a mobility capillary electrophoresis (MCE) and native mass spectrometry (native MS) based method. By analyzing the ellipsoidal geometries of CaM in the solution phase at different Ca²⁺ concentrations, it is interesting to discover that CaM molecules, whether bound to Ca²⁺ or not, possess both closed and open conformations. Moreover, each individual CaM molecule actively “jumps” (equilibrium exchange) between these two distinct conformations on a timescale ranging from milli- to micro-seconds. The binding of Ca²⁺ ions did not affect the structural dynamics of CaM, while the binding of a peptide ligand would stabilize CaM, leading to the observation of a single, compact conformation of the resulting protein complex. A target recognition mechanism was also proposed based on these new findings, suggesting that CaM's interaction with targets may favor a conformational selection model. This enriches our understanding of the binding principles between CaM and its numerous targets.

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Introduction

CaM interacts and modulates the function of hundreds of effector proteins in a Ca²⁺-dependent manner. This unique ability allows CaM to act as a Ca²⁺ sensor, modulating the activity of various proteins involved in processes such as neurotransmitter release, muscle contraction, cell differentiation, apoptosis, and gene transcription.^1–6 It is well known that CaM has two globular domains connected by a flexible linker. Each domain comprises a pair of EF hand motifs that could bind with two Ca²⁺.^7–10 CaM is conventionally believed to adopt different conformations upon binding with Ca²⁺, which then enables further interaction with target proteins. Extensive research studies have been conducted to investigate the relationship between the structure and function of CaM, and a multitude of CaM structures and their associated complexes have been documented in the Protein Data Bank (PDB). Based on these results, it is generally believed that both Ca²⁺-free and Ca²⁺-bound CaM have open conformations, where the N- and C-terminal lobes of CaM are separated by a central linker^8,11–13; besides forming hydrophobic pockets within each lobe, the binding of 4Ca²⁺ would reorganize the intrinsically disordered central linker into an alpha helix^14–16; this central linker transitions back to an intrinsically disordered region (IDR) upon binding to targets;^3,17,18 both open and closed conformations have been observed for CaM within complexes, and all the closed conformations are associated with the addition of 4Ca²⁺.^19–23

Nonetheless, the structure of proteins is not static and can exhibit variability across multiple levels, encompassing local fluctuations to significant conformational changes,^24,25 particularly in the case of intrinsically disordered proteins (IDPs) or proteins featuring IDRs. Ca²⁺ binding and target recognition depend heavily on the structural dynamics of CaM. Techniques, including time-resolved small-angle X-ray scattering,^4,26,27 nuclear magnetic resonance (NMR) relaxation methods,^28–30 single molecule fluorescence resonance energy transfer (FRET)^31,32 and cross-linking mass spectrometry,^33,34 have been applied to explore the structural dynamics of CaM. The intermediate conformational state and a wide range of interdomain distances were sampled. However, these techniques can only provide static structures of CaM. Therefore, this has led to the proposal that the binding of either Ca²⁺ or a ligand may shift a pre-existing structural equilibrium of CaM, instead of following a stepwise structure evolution.²⁵ However, the precise characteristics of protein structural dynamics, including those of CaM, continue to be a complex and actively researched subject, sparking ongoing debates and discussions.

Many new methods have been used to study the dynamics of proteins.^35–39 Native MS enables deep understanding of their structures, interactions, and conformational dynamics.^40–49 More recently, MCE has been developed and coupled together with native MS to study the ellipsoid geometric structures of proteins, as well as protein structure evolutions in different solvent environments.^50–54 In this study, the conformational dynamics of apo-CaM and Ca²⁺-bound CaM, as well as their binding events with a peptide ligand were investigated. Both MCE and native MS experiments were carried out for CaM samples at different Ca²⁺ concentrations. The formation of nCa²⁺–CaM as well as their ellipsoid geometries were characterized. Furthermore, we examined the influence of pH and temperature on these complexes. Finally, stoichiometric and conformational analyses were carried out for mixtures of CaM and the peptide ligand in the presence of Ca²⁺ at various concentrations, offering valuable insights for further research into the mechanisms of CaM–ligand interactions.

Results and discussion

Conformational dynamics of apo-CaM

X-ray and NMR studies have found that the crystal structures of CaM (such as 1CFD, 1CFC, 1DMO) have an open conformation where the N- and C-terminal lobes are separated by a flexible linker.^8,11 To explore the structural dynamics of apo-CaM, MCE and native MS experiments were then carried out. MCE separates ions based on their hydrodynamic radii and effective charges in solvent,⁵¹ and although protein structures may change during the transition from a liquid to a gas phase, the charge-state distributions (CSDs) in native MS experiments still provide valuable protein structure information, specifically solvent-accessible surface areas (SASAs), which can be deduced from the mass spectra.^55–57 Typically, globular proteins exhibit charge-state distributions with a single charge group, while IDPs have charge-state distributions with more charge groups.^58–60 As shown in Fig. 1a, the two peaks in the MCE electropherogram correspond to the neutral marker (phenol) and apo-CaM, respectively. According to MCE results, apo-CaM may only have one conformation, which is consistent with X-ray findings. However, three charge groups, labelled as charge group 1, 2 and 3, were observed for apo-CaM in its native mass spectrum (Fig. 1b), and their average charge states (Z_av) were 7.53, 11.67 and 18.31, respectively (Fig. S1†). Native MS results suggest that apo-CaM has multiple conformations: either three conformations that all behave like globular proteins, or two conformations of which one behaves like a globular protein with a single charge group and the other behaves like an IDP exhibiting two charge groups. Additional size exclusion chromatography (SEC) experiments were also carried out, and a single chromatography peak was observed for apo-CaM (Fig. S2†), which is in agreement with the MCE result but not the native MS result.

Fig. 1 Conformational dynamics of apo-CaM. (a) MCE of apo-CaM, 30 mbar and 15 kV; (b) native mass spectrum of apo-CaM; (c) and (d) ellipsoids of the open and closed conformations corresponding to charge groups 2 and 1, respectively. Ellipsoids are fitted and compared with PDB structures 1CFD⁸ and 1PRW.⁶¹

To elucidate the conflicting experimental results of a single peak in MCE and size exclusion chromatography experiments with a multi charged-state distribution in native MS, based on previous research and our subsequent analysis of experimental results, we propose that apo-CaM possesses two conformations under dynamic equilibrium (Fig. 1c and d): charge group 1 corresponds to a closed conformation that behaves like a globular protein; charge group 2 corresponds to an open conformation that has IDRs, and group 3 results from further unfolding of CaM with IDRs (group 2) during the ESI process. Furthermore, every single apo-CaM molecule would actively “jump” (dynamic equilibrium) between open and closed conformations in the solvent. Several lines of evidence support this hypothesis. First, both single-molecule fluorescence resonance energy transfer^62,63 and cross-linking MS^33,34 experiments found that a wide range of distances are sampled between the N- and C-terminals of CaM, indicating that CaM possesses multiple conformations in solution. By combining double electron–electron resonance electron paramagnetic resonance and molecular dynamic (MD) simulations, it has also been demonstrated that 4Ca²⁺–CaM could adopt multiple conformational substates.⁶⁴ Second, according to NMR studies, the linking region of CaM is flexible before binding to a ligand¹¹ and molecular dynamics simulations demonstrated that this CaM structure with a flexible linking region could further unfold during the ESI process and resulted in dual charge-state distributions in the mass spectrum.⁶⁵ Our subsequent findings also indicate that groups 2 and 3 exhibit similar traits in solution, which distinguish them from group 1 when adapting to dynamic environmental changes. Moreover, the ellipsoids calculated for charge groups 1 and 2 agree well with a closed and an open structure of CaM in the PDB, respectively (Fig. 1c and d, details could be found in Table S1†). The SASA calculated from charge-state distributions of group 3 (23 735.57 Ų) is significantly higher than that of any crystal structure in the PDB, while groups 1 (6729.89 Ų) and 2 (12 523.62 Ų) show good correspondence with the SASA in the PDB. These data explain the origin of the three charge groups: charge groups 1 and 2 correspond to a closed and an open conformation of apo-CaM in solvent, respectively, while group 3 corresponds to the further unfolding of CaM during the ESI process (Fig. S3†). Next, if apo-CaM has two conformational states, why these two conformations are not separatable (Fig. 1a and S2†)? This could be due to the fact that the transition time for CaM molecules ranges from microseconds to milliseconds.^66–69 Within the same transition cycle, all CaM molecules have the same migration distance. Therefore, separation techniques that operate on a timescale of seconds, such as MCE and size exclusion chromatography, cannot differentiate between different conformations of CaM. As a result, different conformational states could be captured/sampled at specific time points using relatively fast techniques, such as fluorescence resonance energy transfer, cross-linking MS and native MS, but they could not be separated in MCE or size exclusion chromatography. Str2str⁷⁰ results further support our conclusion that, regardless of whether the input is 1PRW (Ca²⁺-free) or 1CFD, Apo-CaM is always capable of interconverting between a closed conformation and an open conformation (Fig. S4†).

Conformational dynamics of Ca²⁺-bound CaM

As an important Ca²⁺ signaling process, the binding of Ca²⁺ could induce structure reorganization within CaM, that might enable the following target recognition. With the identification of a closed conformation for 2Ca²⁺–CaM and open conformations for 4Ca²⁺–CaM (such as 3CLN, 1CLL), it has been suspected that the binding events of Ca²⁺ may induce step-wise structure evolution⁷¹ or shift the pre-existing structure equilibrium:²⁵ from an open structure (apo-CaM) to a closed structure (2Ca²⁺–CaM) and back to open structure states (4Ca²⁺–CaM). To investigate the conformational dynamics of nCa²⁺–CaM, MCE and native MS experiments were conducted on CaM samples that had been incubated with varying concentrations of Ca²⁺. First, similar MCE results were acquired, in which single protein chromatography peaks were observed (Fig. 2a). As shown in Fig. 2b, CaM will bind to more Ca²⁺ with increased Ca²⁺ concentrations. apo-CaM, 2Ca²⁺–CaM, and 4Ca²⁺–CaM were found to be dominant peaks when the concentration ratios of CaM : Ca²⁺ were 1 : 1, 1 : 1.5 and 1 : 3, respectively. More data for other concentration ratios were plotted, as shown in Fig. S5.† As shown in Fig. 2b, nCa²⁺–CaM complexes with varying numbers of Ca²⁺ (n ranging from 0 to 4) consistently exhibit three charged groups in their native mass spectra, indicating that nCa²⁺–CaM complexes have similar conformational dynamics to that of apo-CaM. Therefore, similar to apo-CaM, nCa²⁺–CaM all have two dynamically exchanged conformational states: open and closed conformations. The closed conformations act like globular proteins, while the open conformations have IDRs. After applying multi-Gaussian fitting to the MCE electropherogram, the ellipsoid geometries of the closed and open conformations of nCa²⁺-CaM complexes are plotted and compared in Fig. 2c and 2d, respectively. Statistically significant differences in the a, b, and c axes of the ellipsoid geometries of the closed and open conformations of nCa²⁺–CaM were analyzed using GraphPad Prism 8. It was interesting to find that ellipsoid geometries of 2Ca²⁺–CaM and 4Ca²⁺–CaM have no statistical difference with those of apo-CaM (p values: 0.10, 0.81 and 0.98 for a, b and c in Fig. 2c; 0.07, 0.54 and 0.58 for a, b and c in Fig. 2d). As there was no apparent decrease in ion intensities for charge group 3, it raised suspicion that the proportion of IDRs remained unchanged after binding with Ca²⁺. In conclusion, the binding events of Ca²⁺ with CaM do not significantly change the conformational dynamics of CaM or nCa²⁺–CaM complexes.

Fig. 2 Conformational dynamics of nCa²⁺–CaM complexes. (a) and (b) MCE and native MS results of CaM samples with different CaM : Ca²⁺ ratios: 1 : 1, 1 : 1.5 and 1 : 3; (c) and (d) ellipsoid radii (a, b, and c) of the closed and open conformations of nCa²⁺–CaM complexes.

Besides increasing Ca²⁺ concentration, the conformational dynamics of nCa²⁺–CaM in different solvent environments, including varying pH and temperature, were further explored. The relative proportions of nCa²⁺–CaM complexes within the three charge groups under different solvent conditions are illustrated in Fig. 3 (please refer to Fig. S6–7 and Tables S2–S10† for detailed information). Since different charge groups correspond to different CaM conformations, the similarity of the Ca²⁺-binding abilities of the three groups was first characterized using the Manhattan distance method. In all cases, groups 2 and 3 have a smaller Manhattan distance than that between groups 1 and 2 or 1 and 3, confirming that groups 2 and 3 originate from one group of ions with similar conformations in solvents. As depicted in Fig. 2b and 3, CaM possessing the closed conformation (group 1) also exhibits different binding preferences with Ca²⁺, preferring to bind 2 and 4 Ca²⁺. In comparison, CaM having the open conformation (groups 2 and 3) shows no such preferences, and a gradual increase of Ca²⁺ binding numbers was observed by increasing Ca²⁺ concentrations. Fig. S8† presents the K_d fitting curves for different charge states, and the corresponding K_d values are listed in Table S11.† These values were of the same order of magnitude as those reported in the references.^72,73 Furthermore, it is observed that group 1 does exhibit a notably stronger affinity towards Ca²⁺, indicating that 4Ca²⁺–CaM is more inclined to adopt a closed conformation.⁷⁴ As depicted in Fig. S6,† the binding of Ca²⁺ to CaM hindered at lower pH, especially at 3.0, because its isoelectric point is approximately 4.0, resulting in a positively charged state for CaM at this acidic pH level. On the other hand, elevated temperatures, such as 50 °C, enhance the binding ability to Ca²⁺, leading to an increase in the relative ratio of 4Ca²⁺–CaM from 8% to 11%. However, excessively high temperatures (∼90 °C) may lead to protein denaturation. It is worth noting that alterations in Ca²⁺ concentration, temperature and pH did not appear to significantly affect the conformational distribution of CaM (Fig. S9†).

Fig. 3 Relative proportions of nCa²⁺–CaM under different solvent conditions and the Manhattan distances between different charge groups: (a) with increased Ca²⁺ ratio; (b) at different pH; and (c) at different temperatures.

CaM binding with a ligand

The structural flexibility of CaM is believed to account for its ability to recognize various targets, and the concentration of Ca²⁺ is also critical during this process.^75–78 The binding event of CaM with a peptide ligand, Mel, was then explored. The MCE and native MS results of CaM–Mel mixtures (1 : 1) at different Ca²⁺ concentrations are plotted in Fig. 4a and b. At a relatively low Ca²⁺ concentration (CaM : Ca²⁺ = 1 : 1), a fraction of CaM binds with Mel, and both 3Ca²⁺–CaM–Mel and 4Ca²⁺–CaM–Mel complexes are observed in the mass spectrum. As the Ca²⁺ concentration increases, a larger proportion of CaM associates with Mel, and the relative intensities of 3Ca²⁺–CaM–Mel decrease or even become unobservable when the Ca²⁺ concentration is high enough (CaM : Ca²⁺ = 1 : 3, Fig. 4d). As shown in Fig. 4a, nCa²⁺–CaM and nCa²⁺–CaM–Mel were separated in MCE, suggesting that Mel could stabilize the structure of CaM. Fig. 4c illustrates the ellipsoid shape of 4Ca²⁺–CaM–Mel, which was also compared with its crystal structure in the PDB (8AHS).⁷⁹ Instead of “jumping” between two conformations, CaM–Mel complexes adopt a relatively stable, closed conformation.

Fig. 4 CaM binding with a ligand, Mel. (a) and (b) MCE and native MS results of CaM–Mel mixtures at different Ca²⁺ concentrations; (c) ellipsoid shape and radii of 4Ca²⁺–CaM–Mel, as well as its comparison with its crystal structure in the PDB (8AHS, Table S1†); and (d) relative abundance of 3Ca²⁺–CaM–Mel and 4Ca²⁺–CaM–Mel complexes at different Ca²⁺ concentrations.

Based on the presented results, we proposed a target recognition mechanism for the interaction of CaM with Mel. As illustrated in Fig. 5, this mechanism aligns closely with the conformational selection model.^80,81 However, it has been traditionally believed that Ca²⁺ binding induces conformational changes in CaM, facilitating its interaction with target molecules. Our results show that the binding of Ca²⁺ does not markedly alter the conformational dynamics of CaM. Both apo-CaM and nCa²⁺–CaM transition between different conformations. As a result, a target molecule may select and align with its preferred nCa²⁺–CaM conformation. This binding event then shifts the dynamic conformational equilibrium of CaM, which explains the wide range of targets that CaM could recognize. Furthermore, the presence and concentration of Ca²⁺ also play important roles in terms of providing hydrophobic pockets in the C- and N-terminal lobes.^82–85 These pockets can engage with an array of hydrophobic groups present on target molecules, promoting complex formation. This proposed mechanism was also supported by the fact that different CaM complexes harbor varying numbers of Ca²⁺.^{3,17,18,22,86–89} Notably, some ligands, such as the IQ motif of the human cardiac sodium channel Na_v1.5, even demonstrate a preference for binding with apo-CaM.^87,90

Fig. 5 Schematic illustration of the target recognition mechanism of CaM with Mel.

In summary, the conformational dynamics of CaM and its complexes (nCa²⁺–CaM and nCa²⁺–CaM–Mel) were explored using an MCE and native MS based method. Although coarse grained structure information was acquired, MCE–native MS is a relatively rapid technique for characterizing protein conformations under various conditions without the need for crystallization and chemical labeling. Key observations are as follows: 1. CaM molecules, whether bound to Ca²⁺ or not, actively “jump” (equilibrium exchange) between open and closed conformations in liquid environments. 2. In the closed conformation, CaM and Ca²⁺-bound CaM behave as globular proteins that have a single charge distribution in the native mass spectrum. In the closed conformation, CaM prefers to add 2Ca²⁺ and 4Ca²⁺ depending on the concentration of Ca²⁺. 3. In the open conformation, CaM and Ca²⁺-bound CaM have multimodal distributions in their native mass spectra, indicating the presence of IDRs. No preference was observed in terms of the number of Ca²⁺ added to CaM. 4. No discernible conformational change was observed following the binding of various Ca²⁺ (from 1 to 4). The portion of IDRs was not decreased after the binding of Ca²⁺. 5. Besides the 4Ca²⁺–CaM–Mel complex, the 3Ca²⁺–CaM–Mel complex was also observed at a relatively low Ca²⁺ concentration. 6. The coexistence of diverse conformations allows for the recognition of multiple targets, while binding to varying numbers of Ca²⁺ could provide different amounts of hydrophobic pockets, further promoting complex formation. This MCE–native MS based method has revealed the relationship between the conformation of CaM and the number of Ca²⁺ bound, and it has unveiled the possible mechanisms of interaction between CaM and ligands. This coarse grained approach can compensate for the limitations of X-ray, NMR and other techniques in explaining protein dynamics, becoming an important supplementary method for studying protein structure and function.

Materials and methods

Chemicals

Commercial recombinant bovine CaM (Sigma Aldrich, St Louis, MO, USA) was dissolved in 20 mM ammonium acetate at a concentration of 20 μM. The chelating agent, EDTA (25 mM), was then added to dissociate CaM–metal ion complexes, and ultrafiltration was performed to remove Ca²⁺ residues. CaCl₂ was then added with concentration varying from 0 to 80 μM. Calcium chloride (CaCl₂) and ethylenediaminetetraacetic acid (EDTA) were purchased from Thermo Fisher Scientific (Massachusetts, USA). Formic acid and ammonia solution (Dikma, Beijing, China) were used to adjust pH. Melittin (Mel) was purchased from APExBIO (Houston, USA). Ammonia solution (25%) was purchased from Fuchen (Tianji, China). Phenol was obtained from Innochem (Beijing, China). The coated capillary was purchased from Sino Sumtech (Hebei, China). A water bath was used to control the reaction temperature of CaM and CaCl₂. All mixtures were incubated at room temperature for 30 minutes, except for the temperature-controlled experiments, in which samples were pre-incubated at different temperatures for 30 minutes right before the ESI analyses.

MCE experiments

MCE experiments were performed on an Agilent 7100 CE system (Agilent technology, USA). The coated capillary used in the MCE experiment has an inner diameter of 75 μm, a total length of 48.5 cm and an effective length of 40 cm. Phenol was added to a final concentration of 1/6000 (v/v) for each sample as the neutral marker. Before each sample injection, the capillary should be rinsed with 20 mM H₃PO₄ for 3 min and then with 20 mM ammonium acetate (pH 7.0) buffer for 3 min. Samples were injected at a pressure of 50 mbar for 5 s and separated by applying a 10 kV/15 kV DC separation voltage for nCa²⁺–CaM and nCa²⁺–CaM–Mel, respectively, and a 30 mbar driving pressure simultaneously. The samples were detected at a wavelength of 214 nm. The working temperature of the capillary was set at 25 °C in all experiments.

Native MS experiments

Native mass spectra were acquired using an Xevo G2-XS TOF, where 10 μL samples were loaded in the nano electrospray ionization (nESI) glass capillary for analysis. All samples were analyzed under the positive ion mode. The MS parameters used in this work were as follows: spray voltage: 2.8 kV; sampling cone voltage: 60 V; source temperature: 150 °C; desolvation temperature: 400 °C; cone gas flow: 50 L h⁻¹; desolvation flow: 600 L h⁻¹; acquisition rate: 1 spectra per s; MS scan range: 400–5000 Th.

Str2str. Given 1PRW as the initial structure, the backbone frames were sampled through the forward–backward process. The output was a set of global coordinates for the backbone atoms. The side chain conformations were predicted using the backbone-dependent rotamer libraries. The process minimized energy through a Monte Carlo searching scheme, ensuring that the sampled conformations were physically plausible. Open-source implementation is available at https://github.com/lujiarui/Str2Str.

Author contributions

Yi Zhao: data curation, formal analysis, investigation, methodology and writing. Wenjing Zhang: formal analysis and methodology. Jie Hong: data curation. Lei Yang: data curation. Yuanyuan Wang: conceptualization. Feng Qu: project administration. Wei Xu: conceptualization, funding acquisition, methodology, project administration, resources, software, supervision and writing.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by NNSFC (22374009). We also thank the Analysis & Testing Center of the Beijing Institute of Technology for MS instrument support.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4an00378k

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