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Inter-motif communication induces hierarchical Ca2+-filling of Caldendrin Uday Kiran, Phanindranath Regur, Michael R Kreutz, Yogendra Sharma, and Asima Chakraborty Biochemistry, Just Accepted Manuscript • Publication Date (Web): 24 Apr 2017 Downloaded from http://pubs.acs.org on April 24, 2017

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Biochemistry

Inter-Motif Communication Induces Hierarchical Ca2+-Filling of Caldendrin Uday Kiran1, Phanindranath Regur1, Michael R. Kreutz3,4*, Yogendra Sharma1,2*, Asima Chakraborty1* 1

CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad-500007,

India. 2

Academy of Scientific and Innovative Research (AcSIR), New Delhi, India

3

RG Neuroplasticity, Leibniz Institute for Neurobiology, Brenneckestr. 6, 39118 Magdeburg,

Germany 4

Leibniz Group 'Dendritic Organelles and Synaptic Function', University Medical Center

Hamburg-Eppendorf, Center for Molecular Neurobiology, ZMNH, 20251 Hamburg, Germany

Running title: Stepwise Ca2+ binding and conformational hierarchy

*

For correspondence: [email protected]; [email protected]; [email protected]

Funding The work is supported by the CSIR fast-track SRA to AC, CSIR (BSC0115) to YS, and DFG (Kr1879/3-1) to MRK.

ACS Paragon Plus Environment

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Abstract A crucial event in calcium signalling is the transition of a calcium sensor from the apo (Ca2+ free) to the holo (Ca2+ saturated) state. Caldendrin (CDD) is a neuronal Ca2+-binding protein with two functional (EF3 and EF4) and two atypical (EF1 and EF2), non-Ca2+-binding EF-hand motifs. During the transition from the apo to the holo state, guided by the step-wise filling of Ca2+, the protein passes through distinct states and acquires a stable conformational state when only EF3 is occupied by Ca2+. This state is characterized by a Ca2+-derived structural gain in EF3 with destabilization of the EF4 motif. At higher Ca2+ levels, when Ca2+ fills into EF4, the motif regains stability. EF3 controls initial Ca2+ binding and dictates structural destabilization of EF4. It is likely that this unexpected inter-motif communication will have impact on Ca2+-dependent target interactions.

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Biochemistry

Neuronal Ca2+-signaling is organized in micro- and nanodomains, where a plethora of Ca2+-binding proteins (CaBPs) provide the basis for a tight spatial and temporal regulation of Ca2+ signaling. Several neuronal Ca2+-binding proteins share domain organization with their common ancestor calmodulin (CaM); they harbor 2 EF-hand domains, each consisting of 2 EFhand Ca2+-binding motifs. Neuronal calcium sensor proteins are subdivided into two subfamilies, the classical neuronal calcium sensor (NCS) family and the neuronal calcium-binding proteins (nCaBPs).1,2 Caldendrin (CDD) is the founding member of the nCaBP family and is enriched in dendritic spine synapses.3,4 The protein has a unique bipartite structure with a basic and prolinerich N-terminus, and a C-terminus that it shares with two shorter splice isoforms, S-CaBP1 and L-CaBP1 (Supplementary Figure S1), which are much less abundant in brain.3-7 The structural information of CDD is not known, however, based on the structure of S-CaBP1, the C-terminus resembles the structure of CaM with four EF-hand motifs, but in contrast to CaM, the first EFhand motif can probably bind both Mg2+ and Ca2+, while the second EF-hand motif is cryptic.6,8,9 Over the years, a larger CDD interactome has been identified,1 but still very little is known how CDD operates as a Ca2+ sensor that accommodates different binding partners. A frequently neglected aspect in this regard is to understand whether an EF-hand protein has stable conformational states during the transition from the apo to the holo form. Most studies have focused on the apo or the Ca2+-saturated holo protein. However, two modes for Ca2+ filling of EF-hands have been described in the literature: simultaneous and sequential filling.10,11 In the former mode, all sites would be filled at the same time, and thus intermediate equilibrium states are neglected. In the sequential mode, preferred filling of one site over the others induces partially filled, equilibrium-intermediates; depending on the availability of Ca2+.12-16 The structural underpinnings for simultaneous or sequential Ca2+ filling are not clearly known. However, numerous studies on CaBP have been performed for understanding Ca2+ induced structural changes and long-range allosteric effects.17,18 Since many CaBPs supposedly follow a sequential mode of Ca2+ filling, it is likely that a CaBP will also be functional in vivo at Ca2+ levels insufficient to saturate all binding sites or under controlled Ca2+ release as is shown in case of CaM.19 The direct identification of semi-holo states requires the individual monitoring of each EF-hand motif. To address this topic, we harnessed a tryptophan (Trp) EF-hand scanning approach where we introduced a ‘reporter’ Trp in each EF-hand of CDD.

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Experimental Procedures Site-Directed Mutagenesis. The rat CDD cDNA was cloned into the pMXB10 vector (New England Biolabs) in NdeI and XhoI sites.6 Required mutations were introduced by Pfu PCR using mutagenic oligonucleotides following the protocol of QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mutations were confirmed by gene sequencing at our in-house DNA sequencing facility. Protein Over-expression and Purification. The protein over-expression in E. coli BL-21 (DE3) cells was induced by 0.2 mM IPTG followed by 4 hours of incubation at 37 ºC. The overexpressed protein was purified using the IMPACT kit (New England Biolabs), following the manufacturers’ protocol as described earlier (more details are provided in Supplementary Information).6 While NTW, EF1W, EF3W, EF4W and H3W mutants of CDD over-expressed well in E. coli, EF2W mutant of CDD did not over-express. Modelling of Individual Trp Mutants. To rule out any possible structural disturbances due to Trp insertion, we modelled the structures of individual Trp mutants of CDD with the program SWISS-MODEL20 by taking the crystal structure of S-CaBP19 (100% similar sequence with Ca2+ binding domain of CDD) as a template. The calculated solvent accessible surface area (SASA) for individual Phe residues in wild-type and Trp residues in modelled mutant structures are presented below (Table 1): Table 1: Comparison of calculated solvent accessible surface area of the four selected Phe residues of wild-type CDD and Trp residues in mutant CDD S-CaBP1 (PDB ID: 3OX5)

Caldendrin Trp Mutants

Residue

SASA (Å2)

Residue

SASA (Å2)

Phe34

0.196

Trp 165 (EF1W)

0.180

Phe83

2.829

Trp 214 (EF2W)

0.176

Phe111

21.874

Trp242 (EF3W)

20.287

Phe161

4.876

Trp292 (EF4W)

8.001

To further rule out the stearic clashes, we used molprobity software21 and we did not notice any serious steric clashes. These evaluations were further supported by the CD spectra of

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Biochemistry

mutant proteins. We used Protein Interaction Calculator (PIC) software22 to assess different types of interactions between amino acids in CDD. Circular Dichroism (CD) Spectroscopy. Near- and far-UV CD spectra were recorded on a Jasco-810 spectropolarimeter. Near-UV CD spectra were recorded between 350-250 nm using a quartz cuvette of 0.5 cm path length averaged with 4-6 accumulations. A Ca2+ free protein solution of ~1 mg/mL concentration in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 5 mM tris(2-carboxyethyl) phosphine, was used for all the recordings. For Mg2+ and Ca2+ titrations, aliquots from a stock of MgCl2 and CaCl2, each in Chelex-purified buffer, was used. Similarly, far-UV CD spectra were recorded between 250-200 nm using a quartz cuvette of 0.1 cm path length with ~0.1 mg/mL protein solution. Isothermal Titration Calorimetry. Ligand binding for all mutants was carried out by isothermal titration calorimetry (ITC) on a Microcal, VP-ITC system as per the protocol for NCS-1 described earlier.23 Decalcified CDD of 20-30 µM concentration prepared in Chelexpurified 50 mM Tris, pH 7.5, 100 mM KCl buffer. The proteins were saturated with 2 mM MgCl2 followed by titration with a stock of 5 mM CaCl2 until it saturates. ITC experiments were repeated by varying protein concentration to obtain a correct binding isotherm. Association constants, Ka were originated from the data fitting, and global dissociation constant, Kd was derived by the formula: 1/√ (ka1 * ka2). Fluorescence Spectroscopy. Intrinsic Trp fluorescence and extrinsic fluorescence spectra of ANS were recorded on a Hitachi F-7000 spectrofluorometer (Hitachi Inc., Japan) in Chelexpurified 50 mM Tris-HCl, pH 7.5 and 150 mM NaCl. The fluorescence spectra from 310–450 nm were recorded in a quartz cuvette of the path length of 4 mm, at excitation wavelength of 295 nm, with a scan speed of 240 nm/min, and slits being 5 nm open on both ends. For ANS fluorescence experiments, a Ca2+ free 4 µM protein solution was incubated with 10 µM of ANS (prepared 10 mM stock solution in dimethyl sulfoxide/water mixture), and fluorescence emission spectra were recorded between 400-600 nm at excitation wavelength of 370 nm. ANS-protein spectra were corrected for ANS-buffer blank spectra. Aliquots of MgCl2 and CaCl2 from the respective stocks were added to get Mg2+-saturated and then Ca2+-saturated conditions. Equilibrium Unfolding. Equilibrium unfolding of all the CDD mutants in the apo- and Ca2+bound conditions was monitored by change in Trp fluorescence (protocol described earlier).24 For each set (one for apo and another for Mg2+/Ca2+-saturated protein), about 60 samples were

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prepared, each containing 0.1 mg/mL concentration of protein in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 5 mM TCEP and an increasing concentration of GdmCl, ranging from 0-6 M with an average increment of 0.1 M/sample. Similarly, at least two sets for each mutant were performed and the data points were plotted comparatively.

Results Global Unfolding of CDD As CDD does not have Trp residues in its primary sequence, we followed chemical unfolding of the apo-protein and the effect of the saturated level of Ca2+ with GdmCl by monitoring the change in ellipticity at 220 nm by far-UV CD. Unfolding of CDD in the apo and Ca2+-saturated conditions is almost similar till GdmCl concentration of ~3 M, beyond which concentration of the denaturant, we notice a minor stabilization effect of Ca2+ (Figure 1A). We further monitored CDD unfolding by measuring the changes in surface hydrophobicity by employing extrinsic fluorophore, ANS. Unfolding of CDD in the apo and Ca2+-saturated conditions is almost similar in terms of ANS binding, except a minor increase in Ca2+ induced stability (as deduced by emission maxima of ANS fluorescence) is observed at GdmCl concentration of >4 M (Figure 1B). Low [Ca2+] Induces Structural Fluctuation in CDD To monitor the structural changes in CDD at different Ca2+ concentrations (as it is known that intracellular Ca2+ level continuously fluctuates in neurons), we performed unfolding at different Ca2+ concentrations. In case of unfolding monitored by CD, almost no change in unfolding transition of CDD at 20 µM Ca2+ was noticed, as compared to the apo and the holo conditions (Figure 1A). However, as measured by the changes in ANS emission, we observed more red shifted emission at 20 µM Ca2+ than even apo form of CDD, suggesting that the surface hydrophobicity of the protein was reduced at this concentration of Ca2+. This extent of red shift is reduced when [Ca2+] is increased (such as to 100 µM) (Figure 1B). This result highlights the Ca2+ induced flexibility or destabilization of structure in CDD at below saturation level of Ca2+. Whether Ca2+ induces global destabilization (from ANS fluorophore data) or a local structural perturbation, we attempted to understand this phenomenon in detail.

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Biochemistry

Figure 1. (A) Unfolding of CDD (WT) represented with change in ellipticity at 220 nm as a function of increasing GdmCl concentration in apo-, 20 µM Ca2+ and 2 mM (Ca2+ saturated) conditions. (B) Unfolding of CDD (WT) represented by change in emission maxima with increasing concentrations of GdmCl in the apo-, Ca2+-saturated (2 mM), 20 µM and 100 µM conditions probed by extrinsic fluorophore ANS. Result evidently represents that protein is more stable in the apo- and holo-conditions, compared to CDD with lower Ca2+ concentrations. (C) A scheme representing all four EF-hand motifs of CDD and the corresponding reporter. The Trp reporters are: H3W, NTW (F131W), EF1W (F164W), EF2W (F213W), EF3W (F241W), EF4W (F291W). Disabled EF-hand mutants are: EF3Q_EF4W (E253Q, F291W), EF4Q_EF3W (E290Q, F241W). Location of Trp and the site-disabling mutants in the primary structure, are indicated in the scheme. EF1, which binds Mg2+ is blue, cryptic or disabled motifs (EF2, E253Q and E290Q) are hollow, while canonical motifs (EF3 and EF4) are green. Trp Scanning of EF-hand Motifs To investigate local structural changes upon Ca2+ binding to individual EF-hand motifs, we introduced Trp reporters in CDD. CDD harbors no Trp in its primary sequence, but has

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opportune locations of the four Phe residues for Trp scanning, being located either just before (in EF1 and EF3), or immediately following an EF-hand loop (in EF2 and EF4). We, therefore, replaced these Phe (Phe165, Phe214, Phe242 and Phe292) with Trp residues (named EF1W, EF2W, EF3W and EF4W reporter mutants), which then allowed for the monitoring of local changes in conformation around an EF-hand motif during filling with Ca2+ (see scheme in Figure 1C) except for EF2W that did not over-express. The use of Trp to Phe mutations has been described for calcium- and integrin-binding protein 1 for monitoring the role of each EF-hand motif.25 The main purpose of a Trp residue is to probe local structural changes, but it is imperative to examine the effect of replacement of Phe with Trp on protein structure. Based on simulation by SWISS-MODEL (presented in methods) and spectroscopic studies, it is evident that mutations do not lead to significant structural disturbances in protein. Trp mutations did not alter the global Ca2+-binding properties significantly as analyzed by ITC, the secondary as evidenced by far-UV CD spectroscopy, or the overall surface hydrophobicity, as probed by ANS fluorescence (Figure 2A-E, and Table S1, see supplementary information). However, minor changes in near-UV CD spectra in the aromatic region are due to the difference in the environment of Trp in three CDD (Figure 2B). The thermodynamic parameters evaluated for Ca2+ binding by ITC of wild-type protein and Trp mutants manifest with marginal differences in affinities and the average enthalpy changes (Table S1). Further, Ca2+ binding monitored by ITC is exothermic, unlike an initial endothermic reaction followed by exothermic reported previously for S-CaBP1 (C-terminal half of CDD).8 This could be due to the large disordered N-terminal domain of the protein as demonstrated earlier.6 The unfolding profile of EF3W and EF4W matches well with wild-type CDD, demonstrating that Trp mutations have not altered the overall stability of the protein (Figure S2). EF-hand Scanning Reveals Flexibility of EF-hand Motifs Among all the EF-hand motifs, Trp of EF4 in the apo-CDD is shielded more from the polar environment (λmax 334 nm) than that of EF3 (λmax 343 nm), while Trp of EF1 showed red-shifted emission (λmax~348-350 nm) (Figure 2F). The differential dynamicity of the individual EF-hands is also indicated by anisotropy values (r) of each respective Trp. In the apo form, Trp located at the NTD is the most flexible (r ~ 0.047), EF4 is the most rigid (r ~0.074) and those of EF1 and EF3 are in moderately rigid environments (r ~0.062 and 0.059 respectively).

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Biochemistry

Figure 2. (A) Far-UV CD spectra of apo form of NTW (black), EF1W (red), EF3W (blue) and EF4W (green) CDD showing that all mutants have similar secondary structures. (B) Near-UV CD of apo form of NTW, EF1W and EF3W (color scheme is the same as above), depicting the tertiary structure of the mutants; the protein concentration of EF4W was too low to record reliable CD spectra in that range. (C) ANS fluorescence spectra of the mutants in their apo forms, confirming that the overall surface hydrophobicities remain unaltered in spite of the introduced Trp. (D and E) Representative ITC of Ca2+ titration to Mg2+-saturated wt CDD and EF3W-CDD showing the binding isotherm, fitted to two sequential binding sites. (F) The wavelength of maximum emission of all the mutants (except EF2W, which could not be overexpressed) in their apo and holo states, is plotted as a bar representation. Sequential Binding of Ca2+ at Two Distinct Sites We next took advantage of the fact that the Trp residues inserted at each EF-hand motif in CDD are in different environments to monitor local conformational changes in response to Ca2+ binding. While the fluorescence spectrum of EF1W is not altered much even in the presence of excess Ca2+ (2 mM), the spectra of EF3W and EF4W changed noticeably, with the EF3W spectrum undergoing the most prominent alteration (Figure 3). Ca2+ imparts a drastic increase in

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fluorescence intensity of EF3W (starting from [Ca2+]/[protein] ratio as low as ~15) along with a 4-5 nm blue shift of the emission maximum. Ca2+ titration to EF4W, on the other hand, quenches its Trp emission with a moderate (~2 nm) blue shift, but only at higher [Ca2+] (Figure 3). EF1W fluorescence remains nearly unaffected at all Ca2+ concentrations.

Figure 3. Trp fluorescence spectra of CDD reporters: (A) NTW, (B) EF1W, (C) EF3W, (D) EF4W and (E) H3W, upon serial titration with Mg2+ followed by Ca2+. The spectra shown are: apo (black), 1 mM Mg2+ (red), 2 mM Mg2+ (blue), 0.6 mM Ca2+ (green), 1 mM Ca2+ (magenta) and 2 mM Ca2+ (olive). (F) Trp emission intensity maxima plotted as a function of increasing Ca2+: NTW (Green), EF1W (red), EF3W (Black), EF4W (Blue). The differential Ca2+-triggered response of Trp fluorescence led us to hypothesize that with increasing [Ca2+]i, EF3 is the first to sense Ca2+ (Figure 4A). Thus, with elevated [Ca2+]i, EF3 is the first to respond at low Ca2+ concentrations, whereas no other EF-hand (EF1 or EF4) motif binds Ca2+ under these conditions. EF4 (i.e., EF4W reporter) responds to [Ca2+]i only after EF3 (i.e., EF3W reporter) is nearly saturated (Figure 4A). These findings suggest a sequential Ca2+ filling mode of EF3 followed by EF4. Since the Trp of EF1 (i.e., EF1W reporter) was not responsive to added Ca2+, we did not consider this motif in the analysis. This is in agreement

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Biochemistry

with earlier studies on S-CaBP1 where it was shown that EF1 binds Ca2+ with a very low affinity, and no electron density for Ca2+ was noticed in a crystal structure.

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Ca2+ binding

isotherms obtained by ITC are not at variance with the idea of sequential filling (Figure 2D and Table S1). The two macroscopic binding constants obtained by ITC are: Kd1 (7.3 µM) and Kd2 (80 µM), which probably reflect as microscopic constants: Kd1 for EF3 and Kd2 for EF4 (with respect to ITC values from CDD-WT protein).

Figure 4. (A) Normalized change in Trp fluorescence intensity of Trp mutants upon Ca2+ titration (Figure 3F3): EF3W (for EF3 as black circle) and EF4W (for EF4 as red circle) is plotted against [Ca2+]/[Protein] ratio. (B) Equilibrium unfolding curves of all Trp reporters, NTW (Green), EF1W (black), EF3W (blue), EF4W (red) (broken lines for apo and solid lines for holo). Upper panel represents the change in Trp emission maxima, lower panel: curves generated by fitting the fluorescence intensities of the different Trp mutants versus the concentration of GdmCl. (C) Scheme 1. Schematic representations of CDD unfolding in apo (upper panel) and holo (saturated with Ca2+, lower panel) conditions. Protein unfolding starts with disordered Nterminal followed by EF1. No reporter is available to monitor the EF2 (cryptic), as protein did not overexpress; we assume that it unfolds along with EF1. Both EF3 and EF4 unfolds almost simultaneously in apo form. Unfolding of holo form fallowed similarly but EF3 unfolding starts almost after 50% of EF4 unfolding. The graphs represent the global unfolding monitored locally.

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Stable Intermediate States Exist During Ca2+ Filling of CDD The difference in the affinity of Ca2+ between EF3 and EF4 (i.e., between the two high affinity constants) suggests a sequential Ca2+-filling mode in CDD, starting with EF3 and followed by EF4. Moreover, this large difference in the microscopic Ca2+-binding constants indicates that EF3 would be occupied by Ca2+ prior to EF4, which also means that CDD could exist in an intermediate state (conformationally may or may not be different) where only EF3 is occupied by Ca2+. Based on the response of amide NMR intensities of Gly117 residue of EF-hand 3 and Gly133 of EF-hand 4 of S-CaBP1, it is apparent that while following step-wise filling, the protein may exist in one Ca2+-bound state before reaching to saturation.8 Similar stable equilibrium state has also been described in a calcium vector protein (CaVP) by NMR, where Ca2+ occupies site III first during step-wise filling, while site IV remains empty.26 Further prediction from this scheme is: if EF3 is filled, CDD gains an equilibrium intermediate state prior to Ca2+ saturation, which in turn could result in a distinct conformation that is different from both the apo and the holo states. To address this point, we employed ‘segmental monitoring’ of the global unfolding of CDD. This method detects the flexibility changes associated with EF-hand motifs as well as potential unfolding differences associated with intermediate states during sequential Ca2+ filling.27 The unfolding profiles obtained in these experiments indicate that the N-terminal region collapses at sub-micromolar concentrations of GdmCl, both in the apo and holo forms (all unfolding profiles are found in Figure 4B). Among the EF-hand motifs, EF1 unfolds first in the apo protein (beyond 1 M GdmCl), while the rest of the protein remains unaffected. EF3 and EF4 exhibit unfolding at similar GdmCl concentrations, but probably due to cooperativity with EF3 EF4, unfolds prior to EF3. In the holo form again, EF1 initiates unfolding, followed by EF4; however, EF3 starts unfolding only after EF4 is >50% unfolded (unfolding presented both by the change in emission maxima (upper panel) and by fraction unfolded (lower panel), Figure 4B and scheme 1) (This analysis excludes EF2 motif, as EF2W protein did not overexpress). The local C1/2 value of the different motifs of apo CDD (deduced from the unfolding profiles of Trp reporters) varies from 3.3 (for EF1) to 3.84 (for EF3) and 3.7 M (for EF4). For the holo form, the C1/2 value for EF3 rises up to ~5.22 M, while it remains unchanged for EF1, and increases moderately for EF4 (4.14 M), possibly indicating a gain in rigidity/stability of the EF3 (∆C1/2 ~1.38 M) motif, but only a slight gain in EF4 (∆C1/2 ~0.44 M) upon binding of Ca2+ (Figure 4B).

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Biochemistry

Nevertheless, these results were obtained using a single Trp probe within a cooperatively folded domain. Ca2+ Filling at EF3 Follows via Multiple Conformational Steps We next monitored, by unfolding of the EF3W reporter protein, the effect of gradual filling of Ca2+ to EF3. The range of Ca2+ concentrations for this assay was calculated based on segmental fluorescence changes by EF3W as described above (Figure 2). If Ca2+ is bound only to EF3, one would expect that EF3 (i.e., the EF3W reporter) will unfold at higher GdmCl concentrations than its apo form due to acquired rigidity and stability imparted by Ca2+ binding. At the same time, the unfolding transition monitored at EF4 (i.e., EF4W reporter) at those Ca2+ concentrations should remain unaltered. As shown in Figure 5A and 5B (upper panel) unfolding transitions of EF3W lie in between the apo and the Ca2+-saturated forms. Thus, with increasing concentrations of Ca2+ in a range that only allows filling of EF3, we see a noticeable change in unfolding. Based on the amount of protein used for the assay, we started with 20 µM [Ca2+], at which we expected filling of EF3, followed by 50, 100 µM and higher concentrations of Ca2+. We noticed that the unfolding transition profile of EF3W at 20 µM Ca2+ is different from the apo-protein (i.e., filling of Ca2+ reduces stability with respect to C1/2), with obvious differences in the slope of the unfolding transition. Unfolding transitions of EF3W at higher [Ca2+] lie in between the apo and the holo, saturated (2 mM Ca2+) forms, but with a difference in the folding cooperativity, represented by the slope of the transition curve or m-value, which reflects the dependence of the free energy change of unfolding on the GdmCl concentration.28 To highlight this difference, we plotted m-values acquired from unfolding of EF3W at different Ca2+ concentrations against Ca2+ concentrations from 0 to 2 mM (Figure 5C, upper panel). As depicted in the upper panel of Figure 5B for EF3W, conformational rearrangements take place during initial Ca2+ filling that is indicative for increased flexibility (reduced stability) (Figure 5A and 5B). A plot of local C1/2,EF3 value versus [GdmCl] demonstrates a trend where C1/2 increases (C1/2,Ca2+ - C1/2,apo = ∆C1/2 increase to 1.67 M) with increasing [Ca2+] (Figure 6A). A similar pattern was obtained if ∆C1/2 was calculated by the unfolding curve. This clearly indicates that, during gradual filling of EF3, the protein passes through a relatively stable state.

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Figure 5. All upper panels represent EF3W reporter, and the lower panels for EF4W reporter of CDD. (A) Change in Trp emission maxima with [GdmCl]. (B) GdmCl-induced unfolding profiles represented by fraction unfolding calculated at 360/320 nm. (C) Change in m-values of unfolding representing the slope of unfolding transitions performed at varying [Ca2+]: 0, 20, 50, 100, 200, 600 µM, 1 mM and 2 mM). All the unfolding experiments were performed by the inclusion of 2 mM Mg2+. The changes in m-values of protein unfolding during Ca2+ filling are marked by arrows. Graphs represent different conformational states of protein (at EF3W and EF4W reporters) during Ca2+ filling of the sites. Allosteric Ca2+ Filling at EF3 Results in Collapse of EF4 We next monitored the allosteric effect of Ca2+ binding to EF3 on EF4 via unfolding of an EF4W reporter at the above listed Ca2+ concentrations, which allow for gradual filling of EF3 alone. To our surprise, we observed that the unfolding profile of EF4W does not at all follow the apo form. Instead, up to 100 µM [Ca2+]- EF4 actually unfolds at significantly less GdmCl concentrations compared to the apo form (Figure 5A and 5B, lower panels). Unfolding transitions at different [Ca2+] as revealed by the plot of m-values versus [Ca2+] indicated that during initial filling of EF3 a drastic change in folding cooperativity occurs, indicated by a reduced m-value (Figure 5C, lower panel). These data suggest that Ca2+ filling to EF3 causes a collapse of EF4, which is also represented by C1/2,EF4 values calculated for EF4W at increasing [Ca2+] (Figure 6A). The maximum reduction in C1/2 from 3.22 M (apo) to 2.6 M is noticed at 100 µM Ca2+ but with a less cooperative unfolding compared to the apo form. This shows an interesting relationship

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(structural remodeling of Ca2+-free EF4 by the Ca2+ filled EF3) of different motifs in the same protein.

Figure 6. (A) Change in transition midpoint (C1/2) of CDD unfolding performed at different [Ca2+], C1/2,Ca2+- C1/2,apo = ∆C1/2 values (a measure of protein stability) of unfolding for both EF3W (black circles) and EF4W (red circles) calculated at different Ca2+ concentrations are plotted with [Ca2+] (main figure represented with ∆C1/2 values from 0 to 600 µM Ca2+ where protein shows gain (EF3W) and loss (EF4W) of stability at different reporters. Inner panel represent change in ∆C1/2 from 0 to 2 mM Ca2+). While C1/2 increases with increasing [Ca2+] for EF3 (except at 20 µM) motif, it decreases when EF3 is filled, followed by recovery for EF4. (B) High fluctuations in unfolding profile of EF4 (monitored by EF4W reporter). At 100 µM [Ca2+], it destabilizes (gain flexibility); followed by recovery at 1 mM (almost overlap if reported by emission maxima), and stabilizes at saturated but excess 2 mM [Ca2+]. (C) Role of EF3, and motif-specific communication from EF3 filling to EF4. EF3 was disabled for Ca2+ binding and its effects on EF4 were monitored (EF3Q_EF4W mutant). Disabling EF3 destabilizes EF4, with no prominent effect of Ca2+. This mutant though binds Ca2+ (represented in (F) by ITC) with less affinity compared to wild type. (D) On the other hand, disabling EF4 (EF4Q_EF3W mutant), destabilizes the protein to some extent, but Ca2+ increases the stability significantly (though less

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than its wt), suggesting the role of EF3 over EF4. (E) and (F) are the Ca2+ binding isotherms of EF3W reporter and EF3Q_EF4W mutant protein by ITC. Recovery of Partially Destabilized EF4 After Ca2+ Binding Further, at higher concentrations of Ca2+, a significant gain in stability of EF4 was noticed, which is evident by increased C1/2. Interestingly at saturating concentrations like 1 mM Ca2+, the unfolding profile of emission maxima largely matches those of the apo form (Figure 6B). In conclusion, the collapsed EF4 gains structural stability as evidenced by a comparable profile with the apo protein, but with distinct conformational features. The different intermediate transition states are indicated by the change in m-values at different Ca2+ concentrations, occurring prior to the holo- or the fully- saturated state (Figure 5). The Role of EF3 We have described above the existence of intermediate stable conformational states of CDD and that some of these occur when EF3 is occupied by Ca2+ and followed by EF4 filling. In brief, EF3 is first in the hierarchy of sensing Ca2+ and causes a reversible structural collapse of EF4. This inter-motif coupling of EF3 and EF4 also makes it likely that EF3 filling is necessary for EF4 to bind Ca2+ and that the structural disintegration of EF4 caused by Ca2+ filling to EF3 is needed for the Ca2+ -binding properties of EF4. To further address the nature of coupling of both EF-hand motifs, we disabled EF3 by mutating the 12th Glu of the loop (EF3Q mutation) and monitored the effects on Ca2+ binding of EF4 (EF3Q-EF4W mutant). As shown in (Figure 6C), the mutant EF3Q-EF4W, which has only the EF4 motif capable of binding Ca2+, is less stable than the apo form of WT or EF4W (apo ∆C1/2(reporter-WT) = -0.32 M, holo ∆C1/2= 0.83). In addition, EF3Q-EF4W also loses Ca2+dependent conformational changes and loss or gain in stability, as observed during EF3 filling. This is surprising since EF4 in the EF3 disabled protein binds Ca2+ with less affinity (Kd 24 µM of wild type to 101 µM of EF3Q-EF4W) but with similar isotherm as wild-type protein (Figure 6E and 6F, Table S1). This decreased Ca2+ binding affinity to EF4 (EF3Q-EF4W protein) is consistent with having a positive cooperativity between EF3 and EF4 motifs with a Hill coefficient of 1.3.8 In stark contrast, however, disabling EF4 does not influence Ca2+-dependent gain in stability of EF3 greatly (mutant EF4Q-EF3W / apo ∆C1/2 (reporter-WT) = 0.09 M, holo ∆C1/2= 1 M) (Figure 6D). Thus, while disabling EF3 affects the structural properties of EF4 in

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Biochemistry

relation to Ca2+ -binding, the converse is not true: disabling EF4 has comparatively less impact on EF3. Discussion The study of Ca2+-dependent folding pathways and conformational dynamics is an emerging topic and has only been studied with a few EF-hand proteins.16,29-32 In this study, we found stable intermediate steps and inter-motif communication of the EF3 and EF4 sites during Ca2+ filling of CDD that is strikingly similar to those observed previously in NCS-1.16 In NCS-1, the C-terminal domain collapses during Ca2+-filling to an intermediate state with a fully folded Ca2+-bound EF3 and a partially folded, Ca2+-free EF4 site. EF4 motif is filled subsequent to the EF3 and only after proper folding of both EF-hands the N-terminal domain acquires the native state.16 In contrast to NCS-1, we found little cooperative folding and inter-domain communication between the N-terminal and C-terminal EF-hand pairs of CDD. This might be explained by the different domain arrangement of CDD and NCS-1. CDD is with respect to the EF-hand organization the closest homologue of CaM in brain and shares with CaM a flexible linker that in case of CDD contains four additional amino acids, which allow for an even more variable orientation of the domains with respect to each other than in CaM.33 The folding of both domains in CaM is independent and can occur in any order, which might be a general feature of nCaBPs that they share with their common ancestor.34 In members of the NCS family, both domains are separated by an U-shaped linker that imposes a tandem array in which both domains face each other.33 One consequence might be that in the case of NCS-1, folding of EF3 and EF4 is crucial for subsequent folding of the N-terminal domain. Collectively, the studies on inter-domain and intermotif communication point to the existence of stable intermediate conformations in EF-hand proteins. These semi-holo states are based on different folding mechanisms, which probably result from relatively subtle differences in structural architecture. A surprising finding of the present and a previous study16 is that increasing Ca2+ concentrations do not always impart a gain in stability, but might result in increased flexibility of an EF-hand motif that is not filled with Ca2+. It is likely that ligand binding is affected by folding and unfolding transitions and this highlights important differences between simultaneous and sequential Ca2+ filling. The structural underpinnings for simultaneous or sequential Ca2+ filling are not known but a difference in affinities of two EF-hand sites in one domain might favor step-wise filling. It leads to a possibility of the existence of partially filled

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intermediate states which under limited availability of [Ca2+]i in spines and and the existence of common targets, create a highly competitive environment, where EF-hand sensors compete with each other for Ca2+ and target binding.35-38 It is, in fact shown that intracellular Ca2+ levels promote the selective interaction of G protein-coupled receptor heteromer either with NCS-1 or with calneuron-1, which leads to differential modulation of allosteric interactions within the receptor heteromer.39 Therefore, nature of inter-motif communications would have impact on Ca2+-dependent target interactions.

Acknowledgements We acknowledge Syed Sayeed Abdul for his excellent technical support. Abbreviations CDD, Caldendrin, CD, circular dichroism, CaM, Calmodulin, GdmCl, guanidinium chloride. Supplemental Information Supplemental Information includes two figures and one Table can be found with this article online. ORCID Yogendra Sharma: 0000-0003-1345-8478 Author Contributions AC, Conception; AC, PSUK, PR, Design and acquisition of data; AC, YS, PSUK, PR, MRK; Analysis and interpretation of data; MRK, resources; AC, PSUK, YS, MRK; Drafting the article. Competing financial interests The authors declare no competing financial interests.

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