The Differential Response to Ca2+

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Differential Response to Ca2+ from Vertebrate and Invertebrate Calumenin is Governed by a Single Amino Acid Residue Sasirekha Narayanasamy, and Gopala Krishna Aradhyam Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00762 • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Biochemistry

Differential Response to Ca2+ from Vertebrate and Invertebrate Calumenin is governed by a single amino acid residue Sasirekha Narayanasamy and Gopala Krishna Aradhyam* Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai – 600036, INDIA. KEYWORDS Calumenin, EF-hand motifs, intrinsically unstructured protein, switch, Caenorhabditis elegans (C. elegans), Ca2+-binding proteins, Protein folding, Sarcoplasmic reticulum (SR), Spectroscopy

ABSTRACT

Calumenin (Calu) is a well-conserved multi EF-hand containing Ca2+-binding protein. In this work, we focused our attention on the alterations that calumenin has undergone during evolution. We demonstrate that vertebrate calumenin is significantly different from its invertebrate homologs with respect to its response to Ca2+-binding. Human calumenin (HsCalu1) is intrinsically unstructured in the Ca2+ free form and responds to Ca2+ with a dramatic gain in structure. Calumenin from C. elegans (CeCalu) is structured even in the apo form, with no conformational change on binding Ca2+. We decode this structural and functional distinction by identifying a single ‘Leu’ residue based switch located in the fourth EF-hand of HsCalu1, occupied by ‘Gly’ in the invertebrate homologs. We demonstrate that replacing Leu by Gly (L150G) in HsCalu1 enables the protein to adopt structural fold even in the Ca2+ free form, similar to CeCalu, leading to ligand compensation (adoption of structure in the absence of Ca2+). The fourth (of seven) EF-hand of HsCalu1 nucleates the structural fold of the protein depending on the switch residue (Gly/Leu). Our analyses reveal that Leu

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replaced Gly from fishes onwards is absolutely conserved in higher vertebrates, while lower organisms have Gly, not only enlarging the scope of Ca2+-dependent structural transitions but also drawing a boundary between the invertebrate and vertebrate calumenin. The evolutionary selection of the switch residue corroborates well with the change in the structure of the protein, its pleiotropic functions and seems extendable to the presence or absence of a heart in that organism.

INTRODUCTION In the event of speciation, orthologous genes that are separated evolutionarily far (in time) 1,2

from each other are likely to function identically.

Despite significant differences in amino

acid sequences, the orthologous proteins retain domain architecture when the function of the protein is indispensable to evolutionary fitness.

2,3

Deviations from such a pattern, in the

protein conformation, are due to synonymous/non-synonymous mutations and nucleotide insertions/deletions in the “hot-spot” sites of the protein.

4,5

Studying the effect of such

modifications in orthologs, in a highly conserved domain, such as EF-hands, that alter the protein’s structure and function assume extreme evolutionary and functional significance. In this study, we demonstrate this phenomenon explicitly in calumenin, a Ca2+-binding protein (CaBP) involved in many functions. Calumenin is an acidic, low-affinity CaBP that belongs to the CREC family (Cab45 6, Reticulocalbin 7, ERC-55

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and Calumenin

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) of EF-hand-containing CaBPs.11,12 The

primary sequence of HsCalu1 consists of seven predicted EF-hand domains (296 amino acids, ~34 kDa). Calumenin interacts with diverse proteins such as G551D-Cystic fibrosis transmembrane conductance regulator (CFTR) thrombospondin-1

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, Serum amyloid component P

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and

and is involved in the fundamental phenomenon of muscle contraction

and relaxation processes in vertebrates.16-18 Invertebrate homologue of calumenin, such as


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Biochemistry

that from C. elegans (CeCalu), shares 64% similarity and 48% identity with the human ortholog (Figure 1, upper panel). The only available report on CeCalu deals with its role in body size, muscle function, and cuticle formation with no mechanistic details.19 A comparative study, therefore, of CeCalu vis-à-vis HsCalu1 would provide insight into their functional similarities despite the divergent structures. The comparative study led to the identification of a key difference between HsCalu1 and CeCalu with respect to the response to Ca2+. HsCalu, like many other CaBPs, is intrinsically unstructured and gains structure upon binding Ca2+, whereas CeCalu is structured even in the apo form. We have identified the epicenter of this phenomenon in the protein, which is controlled by an amino acid residue (Leu150) that alters the mechanism of Ca2+-mediated structural switching in human calumenin. Mutating the specific Leu to a conserved residue (Gly) in HsCalu1 turns the switch off. Leu residue, in higher organisms, helps in nucleating the Ca2+-mediated structural fold of the protein. Our analysis provides evidence of Gly at the sixth position in the EF-hand loop of lower metazoans (lower than fish) that is substituted to a Leu residue in the EF-hand 4 in chordates and its correlation to the type of heart in the organism. The acquisition of a non-synonymous change (Gly to Leu) is a key evolutionary event engineered in calumenin during its recruitment in higher metazoans leading to its existence in an unstructured conformation.

Experimental methods Sub-cloning of HsCalu1, generation of deletion constructs and mutants. cDNA of human calumenin isoform1 (MGC: 3507961; UniProt Accession ID:- O43852) was obtained in pOTB7 non-expression vector as glycerol stock from Saf Labs Pvt. Ltd., Mumbai, India. HsCalu1 (891 bp) sequence was amplified using gene-specific primers containing NdeI and XhoI restriction adaptors at 5’ and 3’ ends respectively, making sure the 19 amino acids signal ACS Paragon Plus Environment

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sequence was removed. The amplified product was ligated into pTYB12 expression vector and clones were confirmed by nucleotide sequencing. Mutants (A32G, L150G, L150G/G187L) of HsCalu1 and a single mutant (G146L of CeCalu1) were generated by sitedirected mutagenesis. Serial deletion constructs of HsCalu1 gene from the N-terminal region (EF1, EF12, EF123, EF1234, EF12345 and EF123456) were generated by site-directed mutagenesis (insertion of a stop codon progressively after each of the EF-hand motifs). The following deletion constructs (EF4567, EF567 and EF234567) were generated by PIPE cloning method.20 All the mutants were confirmed by nucleotide sequencing. Protein expression and purification of HsCalu1, mutants, and fragments. The clones were transformed into E. coli (BL21 (DE3)) cells and grown in LB media containing ampicillin (100 µg/mL) at 37 °C for the overexpression of the chitin-binding domain-fused protein. At OD600 = 0.6, cells were induced with 100 µM of isopropyl-thio-β-galactoside (IPTG) and incubated at 23 °C for 18 hours. Cells were harvested, lysed in lysis buffer containing 50 mM Tris, 400 mM NaCl, 10% (v/v) glycerol and 5 mM CaCl2 (pH 8) by sonication (Vibracell Sonics and Materials, Inc. Newtown, CT, USA). The soluble fraction was loaded onto “chitin column” (New England Biolabs, MA, USA) pre-equilibrated with lysis buffer. The column was washed with the same buffer and incubated with the buffer containing 50 mM Tris, 400 mM NaCl, 10% (v/v) glycerol, 5 mM CaCl2 and 50 mM DTT (pH 8) for 20 hours at 4 °C to cleave chitin-binding domain tag. After incubation, the “tagless” proteins were eluted, concentrated and purified by gel filtration chromatography. The homogeneity of the concentrated proteins was assessed by SDS-PAGE (12%) and TricinePAGE (14%) (for EF1). Cloning, expression, and purification of calumenin from C. elegans (CeCalu). Calumenin gene (897 bp) from C. elegans (UniProt Accession ID:- G5EBH7) was amplified from total cDNA using gene-specific primers containing NdeI and EcoRI restriction adaptors ACS Paragon Plus Environment

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Biochemistry

at 5’ and 3’ ends respectively and cloned into a pTYB12 bacterial expression vector. The clone was confirmed by nucleotide sequencing. The plasmid containing CeCalu gene was transformed into E.coli (BL21 (DE3)) cells and grown in LB media containing ampicillin (100 µg/mL) at 37 °C for the expression of chitin-binding domain fused CeCalu. At OD600 = 0.6, cells were induced with 100 µM of IPTG and incubated at 23 °C for 18 hours. CeCalu was purified as “tag-less” protein using the same protocol mentioned for HsCalu1 purification. Fluorescence spectroscopy studies. All the buffer solutions used for Ca2+-binding studies were prepared using deionized water purified by Chelex-100 resin (Bio-Rad, Richmond, CA, USA). Intrinsic tryptophan (Trp) fluorescence emission spectra for all the proteins were recorded on a Fluorolog fluorimeter (HORIBA Jobin Yvon, NJ, USA) in 50 mM Tris and 50 mM NaCl (pH 8) by exciting the samples at 295 nm. The emission spectra were recorded from 310 to 400 nm with a slit width of 5 nm and a scan speed of 100 nm/min, each spectra being an average of 3 accumulations. Ca2+-binding to the proteins were monitored by titrating with CaCl2.

For all spectra, the background spectra of the buffer were recorded and

subtracted. Each spectrum represents independent experiments, which was repeated several times. Circular Dichroism Spectroscopy. CD spectra for all the proteins were recorded on a Jasco J-815 spectropolarimeter at room temperature in 0.1 cm and 1 cm path length quartz cell for far-UV (198-250 nm) and near-UV CD (250–350 nm) respectively. The spectra were recorded in 50 mM Tris, 50 mM NaCl (pH 8) buffer and increasing concentrations of CaCl2 (0.01 mM–5 mM) were used. Far-UV CD spectra were also recorded for the fragments (EF1, EF12, and EF123) in a hydrophobic buffer containing 50 mM Tris, 50 mM NaCl (pH 8) in the presence of either methanol (90%) or trifluoroethanol (70%). All the spectra were corrected for the solvent baseline. ACS Paragon Plus Environment

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Far-UV CD readings at 222 nm were recorded for HsCalu1 and L150G mutant of HsCalu1 from 20 to 95 °C with a scan rate of 1 °C/min controlled by a Peltier element. The spectra were recorded in 10 mM HEPES pH 7.2 containing 1 mM CaCl2. The dichroic activity at 222 nm as a function of temperature was recorded and corrected for the solvent baseline by subtraction of the CD spectra of the buffer. Ellipticity values at 222 nm were normalized to obtain fraction of protein unfolded using the standard equation and described as, ( ) = ( − )/( − ) θN and θD represent ellipticity of the native and fully unfolded forms respectively. The fraction unfolded values were calculated and fit to Boltzmann sigmoidal equation. Isothermal titration calorimetry (ITC) studies. All the ITC experiments were performed in 50 mM Tris and 100 mM NaCl (pH 8) at 25 °C in a VP-ITC calorimeter (MicroCal Inc., Northampton, MA, USA). The buffers and protein samples used for ITC experiments were centrifuged and degassed before loading onto the syringe and sample cell. In the case of HsCalu1, a typical reaction in which 3 µl aliquots of 4 mM CaCl2 were injected into the 45 µM of HsCalu1 in the sample cell (1.43 mL). A total of 70 injections were carried out for HsCalu1. For L150G mutant of HsCalu1 (10 µM), 3 µl aliquots of 2 mM CaCl2 was titrated. In the case of CeCalu, the protein concentration of 12 µM was titrated with 3 µl aliquots of 1 mM CaCl2. A total of 30 injections were performed for L150G mutant of HsCalu1 and CeCalu. Each peak in the thermogram corresponds to the injection of CaCl2 into the protein solution. The obtained data were subtracted from the heat of reaction obtained for the buffer. The data were best fit using Origin software package (MicroCal, Inc.). For every ITC experiment, the thermodynamic parameters, such as the association constant (KA), the stoichiometry (N), enthalpy (∆H) and entropy (∆S), were obtained from the ITC fit data whereas the dissociation constant (KD) was calculated from the association constant (KA) using the formula KD=1/KA. ACS Paragon Plus Environment

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Biochemistry

Prediction of protein disorder. The prediction of disordered regions in HsCalu1 was obtained by MetaPrDOS

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using the web server (http://prdos.hgc.jp/cgi-bin/meta/top.cgi).

The software predicts the disordered region in the input amino acid sequence by combining the prediction results of several methods. The predictors used in the meta approach are DISOPRED2, DISPROT (VSL2P), DISpro, POODLE-S, and PrDOS. Each predictor draws an individual score for the input amino acid residues. A graphical plot, which is shown as the output, demonstrates the tendency of disorder for each amino acid residue indicating the probability of being folded (below 0.5) or disordered (above 0.5). A false positive rate of 5% was applied for the prediction of the disordered regions in the sequence. The consensus drawn by the MetaPrDOS indicates two shorter disordered segments (less than 12 amino acids in length) and a long disordered region (EF1; 48 amino acids) in the N-terminal region. Only the predicted disordered region which is above ~12 amino acid residues were considered for experimental verification. In order to validate the prediction results, the predicted N-terminal disordered region (EF1) was generated by inserting a stop codon after the EF1 in the full length protein. On the other hand, the disordered region (EF1) was removed from the fulllength protein (EF234567) by PIPE cloning method and both the constructs were purified in isolation. The native conformation of both the fragments was assessed by tryptophan fluorescence (only for EF234567) and far-UV CD studies. Results and Discussion One protein, two species, and two Ca2+ related conformations. We present here the case of a protein from two evolutionarily distant species that responds with completely opposite features towards Ca2+-binding despite sharing closely similar primary sequence: CeCalu, 298 amino acid residues, with five putative EF-hand motifs shares 48% identity and 64% similarity with HsCalu1. This implies that the nature and mode of Ca2+ signaling by


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calumenin in lower vertebrates is different, and the same protein adapts to a different mode of signaling events upon binding to Ca2+ in higher organisms. Human ‘calumenin’ is natively unstructured in the apo form and gains structure upon Ca2+-binding. Tryptophan (Trp) fluorescence emission spectra of the hepta EF-Hand apoHsCalu1 when excited with 295 nm light lead to the emission maxima being at ~353 nm (Figure 1a). Far-UV Circular Dichroism (CD) spectra of HsCalu1, recorded in the 198-250 nm range depicts a negative peak in the 200-205 nm range, indicative of the protein structure being random coil nature (Figure 1b). Additionally, the tertiary structure content monitored in the 250-350 nm range (by near-UV CD), demonstrates minimal spectral signatures indicating the random disposition of the aromatic amino acids (Figure 1c). These results collectively demonstrate the intrinsically unstructured nature of the apo-HsCalu1. Further examination of the Ca2+-related changes in protein conformation demonstrated very interesting observations. The addition of Ca2+ in the protein solution leads to a blue shift of ~11 nm (λem, max 341 nm) of the fluorescence emission peak (Figure 1a). Ca2+ also induces changes both in far- and near-UV CD spectra indicating that the Ca2+ bound protein adopts a largely α-helical conformation (Figure 1b and 1c). These Ca2+-induced prominent changes in HsCalu1, thus, demonstrate that the protein adopts a structured conformation in the Ca2+-bound form as also demonstrated previously.

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It is to be mentioned that the conformation of HsCalu1 is extremely specific to

Ca2+ and Mg2+ does not induce a significant change in the unstructured nature of the protein (Figure S1 and S2).


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Biochemistry

Figure 1. The difference in the native conformation of calumenin from human and nematode. Upper panel represents the pairwise alignment of HsCalu1 and CeCalu protein sequences. The predicted secondary structures are shown on top and bottom of the pairwise alignment for HsCalu1 and CeCalu respectively. The Ca2+-binding sites are indicated as solid lines and the coil regions shown as dashed lines. The first EF-hand of HsCalu1 lacks one of the flanking helices shown as dashed rectangle. The ‘similar’ and ‘identical’ amino acids between HsCalu1 and CeCalu are highlighted in grey and black respectively. The single amino acid switch in HsCalu1 ((150Leu) at the sixth position in the loop region of EF-hand 4) and the corresponding amino acid ACS Paragon Plus Environment

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(Gly) at the sixth position in CeCalu (shown in the red box) are marked by an asterisk. Lower panel, (a) Ca2+-induced conformational changes in HsCalu1 were monitored by the intrinsic Trp fluorescence emission spectra of the protein (1 µM). (b) Far-UV CD spectra of HsCalu1 (4.5 µM) were recorded in the λ198-250 nm range. The apo form of HsCalu1 is unstructured and upon binding to Ca2+, undergoes a conformational change. In both (a) and (b) black and red lines represent apo and holo forms (recorded in the presence of 1 mM CaCl2) of HsCalu1 respectively. (c) Conformational changes in the tertiary structure of HsCalu1 (14 µM) upon Ca2+-binding as determined by near-UV CD spectra. Apo-HsCalu1 (black line) shows a major band at 262 nm, characteristic of phenylalanine residues that remains unaltered upon Ca2+-binding. The loss of CD signal at 280 and 292 nm wavelengths is indicative of the highly mobile side chains of the Tyr and Trp residues demonstrating unfolded structure. Upon binding to Ca2+ (1 and 5 mM CaCl2, dashed red line and solid red line respectively), the bands corresponding to tryptophan and tyrosine residues were well resolved indicative of the protein adopting a folded conformation. (d) Conformational changes of CeCalu (1 µM) in both apo and holo form were monitored by Trp fluorescence. CeCalu shows fluorescence emission maxima at 348 nm and no further fluorescence shift was observed upon Ca2+-binding. (e) Far-UV CD spectra of purified CeCalu (4.5 µM) was recorded in the λ198-250 nm range. Apo form of the protein is structured and Ca2+-binding does not lead to any conformational changes. In the spectra (d) and (e), black and red lines represent apo and holo (1 mM CaCl2) form of CeCalu respectively. (f) Changes in the tertiary structure of CeCalu (14 µM) upon Ca2+-binding was determined by near-UV CD spectra. The spectra for the apo-CeCalu (black solid line) show the peaks corresponding to Phe and Trp residues in the absence of Ca2+ indicative of the folded structure. No significant conformational changes in the tertiary structure of CeCalu were observed upon binding to 1 mM (red dashed line) and 5 mM CaCl2 (red solid line).

C. elegans ‘calumenin’ is structured in the apo form and Ca2+ has no influence on its conformation. As we were interested in understanding how Ca2+ signaling propagates in evolution, we now compare the nature of Ca2+-mediated structural changes in invertebrate calumenin with those observed in human protein. For this purpose, we chose calumenin from C. elegans, a nematode, which (hierarchy-wise) is placed much below in the evolutionary tree. CeCalu consists of 298 amino acids (without the signal sequence, ~34 kDa). For our study, we cloned CeCalu gene (897 bp) into the bacterial expression vector and purified the protein as mentioned in the experimental procedure. Apo-CeCalu shows an emission maximum at ~348 nm in its fluorescence emission spectrum, which is almost 5 nm blueshifted than apo HsCalu1 (Figure 1d), and two negative peaks at ~208 and 222 nm in far-UV CD spectrum, both observations being indicative of helical conformation (Figure 1e). The tertiary structure signature obtained in near-UV CD spectra in the 250-350 nm range elicited defined peaks in the aromatic region (290, ~275 and ~264 nm) (Figure 1f). Thus, unlike


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Biochemistry

HsCalu1, apo-CeCalu adopts an ordered/folded structure even in the absence of Ca2+. We next studied the effect of Ca2+ on the conformation of CeCalu and notice that Ca2+ does not affect the secondary and tertiary structure of CeCalu (Figure 1e and 1f), as well as Trp fluorescence significantly (Figure 1d), suggesting that unlike HsCalu1, the mode of Ca2+ related signaling mediated by CeCalu is entirely different.

Identification of structural Pleiotropic Centre(s) in HsCalu1. To understand the mechanistic aspects of why HsCalu1 is unstructured in the apo form, while CeCalu is not, we used a variety of informatics tools to identify the region(s) in the human protein that is causative for the disordered conformation. The consensus drawn by MetaPrDOS analysis is a long disordered region at the N-terminus and two shorter disordered segments (Figure S3). In our study, we generated a construct (1-48 amino acids) called “EF1” (~5.5 kDa), the domain that is predicted as a disordered region and studied its intrinsic structural properties. Far-UV CD measurements indicate unstructured/random conformation (as predicted), and unlike HsCalu1 (full-length protein), Ca2+ did not elicit any structural changes in the peptide (Figure S4). On the other hand, the N-terminal truncated mutant/fragment, HsCalu1∆1-48 (wherein the predicted disordered region is removed from the full-length protein) is also disordered in the apo form (Figure 2k and 2l), a fact that does not correlate with the computationally predicted result. Similar to its full-length protein, HsCalu1∆1-48 also folds only in the presence of Ca2+, which is well evident from far-UV CD studies. Based on these results, we conclude that HsCalu1 is an intrinsically disordered protein and folds upon binding to Ca2+. Fold initiator EF-hand motif in HsCalu1. We generated a number of overlapping constructs (Figure 2, upper panel) namely EF12 (~10 kDa), EF123 (~14 kDa), EF1234 (~20 kDa), EF12345 (~24 kDa), EF123456 (~29 kDa), EF4567 (~20 kDa), EF567 (~15 kDa) and EF234567 (~30 kDa) to pinpoint the specific EF-hand motif that enables Ca2+-dependent ACS Paragon Plus Environment

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Figure 2. EF-hand 4 initiates protein folding in HsCalu1. Upper panel depicts the schematic representation of HsCalu1 and all the fragments generated. HsCalu1 (296 amino acids, without signal sequence) consists of seven predicted EF-hand motifs. The label at the left-hand side of each fragment denotes the combination of EF-hand motifs as per occurrence in the primary sequence. These fragments were generated for determining the EF-hand motif which initiates Ca2+-induced protein folding processes. Lower panel demonstrates Trp fluorescence (excitation: 295 nm; emission spectra: 310-400 nm) and far-UV CD spectra (198-250 nm) for all the constructs (a,b; EF12, c,d; EF123, e,f; EF1234, g,h; EF12345, i,j; EF123456, k,l; EF234567, m,n; EF4567 and o,p; EF567). In all the panels, the black and red line represents apo and holo (recorded in the presence of 1 mM CaCl2) forms respectively.


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Biochemistry

structural transition (“fold initiator EF-hand motif”). Each construct was purified and Ca2+induced folding was studied (Figure 2, bottom panel). As seen by the effect of Ca2+ on fluorescence emission (blue shift in λmax and increase in quantum yield) and ellipticity values upon binding Ca2+ (1 mM), the fragment comprising EF1234 seems to initiate folding (Figure 2e and 2f). This folding process is prominent in the fragment EF12345, implying that Ca2+coupled protein folding is more pronounced when the EF-hand motifs 4 and 5 form “pairs” (Figure 2g and 2h). The fragments that do not have EF4 and EF5 (i.e., EF12 (Figure 2a and 2b), EF123 (Figure 2c and 2d)) lack the ability to adopt a structure upon addition of Ca2+. However, these fragments (EF12 and EF123) adopt a helical conformation in the presence of methanol as observed by far-UV CD studies (Figure S5). The C-terminal fragments (EF4567 and EF567) that have EF-hands following EF4 and EF5 are also unresponsive to adopting a more defined structure (Figure 2m and 2n for EF4567; 2o and 2p for EF567). These observations demonstrate that Ca2+ binding to EF-hand 4 and 5 in HsCalu1 “nucleate” the formation of structure in the core of the protein followed by the Ca2+-binding to EF2/EF3, aiding the protein to adopt an initial structure. Later, the presumably partially folded structure allows EF-hand motif 6/7 to bind Ca2+ and eventually helps the rest of the protein to fold into a well-ordered conformation (Figure 6 as part of the discussion). Based on the experimental results on EF-hand 1 peptide (Figure S4) and from ITC studies for full-length HsCalu1 identifying the number of Ca2+-binding sites as seven (Figure 5a), we propose that EF1, despite being unpaired, binds Ca2+ when all the other six EF-hands are occupied with Ca2+ during the folding process.

Choice of a “Leu (in vertebrates) or a Gly (in invertebrates)” residue in the corresponding EF-hand. We compared the sequence of the 12 amino acid loop regions of EF-hand motifs of HsCalu1 with the consensus loop sequences (amino acid preference at each ACS Paragon Plus Environment

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position in the EF-hand loop) of other EF-hand CaBPs. The EF-hands 1 and 4 have Ala and Leu residues at the sixth position respectively (Figure 3, Upper panel), which is more favored by Gly (96%). Gly residue at this position helps in the required bending of the loop and facilitates the unusual main chain conformation.23,24 Therefore, we ‘zeroed in’ our attention at the sixth position in EF-hand 1 and 4 of HsCalu1 and mutated these residues to Gly. Fluorescence and far-UV CD spectral studies on A32G mutant of HsCalu1 demonstrate characteristics similar to those of the wild-type: largely unstructured in the absence of Ca2+ and gain of the structure upon binding Ca2+ (Figure 3a and 3c). Remarkably, mutating Leu to Gly (L150G) in HsCalu1 leads the protein into an ordered conformation even in the apo (Ca2+ free) form and changes its energy landscape for folding (Figure 3d), unlike the natively unstructured conformation of its wild-type protein (Figure 1b). Further, no significant structural changes are elicited upon binding Ca2+ (Figure 3b and 3d), intriguingly the behavior that is demonstrated by CeCalu (Figure 1e).


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Biochemistry

Figure 3. Native conformation of A32G and L150G mutants of HsCalu1 in the apo and holo form. Upper panel shows the alignment of seven EF-hand motifs of HsCalu1. Gly at the sixth position in an EF-hand loop is highly conserved (highlighted in green). In EF-hand 1 and 4 respectively Gly at the sixth position in HsCalu1 is substituted to Ala and Leu (highlighted in red). Bottom panel shows the Trp fluorescence (310-400 nm) and far-UV CD spectra (198-250 nm) for the mutants. Ca2+-binding to A32G mutant of HsCalu1 leads to a blue shift from 352 nm to 345 nm (a) and induces helical secondary structure (c). (b) Apo form of L150G mutant of HsCalu1 shows λem, max at 347 nm and no further shift was observed upon Ca2+-binding. (d) Far-UV CD spectra of L150G mutant of HsCalu1 demonstrate structured conformation in the absence of Ca2+ and Ca2+ -binding does not lead to any conformational changes. In both spectra, black and red lines represent apo and holo form (recorded in the presence of 1 mM CaCl2) respectively.

These results indicate that the EF-hand 4 of HsCalu1 selected Leu over Gly at the sixth position in order to remain in a dormant disordered conformation. To examine whether the position of this switch residue can be moved in the protein (for HsCalu1 to adopt nativelyACS Paragon Plus Environment

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unstructured conformation), we substituted Gly at the sixth position in the loop of the fifth EF-hand to Leu in the L150G of HsCalu1 (L150G/G187L). This double mutant exists in a structured conformation, and replacement of Gly in EF-hand 5 to Leu does not yield results similar to that of its wild-type protein as observed by far-UV CD studies (Figure S6). Similarly, mutating Gly at the sixth position in the corresponding EF-hand 4 of CeCalu to Leu did not revert back to the unstructured conformation (Figure S7) implicating the importance of the residues in the EF-hand 4 loop of HsCalu1. Based on these results, we conclude that there exists a binary switch in HsCalu1, which is controlled by the choice of a single amino acid that is

150

Leu. We further quantitated the effect of Ca2+ on the intrinsic stability of the

protein fold by recording changes in ellipticity value at 222 nm (θ222), plotted as a function of temperature on HsCalu1 and L150G mutant of HsCalu1 (Figure 4). These experiments indicate that the Ca2+-bound HsCalu1 (Tm is 68 °C) is thermostable (Figure 4a) whereas the L150G mutant of HsCalu1 (Figure 4b) loses its stability (Tm, 58 °C for holo form) thus differs in biophysical properties compared to wild-type.

Figure 4. Thermal unfolding studies of HsCalu1 and L150G mutant of HsCalu1. Far-UV CD ellipticity value at a single wavelength (λ222 nm) was recorded for HsCalu1 and L150G of HsCalu1 with increase in temperature, ranging from 20 °C-95 °C at an increment rate of 1°C per min. HsCalu1 (4.5 µM) and L150G mutant of HsCalu1 (4.5 µM) were prepared in 10 mM Hepes buffer pH 7.2 containing 1 mM CaCl2. Panel (a) and (b) demonstrate the change in the ellipticity value at 222 nm with increase in temperature shown as a fraction of unfolded protein, as a function of temperature for HsCalu1 and L150G mutant of HsCalu1 respectively. The calculated melting temperature (Tm) for both proteins demonstrate that HsCalu1 is highly stable (Tm: 68 °C) than L150G mutant of HsCalu1 (Tm: 58 °C). ACS Paragon Plus Environment

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Biochemistry

Choice of a “Leu (in vertebrates) or a Gly (in invertebrates)” residue and Ca2+binding affinity. We investigated the energetics of ion binding of wild-type HsCalu1, CeCalu and also sought to determine the impact of the L150G mutation on Ca2+-binding affinities. Calorimetric changes upon addition of Ca2+ to apo-HsCalu1 demonstrates a typical calorimetric reaction in which the heat evolved per injection increases at low Ca2+ concentrations till the molar ratio (of the interacting molecules) of ~3 is reached and then gradually decreases till the background signal is attained (Figure 5a). The ITC data fit best to “sequential five sets of binding sites” model with stoichiometry (N value) of seven (corresponding to seven EF-hands). The affinity calculations of HsCalu1 revealed low to moderate affinity Ca2+-binding sites with dissociation constants (KD) ranging from 2 µM to 172 µM. These KD values (Table 1) and the number of binding sites are consistent with the previous report.10 A typical binding isotherm for Ca2+ binding to L150G mutant of HsCalu1 is shown in Figure 5c. Ca2+ binding to L150G mutant of HsCalu1 is an exothermic process and titration data best fit to “one-set of sites model” with an N-value of 4.5 (number of binding sites) (Figure 5c). The derived KD value is about 24 nM, which is lower by several orders of magnitude (103) than its wild-type, suggesting that the mutation mediated structural change leads to enhance Ca2+ binding affinity.


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Figure 5. Isothermal titration calorigram of Ca2+-binding to calumenin orthologs. (a) Isothermogram of Ca2+-binding to HsCalu1. HsCalu1 in the sample cell (1.43 mL) was titrated with 70 injections, 3 µl aliquots of 4 mM CaCl2 into 45 µM of the protein. The upper trace in all the panels demonstrate the heat changes upon titration of CaCl2 into the protein solution. The lower trace demonstrates the plot of kcal/mol of heat absorbed/released per injection of CaCl2 as a function of ligand to protein molar ratio and the best ‘least-squares fit’ of the data. Ca2+-binding to HsCalu1 best-fit to the ‘sequential binding model’. (b) Ca2+-binding to CeCalu as demonstrated by ITC. CeCalu (12 µM) in the sample cell was titrated with 30 injections, 3 µl each of 1 mM CaCl2. The data fit best to ‘one set of sites’ model. (c) Isothermal titration calorigram of Ca2+-binding to L150G mutant of HsCalu1. L150G mutant of HsCalu1 (10 µM) in the sample cell was titrated with 30 injections, 3 µl each of 2 mM CaCl2 stock solution. The data best fit to ‘one set of sites’ model and the calculated thermodynamic parameters for all the experiments are described in Table 1. For all the ITC experiments, the protein and ligand were prepared in 50 mM Tris pH 8 containing 100 mM NaCl and the titrations were carried out at 25 °C. The data were analyzed by Origin software.

On the other hand, Ca2+ binding to CeCalu is an exothermic process and the data best fit to “one-set of sites model” with N-value of 2.45 (number of binding sites, Figure 5b). Similar to L150G mutant of HsCalu1, CeCalu also binds Ca2+ with comparatively a significantly high affinity with KD of 35 nM. The thermodynamic parameters obtained for all the ITC experiments are given in Table 1. Thus, both orthologs also differ in Ca2+ binding affinities. Together, these results suggest that the choice of a single amino acid (150Leu or

150

Gly)

dictates not only the conformational state of the protein but also Ca2+-binding affinity.


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Biochemistry

The choice of Leu (in vertebrates) or Gly (in invertebrates) and the evolution of Ca2+ signaling. Calumenin is ubiquitously expressed in metazoans but is absent in prokaryotes and fungi. To explore whether the switch residue (Leu) of calumenin orthologs is conserved over deep evolutionary time and also to get an evolutionary history of the choice of either Leu or Gly is present in calumenin among metazoans, we aligned protein sequences from different organism (Figure S8). Since calumenin is maximally expressed in the heart

9,10

, the choice of

either ‘Gly’ or the ‘Leu’ also seems to be a signature of the type of heart that an organism develops. Multiple sequence analysis reveals that Gly residue invariably exists in the basal metazoans, such as poriferans and cnidarians which lack heart (Table 2). Acoelomates (Platyhelminthes), pseudocoelomates (e.g., C. elegans) and some of the deuterostomes (hemichordata and echinodermata) which have a Gly residue also lack a well-defined heart though/but have a specialized type of blood vascular system (which can be either ‘open’ or ‘closed’). So far as the protein sequences we could obtain from the databases, the amino acid


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at the sixth position in the loop region of all the EF-hand motifs of calumenin from lower organisms is never occupied by Leu residue, with Gly being well conserved. Table 2. Demonstrates the distribution of calumenin among metazoans, drawn as per the

‘tree of life.’ The amino acids at the sixth position in the corresponding EF-hands loop of all the calumenin orthologs are indicated. Gly at the sixth position is highly conserved in lower metazoans which have a poor vascular based circulatory system and the amino acid Leu at the sixth position is evolved from fishes.

Following them in the evolutionary tree are the early chordates viz. fishes (Sarcopterygii and Actinopterygii) that have well-defined heart regulated by CICR process (Ca2+-induced Ca2+ release) and form a ‘border’ between the higher and lower organisms. These organisms have Leu/Gly/Thr/His/Met residue at the same position. Among chordates, the source of Ca2+ utilization for myocyte contraction differs among species, depending on the lifestyle of the organism.25, 26 In these organisms the choice of the residue seems to depend on the lifestyle of


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Biochemistry

the organisms, whether it is sedentary (Gly) or active (Leu). Starting from Aves and mammals (the heart of endothermic animals), SR-mediated Ca2+ cycling are indispensable for their strong and fast contracting hearts.26 Meanwhile, analyzing the corresponding EF-hand loop sequences in chordates (amphibians, reptiles, aves, and mammals), we have identified a 100% conservation of Leu residue in all the organisms, revealing a distinct “switch over” to Leu from Gly (Table 2). Calumenin being expressed strongly in cardiac muscles of mammals9,10 associates with SERCA

16,17

and ryanodine receptor

18

suggesting its role in the regulation of Ca2+

storage/shuttling in SR. Thus, the “Leu” form (i.e., wild-type HsCalu1) functions as an efficient regulator of Ca2+ homeostasis (as it can help in sequestering and release Ca2+ easily, because of its low-affinity Ca2+-binding sites) compared to the Gly variant (i.e., C. elegans type). Therefore, it is plausible that the presence of Leu at the sixth position in EF-hand 4 of calumenin may be a contributing factor in the evolution of higher resting and maximal heart rates in vertebrates. Hence, we hypothesize that calumenin has adopted time-dependent changes in an evolutionary scale and functions according to the organism’s physiological requirement.


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Figure 6. The proposed model of Ca2+ induced folding of HsCalu1. HsCalu1 exists as unstructured protein in the absence of Ca2+ (Step 1). According to our data, EF-hand 4 forms the ‘nucleation site’ for Ca2+ induced folding (Step 2) and pairs with EF-hand 5. Ca2+-induced conformational changes in EF-hand 4 and 5 facilitate EF-hands 2 and 3 to bind Ca2+ followed by EFhands 6 and 7 (Step 3 & 4). EF-hand 1 is unpaired and binds Ca2+ last indicated by a dashed circle.

In conclusion, we demonstrate a rare example of how a single point mutation, L150G, endows HsCalu1 with an ability to exist as natively structured conformation and binds Ca2+ tightly, similar to its ancestral CeCalu. Thus, our study illustrates how structural diversity arises rapidly among calumenin orthologs through a non-synonymous mutation during the evolution of higher organisms. We, therefore, hypothesize that a Leu dependent structural switch is at the heart of the multiple functions for this molecule, which offers an evolutionary advantage to perform multiple functions in chordates. Large conformational changes as a consequence of a point mutation in a protein are infrequent. We demonstrate here an excellent


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Biochemistry

example of such a large structural change in a protein from the smallest change possible, for function.

Table of Contents Graphics (TOC)

ASSOCIATED CONTENT Supporting Information. Mg2+ binding to HsCalu1 as monitored by Trp fluorescence and far-UV CD spectra (Figure S1), Mg2+-binding to HsCalu1 as determined by ITC (Figure S2), Prediction of structural disorder in HsCalu1 by MetaPrDOS (Figure S3), Far-UV CD spectra of EF1 (Figure S4), Effect of methanol on the secondary structure of fragments EF12 and EF123 (Figure S5), Far-UV CD spectra of L150G/G187L double mutant of HsCalu1 (Figure S6), Far-UV CD spectra of G146L mutant of CeCalu (Figure S7), Multiple sequence alignment of calumenin orthologs (Figure S8). AUTHOR INFORMATION Corresponding Author * Email: [email protected]


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Author Contributions GKA and SN conceived and designed the study. SN performed the experiments. GKA and SN analyzed the results and wrote the paper.

Funding Sources SN thanks Council of Industrial and Scientific research (CSIR) for fellowship during her Ph.D. This work was supported by funding from IIT Madras and Board of Research in Nuclear Sciences (BRNS) (Grant no. 2012/37B.52.BRNS), Dept. of Atomic Energy (DAE) Govt. of India. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Prof. K. Subramanian, Department of Biotechnology, IIT Madras, for providing initial help in cloning the CeCalu gene. We thank Ms. Jayashree Aradhyam for editing the manuscript. We thank DST-FIST facility, Department of Biotechnology, IIT Madras for the instrumentation facility.

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The Differential Response to Ca2+

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