Structural and biochemical characterization of neuronal calretinin ...

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domain I–II (residues 1–100) ... domain (residues 1–100) of rat calretinin (CR). CR, with ... considered important in maintaining nontoxic levels of free.
Eur. J. Biochem. 268, 6229–6237 (2001) q FEBS 2001

Structural and biochemical characterization of neuronal calretinin domain I– II (residues 1– 100) Comparison to homologous calbindin D28k domain I–II (residues 1 –93) Małgorzata Palczewska1, Patrick Groves1, Attila Ambrus2,*, Agata Kaleta1, Katalin E. Ko¨ve´r3, Gyula Batta4 and Jacek Kuz´nicki1,5 1

Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland; Department of Biochemistry and Molecular Biology, University of Debrecen, Hungary; 3Department of Inorganic Chemistry, and 4Research Group for Antibiotics, Department of Chemistry, University of Debrecen, Hungary; 5 International Institute of Molecular and Cell Biology, Warsaw, Poland 2

This study characterizes the calcium-bound CR I – II domain (residues 1–100) of rat calretinin (CR). CR, with six EF-hand motifs, is believed to function as a neuronal intracellular calcium-buffer and/or calcium-sensor. The secondary structure of CR I–II, defined by standard NMR methods on 13C,15N-labeled protein, contains four helices and two short interacting segments of extended structure between the calcium-binding loops. The linker between the two helix–loop–helix, EF-hand motifs is 12 residues long. Limited trypsinolysis at K60 (there are 10 other K/R residues in CR I–II) confirms that the linker of CR I–II is solvent-exposed and that other potential sites are protected by regular secondary structure. 45Ca-overlay of glutathione S-transferase (GST)–CR(1–60) and GST–CR(61–100) fusion proteins confirm that both EF-hands of CR I–II have intrinsic calcium-binding properties. The primary sequence and NMR chemical shifts, including calcium-sensitive glycine residues, also suggest that both EF-hand loops of CR I–II bind

calcium. NMR relaxation, analytical ultracentrifugation, chemical cross-linking and NMR translation diffusion measurements indicate that CR I –II exists as a monomer. Calb I–II (the homologous domain of calbindin D28k) has the same EF-hand secondary structures as CR I–II, except that helix B is three residues longer and the linker has only four residues [Klaus, W., Grzesiek, S., Labhardt, A. M., Buckwald, P., Hunziker, W., Gross, M. D. & Kallick, D. A. (1999) Eur. J. Biochem. 262, 933–938]. In contrast, Calb I– II binds one calcium cation per mono-meric unit and exists as a dimer. Despite close homology and similar secondary structures, CR I–II and Calb I–II probably have distinct tertiary structure features that suggest different cellular functions for the full-length proteins.

Calretinin (CR) and calbindin D28k (Calb) are homologous calcium-binding EF-hand proteins with 59% sequence identity (rat forms) [1 – 3]. The proteins contain six

helix –loop–helix motifs (EF-hands) in which the loops carry the calcium-binding ligands. The rat sequences are 271 (CR) and 261 (Calb) amino acids long [4,5]. Both proteins have distinct, predominantly neuronal cellular distributions [3,6 –9] and Calb is additionally found in the digestive system [10]. Both proteins are used as markers for a subset of neurons and several neurodegenerative diseases [11 –13]. Immunohistochemistry of CR is used to distinguish adenocarcinomas from mesothelioma [14,15], with CR playing a role in the early stages of mesothelioma [16]. In contrast, Calb is found in subpopulations of neuroendocrine phenotypes of some carcinoids and small-cell carcinomas [17]. EF-hand proteins act as intracellular calcium-sensors, linked to protein signaling cascades, and/or calcium-buffers [3]. For example, calmodulin binds to more than 100 different proteins in a calcium-specific manner, while parvalbumin is considered important in maintaining nontoxic levels of free intracellular calcium through its buffering ability [3]. The function of CR is unclear 2 there is evidence for both buffer and sensor roles (reviewed in [18]) but no definite target protein has yet been identified for CR to support a sensor role. Calb appears to play a buffer role in neurons [19] and possibly facilitates calcium uptake through the digestive system [10]. However, there is evidence that intestinal brush border membrane alkaline phosphatase

Correspondence to J. Kuznicki, Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland. Fax: 1 48 22 822 53 42, Tel.: 1 48 22 659 31 43, E-mail: [email protected] or to G. Batta, Research Group for Antibiotics, Department of Chemistry, Egyetem te´r. 1, University of Debrecen, PO Box 70, H-4010 Debrecen, Hungary. Fax: 1 36 52 512 914, Tel.: 1 36 52 512 900 extn 2370, E-mail: [email protected] Abbreviations: CR, calretinin (rat isoform, unless otherwise stated); CR I–II, calretinin residues 1 –100 consisting of the first pair of EF-hand motifs; Calb, calbindin D28k (rat isoform, unless otherwise stated); Calb I–II, calbindin D28k 1–93 consisting of the first pair of EF-hand motifs; Calb III –IV, calbindin D28k 79– 193, consisting of the second pair of EF-hand motifs; CSI, chemical shift index; GST, glutathione S-transferase; DOSY, diffusion ordered spectroscopy. *Present address: The University of Arizona, Department of Chemistry, 1306 E University, Tucson, AZ 85721–0041, USA. Note: a web page is available at http://www.nencki.gov.pl/labs/cbplab/kuzhome.htm (Received 28 June 2001, revised 24 September 2001, accepted 4 October 2001)

Keywords: calretinin; calcium; calbindin D28k; EF-hand; NMR secondary structure.

6230 M. Palczewska et al. (Eur. J. Biochem. 268)

and caspase-3 might serve as functional targets for Calb [20,21]. The biochemical characterizations of CR [22–25] and Calb [26 –29] suggest both proteins undergo significant calcium-dependent structural changes. In particular, calcium-modulated exposure of hydrophobic surfaces, perhaps the most important sensor characteristic, has been reported for both CR and Calb [22,24,30]. The biochemical properties of CR and Calb are similar to those of calmodulin, the best-known EF-hand calcium sensor. It is difficult to localize the biochemical properties of CR and Calb to particular domains but this information can be obtained by studying protein fragments. For example, the structure of the calcium-bound first domain of Calb, comprising the first two EF-hands (Calb I–II, residues 1–93), was recently reported [31]. Calb I–II binds a single calcium and forms a homodimer [31]. We have expressed and purified the homologous domain of CR (CR I–II, residues 1–100) [23,32]. This has allowed us to analyze the biochemical and structural properties of CR I– II and to compare them with Calb I–II. Our data show that CR I –II and Calb I –II share virtually the same EF-hand structures. However, a shorter helix B in CR I–II accentuates a longer linker loop between the EF-hands of CR I–II compared to Calb I–II. In contrast to Calb I –II, CR I–II shows no tendency to dimerize and both EF-hands of CR I–II bind calcium. We conclude that the significant structural and biochemical differences between CR I –II and Calb I –II are related to a region of poor sequence identity (residues 54–79 of CR). We expect the differences between CR I–II and Calb I –II will be present in the full-length proteins and that these differences may play a role in the distinct cellular functions of CR and Calb.

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Sequence/structure analysis Protein sequences for rat CR and rat Calb were retrieved from GenBank (accession numbers X66974 and M31178). The relevant parts of the N-terminal modified CR I– II and Calb I –II sequences were aligned with SeqWeb (Oxford Molecular Group) using the BESTFIT algorithm. NMR determination of secondary structure A standard method of assigning protein backbone HN, NH, CA, HA, CB, HB and CO chemical shifts was used, based on protocols described in Cavanagh et al. [35] and Sattler [36]. The sample contained 1 mM 13C,15N-labeled CR I–II, 50 mM Tris pH 7.7, 25 mM NaCl, 10 mM CaCl2. The experiments included HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, CBCANH, CBCA(CO)NH (H)CC-TOCSY(CO)NH, H(CC)-HEHAHA(CO)NH, HNHA, and HSQC. The experiments were processed using FELIX (MSI, San Diego). The AUTOASSIGN program [37] was used to obtain automatic assignments. All assignments were manually checked for residue type and compared to Calb I–II assignments [31] in homologous regions using FELIX (MSI software) or SPARKY [38]. Full experimental details are included in BMRB entry 5156. Spectra at pH 5.6 were more complete but of poorer quality (more overlapping peaks in the area of the HSQC corresponding to unstructured peptide). The assignment at pH 7.7 was extended by a series of experiments at pH 6.7 as few of the signals undergo large pH dependent changes in chemical shift. A restricted set of triple resonance data was collected on a sample in which the pH was lowered to 6.7, leading to a more complete assignment. Limited trypsinolysis of CR I –II

E X P E R I M E N TA L P R O C E D U R E S Expression and purification of CR and CR fragments Recombinant CR and CR fragments were expressed from Escerichia coli as glutathione S-transferase (GST) fusion proteins and were purified as described previously [23,33]. GST–CR(1–60) and GST–CR(61 –100) were prepared using the same techniques as described for the preparation of other GST – CR fragments [23,33]. Extracellularly expressed CR I–II using Pichia pastoris was purified by DEAE chromatography [32] and the proteins eluted were desalted by dialysis. The resulting protein solutions were lyophilized to dryness. Reconstituted protein concentrations were established using the Bradford method, with BSA as standard [34]. CR I–II from P. pastoris was used in all experiments unless stated otherwise. 15 N-labeled CR I– II was produced from P. pastoris using 98% 15N-ammonium sulfate (Martek Biosciences) and the same protocols as for unlabeled material [32]. 13 15 C, N-labeled CR I–II was prepared as for 15N-labeled material but with 5 g:L21 glucose (day 1) and 2 g:L21 98% 13 C-glucose (day 2) (Martek Biosciences) as the sole carbon source during the growth phase and the addition of 0.5% 13 C-methanol each day (Euris-top, France) during the expression phase (additional 5 days). Protein purification followed the same steps as for unlabeled protein.

Tryptic digests of CR I – II were investigated using conditions and protocols previously described for CR [39]. Briefly, 40 mg protein in 80 mL 150 mM KCl, 50 mM Tris pH 7.5 was subjected to 1 : 100 trypsin (w/w) for 0 –50 min. Uncleaved (as control) and cleaved proteins were then separated by Tris/tricine/PAGE on 15% acrylamide gels [40]. The identity of the CR polypeptides were confirmed by MALDI-TOF MS (CR (1 –60), 7382 Da with 7377 Da expected, and CR(61– 99) 4570 Da with 4572 Da, unknown loss of last residue) and N-terminal amino acid sequencing (GSMAG for CR(1–60), residues 22 to 1 3, and SDNFG for CR(61 –100), residues 61 –65). Calcium-binding to the CR II EF-hand motif Samples of GST, CR, GST–CR I, GST –CR II and standards were separated on 10% acrylamide Tris/tricine/gels in the presence of SDS and then electroblotted to nitrocellulose (0.45 mm, Bio-Rad) in 20% methanol, 48 mM Tris, 39 mM glycine, 0.37% SDS buffer (pH 8.3) for 60 min at 0.1 A. The method of Maruyama et al. [41] was followed with the blot incubated in 2 mM 45Ca (10.5 Ci:g21 Ca; ICN) in 5 mM MgCl2, 60 mM KCl, 10 mM imidazole pH 6.8 for 10 min followed by washing in 50% ethanol (1 min). Subseqeuntly, blots were subjected to autoradiography on Hyperfilm (Amersham Life Science) and developed after 24 h.

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Characterization of calretinin domain I – II (Eur. J. Biochem. 268) 6231

Measurement of correlation time and translation diffusion of CR I –II

R E S U LT S

The 15N T1, T2 and NOE relaxation data in a 1.5-mM 15 N-labeled CR I–II sample with 10% D2O (for field lock), 33 mM NaCl, 10 mM CaCl2, 50 mM sodium acetate pH 5.4 at 310 K were measured in a series of two-dimentional heteronuclear correlated spectra using the sensitivityenhanced gradient pulse schemes of Farrow et al. [42]. Spectral widths were 13 and 28 p.p.m. in F2 and F1, respectively, on a Bruker DRX 500 spectrometer. The 1H carrier frequency was placed on the water resonance and at 118 p.p.m. for nitrogen. An in-house written MATLAB routine was applied to fit NMR relaxation data to experimental parameters. A sample of unlabeled 0.25 mM CR I–II was prepared in 50 mM deuterated Tris, 25 mM NaCl pH 8.1. The stimulated-echo with longitudinal eddy current delay (STE-LED) method was applied to obtain diffusion ordered spectroscopy (DOSY) spectra at 278 K [43,44]. We implemented Bruker’s LEDGS2S pulse sequence [45] with a combined watergate and presaturation sequence which allowed measurements in H2O/D2O solution. The mean of the diffusion profile arising from CR I –II protons between 0 and 4 p.p.m. was determined. The relative diffusion rate measurements were carried out using known proteins as external references or internal TSP [3-(trimethylsilyl)propionic acid, sodium salt]. Calibration proteins included ubiquitin (8.7 kDa), Spo0F (14.4 kDa) and HP-RNAse dimer (24.7 kDa).

Definition of CR I –II structure

Analytical ultracentrifugation of CR I –II Experiments were carried out on a Beckman XL-A instrument in a sedimentation equilibrium experiment at 39 000 g as described for Calb I–II [31]. Analytical ultracentrifugation was performed at 293 K with a sample concentration of 0.233 mg:mL21, < 20 mM as calculated by a molar extinction coefficient of 1 ¼ 11 460 cm21:M 21. The buffer used was 1 mM CaCl2, 40 mM potassium phosphate pH 6.4. Chemical cross-linking of CR I –II Reactions were carried out in 20 mL volumes modified from the method described by Staros et al. [46]. six micrograms of protein and 1 mM CaCl2 were initially prepared in 10-mL. A stock solution of 10 mL 100 mM NaCl, 100 mM Tris pH 8.0, with 20 mM N-hydroxysuccinimide and 8 mM 1ethyl-3-(3-dimethylaminopropyl)-1-napthalene-sulfonic acid (EDC), was then added. After 60 min in the dark at room temperature, the reactions were terminated by the addition of 4  SDS sample buffer and heating at 95 8C for 5 min. The reaction mixtures were separated by Tris/tricine/ SDS/PAGE [40], together with a low-range molecular weight marker (Promega), on 10% acrylamide gels and stained with Coomassie blue. Bovine S100B, bovine a-lactalbumin and horse skeletal myoglobin (all from Sigma) were used as control protein samples. Myoglobin and a-lactalbumin produce single bands of monomeric weight and S100B provides a positive control with a yield of cross-linked dimer of < 30%.

The CR I – II and Calb I – II sequences have high sequence identity, Fig. 1, and many other residues display conservative changes. The region between residues 54 and 79 of CR I –II, encompassing the linker, helix C and start of the second calcium-binding loop, has least identity to Calb I –II. In this region, identity persists only for hydrophobic residues of helix C that are expected to form part of the hydrophobic core. Standard protocols were followed to obtain spectra suitable for backbone assignment of calcium-bound CR I –II (full sample details, acquired NMR experiments and assignments at pH 6.7 and 7.7 are available from BMRB entry 4749). The AUTOASSIGN program [37] was used to obtain automatic assignments. By running the program with several sets of parameters, we were able to obtain a consistent auto-assignment for residues T17-I36, E70-I83 and T94-N97 that were subsequently confirmed manually. Other assignments, most notably for helix B, were obtained by manual assignment. The assignment was extended at pH 6.7 for residues between L13 and F98, except for K39, E40, K53, M58 and S61. Fig. 2 shows an assigned HSQC spectrum of CR I–II at pH 7.7. A summary of the secondary structure elements defined by pH 7.7 NMR data is given in Fig. 3. Four helices are defined by chemical shift index (CSI), medium-range NOEs and measured 3JHNHa values smaller than 5 Hz (A18– D31, G38 –K53, E66–K77 and A86–L92). Three short stretches of extended structure (E15–T17, Y35–E37 and K82 –E84) are suggested by CSI. However, the extended structure is supported only by consistently very strong HNi,Ha(i-1) NOEs and 3JHNHa values . 7 Hz for the latter two segments. The short segments of extended structure found

Fig. 1. Pairwise alignment of CR I–II (1–100) and Calb I–II (1–93) sequences (both of rat origin). Additional residues at the N terminus, that are the result of molecular biology manipulations, are underlined. An EF-hand consensus sequence is placed between the aligned EF-hand I and EF-hand II sequences [3]. The helices are shown as boxes with conserved hydrophobic (h) and other amino acids (.), the calcium-binding ligands are designated by their coordinates: x, y, z, -y, -x and -z [60]. Residues discussed in the text are given in bold (G34, K60 and G81 of CR I –II and G29, G66, R68, G71 and G74 of Calb I –II). The SeqWeb alignment is shown between the aligned EF-hand sequences: identical residues (|), homologous residues (:) and similar residues (.) are marked, as scored by the program. The helices defined by Klaus et al. for Calb I –II are boxed [31] and the secondary structure elements are labeled (helices A– D; helices pre-A and D0 found in Calb I –II; the linker between EF-hand motifs; Cal I and Cal II denote the two calcium-binding loops). For comparison, the position of helices A –D is also boxed for the CR I –II sequences, as determined by the NMR data described in this paper (see Fig. 3). Five asterisks in the linker at the start of EF-hand II of the Calb I –II sequence denote a five-residue long gap compared to the CR I –II sequence.

6232 M. Palczewska et al. (Eur. J. Biochem. 268)

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Fig. 4. Limited trypsinolysis of CR I–II. Incubation times are given above the lanes. The two proteolytic fragments correspond to CR(1–60) and CR(61–100) for the upper and lower bands, respectively.

Fig. 2. 1H,15N HSQC of 15N-labeled CR I –II with assignments. Of note are residues G34 and G81, the ‘position 6’ glycine residues. Residues I36, E37, I83, E84 and M85 have 1H and 15N chemical shifts characteristic of extended conformation (confirmed by other assignments). Other resonances were assigned at pH 6.7. These assignments are missing from this figure as they fall in the crowded part of the spectrum and their assignments could not be transferred unambiguously, even though some of these peaks are still observed in this HSQC. A pH titration indicated that the majority of resonances did not undergo pH-induced changes in chemical shift . 0.1 p.p.m. (1 H) or . 0.5 p.p.m. (15N). Lines connect the unassigned glutamine and asparagine side-chain resonances.

in the calcium-binding loops interact in a b-sheet-like manner. This interaction between residues Y35–E37 and K82–E84 is supported by interstrand NOEs (I36HN – E84HA and I36HN – I83HN). The linker could not be assigned at pH 6.7. The CSI scores at pH 6.7 are added to Fig. 3 as this data could not be confirmed unambiguously at pH 7.7. Our secondary structure assignment is consistent with known capping motifs. T13 and Q16 provides an N-terminal helix capping motif in ShK toxin, which plays a role in helix

Fig. 3. Summary of the secondary structure assignments extracted from NMR data at pH 7.7. Medium-range NOEs are given as lines connecting assignments between HA and i 1 3 or i 1 4 HN resonances (marked as NOE i 1 3 and NOE i 1 4, derived from a NOESY-HSQC experiment). CSI parameters, scored as 1 1, 0 or 2 1, are calculated from the backbone assignment according to Wishart et al. [61]. Note: the CSI data for the linker region was determined from NMR data at pH 6.7. 3JHNHa values (marked as 3J) are calculated from a HNHA spectrum using a MATLAB routine.

stabilization [47]. CR I –II (T17 and Q20) and Calb I –II (T12 and Q16) contain analogous residues at the N-terminus of helix A. The residues around G54 suggest that it forms the C-capping motif of helix B [48], reinforcing the NMR data that helix B of CR I –II is three residues shorter than that of Calb I–II. The residues around G65 are also consistent with an N-capping motif and agree with the Calb I –II assignment, although N63 could provide an alternative capping site [48]. A C-capping motif for the L92P93 pair of residues, as suggested by Prieto and Serrano [49], agrees with our secondary structure determination of CR I –II and is also found in Calb I–II. Fig. 4 shows the result of limited trypsinolysis of CR I –II. Using the conditions used previously to cleave CR, we found that CR I–II was fully digested in < 5 min, similar to that reported previously for CR [39]. The resulting tryptic fragments remained relatively resistant to further cleavage (. 30 min) in the presence of calcium. MALDI-TOF and N-terminal amino acid sequencing confirmed that the upper band is CR(1–60) and the lower band is CR(61–100). Preferential cleavage of CR I–II at K60 is consistent with the secondary structure presented in Fig. 3 and the previous results for CR [39]. All other potential trypsin cleavage sites are found either in helical regions (K26, K50, R52, K53, K67, K69, K74) or in the calcium-binding loops (K39, K77, K82). Both EF-hands of CR I –II bind calcium The 12-residue long calcium-binding loops of EF-hands are readily identified from their primary sequence. Significant deviations from the consensus sequence usually lead to a low-affinity calcium-binding site. Here we provide experimental evidence that both EF-hands of CR I–II bind calcium. CR fragments corresponding to the first (residues 1 – 60) and second (residues 61 – 100) EF-hands were expressed as GST fusion proteins. We found thrombin cleaved CR fragments difficult to purify and blot. Therefore, 45 Ca-overlay experiments were performed on the fusion products together with CR and GST as controls, Fig. 5. Ponceau red staining of the electroblot indicated that some cleavage of the fusion proteins occurred and a lower band, most probably GST, was observed. We observed that CR, but not GST (also the lower bands of lanes 2 and 3), binds 45 Ca under the conditions used. GST–CR(1–60) and GST– CR(61 –100) provide 45Ca-overlay bands of similar intensity when blotted in similar quantities. This is consistent with both EF-hand sequences containing a single calciumbinding site. NMR data also provides evidence suggesting that both EF-hands of CR I–II bind calcium. The calcium-bound

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Characterization of calretinin domain I – II (Eur. J. Biochem. 268) 6233

several reference EF-hand proteins are given in Fig. 6. The chemical shifts agree with biochemical data that only certain calcium-binding sites of the reference proteins are filled [31,50 –56].

CR I– II is monomeric Fig. 5. CR(1–60) and CR(61–100) bind calcium. Proteins were separated by 10% Tris/tricine/SDS/PAGE and electroblotted to nitrocellulose as described in Experimental procedures. (A) Ponceau red stain of blotted proteins. Lane 1, GST; lane 2, GST–CR(1–60); lane 3, GST–CR(61–100); lane 4, CR. The fusion proteins in lanes 2 and 3 appear to be partly digested; the lower band corresponds to GST. (B) 45 Ca-overlay of the blot shown in part A indicating that CR and GST fusion products of CR fragments bind 45Ca.

NMR assignments of conserved glycine amide protons, at position six of the 12-residue long calcium-binding loops of EF-hand proteins, have chemical shifts . 10 p.p.m. for filled loops and chemical shifts of < 8 p.p.m. for empty loops [50]. The chemical shifts of CR I–II, Calb I– II and

Fig. 6. ‘Glycine 6’ of the calcium-binding loop is a sensitive indicator of calcium loading. Filled bars denote EF-hand loops that bind calcium and empty bars those that do not, as determined by biochemical or crystal structure characterization [31,50–56]. (A) Frequenin (G33, G78, G114, G162) [51,52]. (B) GCAP-2 (G38, G72, G110, G163) [53,54]. (C) Calb I –II (G29, G71) [31]. (D) Nereis diversicolor sarcoplasmic Ca21-binding protein (G21, G55, N109, G143) [55,56]. (E) Calerythrin (G22, G73, G117, G151) [50]. (F) CR I–II (G34, G81).

Fig. 7. CR I–II is monomeric. (A) Plot of global correlation time determined from NMR relaxation data for three reference proteins vs. their known molecular weights (X). The reference proteins are ubiquitin (Martek Biosciences Corporation, Columbia), Spo0F [62] and the dimeric HP-RNase [63]. The experimentally determined correlation time of CR I –II is plotted against its predicted monomeric mass (B) and the predicted dimeric mass (A). (B) Plot of relative diffusion coefficients for several reference proteins, as in (A) vs. known molecular weight (X). CR I –II experimental DOSY data is plotted against its predicted monomeric mass (B) and predicted dimeric mass (A).

15

N NMR relaxation experiments can be used to determine global correlation times of proteins that are related to the size of the studied molecule. Using the isotropic form of the Lipari –Szabo analysis [57], a global correlation time of 6.7 ^ 0.5 ns was determined for calcium-bound 15 N-labeled CR I–II at pH 5.4 and 310 K (Fig. 7A). Also, the diffusion rate of an unlabeled CR I–II sample was evaluated under different conditions on an arbitrary log10 scale at pH 8.1 and 278 K (Fig. 7B). Both sets of NMR data for CR I –II clearly fit with a monomeric mass (12 kDa) rather than a dimeric mass (24 kDa) when compared to reference proteins (Fig. 7A,B). The NMR data indicate that CR I –II exists as a monomer even at protein concentrations as high as 1.5 mM .

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Fig. 8. 10% Acrylamide gel run under Tris/tricine/SDS/PAGE conditions and stained with Coomassie blue showing the products of chemical cross-linking of 0.3 mg:mL21 protein in the presence of 1 mM CaCl2. Lane 1, S100B; lane 2, a-lactalbumin; lane 3, myoglobin; lane 4, CR I –II; molecular weight standards are indicated to the left.

Analytical ultracentrifugation showed earlier that calciumbound Calb I –II is dimeric [31] and this method was used under identical conditions to establish the extent of dimerization of CR I– II. Analytical ultracentrifugation revealed that $ 90% of 20 mM CR I–II exists as a monomer in the presence of 1 mM CaCl2. Possible dimer, trimer and tetramer species accounted for , 4% each sample. In cross-linking experiments, a single band is observed for CR I–II (lane 4, Fig. 8) at the molecular mass expected for monomeric protein. A small amount of cross-linked CR I–II, < 3% according to densitomeric analysis, is probably the result of nonspecific cross-linking as the same amount of cross-linked dimer species was detected for the monomeric proteins a-lactalbumin and myoglobin. S100B, which forms both covalent and noncovalent dimers, provides a yield of 30–40%. This is an acceptable yield for this method [46].

DISCUSSION CR I– II EF-hand structure: comparison to Calb I –II Only a partial backbone assignment of CR I –II was possible at pH 7.7 because the intensity of some resonances are diminished or unobserved in HSQC-based spectra (Fig. 2). Moreover, several weak resonances do not provide sufficient intensity in three-dimensional NMR experiments to enable positive assignment. It is difficult to assign spectra of CR I–II at lower pH due to the overlap of broad resonances. When the pH was lowered to pH 6.7, it was possible to assign most of the resonances between L13 and F98. Many of the new pH 6.7 assignments resonated in the region where residues in unstructured segments of protein are usually found and their CSI scores confirmed this. The N terminus of CR I –II could not be assigned even at pH 6.7. However, NMR data for Ala2 and Gly3 at pH 6.7 suggests that these residues are in an unordered state. The NMR data of CR I– II is consistent with an unordered N terminus and linker. Klaus et al. described the N-terminal region as containing a pre-A helix and noted that it is poorly formed [31]. This region was not detected in the NMR spectra of CR I–II (Fig. 3). There is also no sequence homology between CR I–II and Calb I– II for this helix (Fig. 1). The C termini of the CR I–II and Calb I–II domains contain a consensus sequence (ENFLLXF, where X is a variable residue), which is only partly observed in Fig. 1. This segment is present in Calb and CR sequences of all species as well as the recently

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described secretagogin sequence [58]. We do not observe helix D0 in CR I –II (Fig. 1) possibly because CR I–II is three residues shorter than Calb I–II. The secondary structures of CR I –II and Calb I –II are almost identical within the EF-hand regions (see Fig. 1). Helices A, C and D, together with the three-residue b-type cross-strand interaction between the two calcium-binding loops, are in identical positions. However, helix B of CR I –II is three residues shorter than helix B in Calb I–II. A number of identified helix capping motifs are consistent with the determined secondary structure of CR I–II, including the shorter helix B of CR I–II. Limited trypsinolysis of CR I– II leads to fragmentation that is consistent with the defined secondary structure: K60 is in the linker whereas the other 10 potential cleavage sites are found in helical regions or in the calcium-binding loops that scaffold the calciumbinding ligands and are consequently protected. Helix B of CR I –II is still longer than predicted by the EF-hand consensus sequence, an unusual feature first described for Calb I–II. The CR I–II sequence also has a five-residue insertion in the linker compared to Calb I–II (see Fig. 1). Therefore, the linker of CR I–II is 12 residues long compared to only four residues in Calb I –II.

CR I– II binds two calcium cations Based on a comparison with the consensus sequence, both EF-hands of CR I–II are predicted to bind calcium. On the other hand, the residues at positions 66, 68 and 74 (G66, R68 and G74) of Calb I–II lack the side-chain oxygens expected to provide calcium-binding ligands. The absence of calcium-binding to the EF-hand II motif of Calb has been shown in the individual polypeptide, Calb I–II, Calb mutants and full-length Calb using a number of experimental techniques in several laboratories [26 –29]. The analogous residues of CR I– II (D76, N78 and E84), as well as the whole second loop sequence, have suitable ligands with side-chain oxygens where the consensus sequence requires them for calcium binding. The second EF-hand of CR I–II has a potential high calcium affinity. The principal limited proteolysis products of CR I –II conveniently correspond to individual EF-hand motifs (residues 1–60 and 61–100). We found that the PAGE separated limited trypsinolysis products of CR I –II bound 45 Ca. However, GST fusion products of the same fragments were produced in order to clarify our data due to the poor blotting efficiency of the small CR fragments. Both CR(1 –60) and CR(61 –100) bind 45Ca as GST fusion products. Individual synthetic Calb EF-hands I, III, IV and V ˚ kerfeldt et al. [29] agrees bind calcium [29]. This data of A with the large body of independent data collected on Calb and truncated Calbs by Kumar’s group. This latter work revealed that EF-hands II and VI of Calb do not bind calcium and that Calb binds a total number of four calcium cations [26 – 28]. Calcium-dependent NMR data for ‘position 6’ glycine residues also support calcium-binding to both EF-hands of CR I–II (Fig. 6) but only to EF-hand I of Calb I– II. These glycine chemical shifts report the formation of a strong interaction between the glycine amide and a carboxylate side-chain. This has been shown empirically to occur when calcium ions are bound to the site. In summary, the 45Ca-binding data for CR fragments and

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Characterization of calretinin domain I – II (Eur. J. Biochem. 268) 6235

NMR data for CR I– II indicate that the stoichiometry of calcium binding in CR I–II is 2 : 1. Calcium-bound CR I –II is monomeric We speculate that the Calb I –II homodimer structure mimics the interaction between Calb I–II and other parts of the Calb sequence [31], in accordance with the Linse model where all six EF-hand motifs of Calb interact within a single globular domain structure [59]. In contrast to Calb I –II, CR I–II is monomeric under various conditions. We have not observed an interaction between CR I– II and various complement CR fragments using fluorescence, chemical cross-linking and size exclusion chromatography (M. Palczewska, P. Groves & J. Kuz´nicki, unpublished data). Therefore, we believe that CR does not form a single globular domain. Perhaps the different oligomeric states of CR I –II and Calb I–II are an indication of different organizations of EF-hands within full-length CR and Calb. CR I– II and Calb I– II may have different structures CR I – II and Calb I – II have different biochemical properties, despite their similar secondary structures. The four-residue linker of Calb I–II could restrict the relative positions of helices B and C to a greater degree than the 12-residue linker of CR I –II. The low-resolution NMR structure of calcium-bound Calb I–II suggests that the second calcium-binding loop may contain a distorted conformation [31]. We show that the second calcium-binding loop of CR I–II binds calcium and is therefore expected to adopt a characteristic conformation found in other EF-hand protein structures. We predict that an overlay of future high-resolution CR I –II and Calb I –II structures will reveal that the largest structural differences lie in the orientation of helix C, together with the adjacent linker and second calcium-binding loop structures. There is a region of poor sequence identity localized to residues 54– 79 of CR I –II that supports this prediction (Fig. 1). High resolution structures of both apo- and calcium-bound domains are essential to understand fully the differences in CR I–II and Calb I–II structure and properties. Are the functions of CR and Calb distinct? A large body of physiological data supports a calcium-buffer role for both Calb and CR. However, biochemical (and some physiological) data suggest sensor roles, as do the interaction of Calb with at least two other proteins [20,21]. The ongoing high-resolution structure projects on both CR I –II and Calb I–II will provide a clearer answer as to the nature of the calcium-induced structural changes, differences in structure and clues to the function of the full-length proteins. The present work establishes that the first domains within CR and Calb have distinct structural features and biochemical properties, and potentially perform different functions within the full-length proteins.

ACKNOWLEDGEMENTS We are greatly indebted to Werner Klaus (F. Hoffmann-LaRoche AG, Basel) for discussions about the different oligomeric states of Calb I –II, the subject of his earlier studies, and CR I –II. Francis Mueller and

Eric Kusznir (F. Hoffmann-LaRoche AG, Basel) determined the analytical ultracentrifugation data on CR I –II under the same conditions as used earlier for Calb I –II. Sa´ndor Ke´ki (Chemistry Department, Debrecen) determined MALDI-TOF data for the limited proteolysis products of CR I –II. We thank Walter Chazin (Vanderbilt University, Nashville) for critical evaluation of the manuscript and Barbara Zarzycka (Warsaw) for her technical assistance. This work was supported by an International Center for Genetic Engineering and Biotechnology (ICGEB) grant to J. K. [CRP/Pol97-01(t1)] and G. B. [CRP/Hun97–01(t1)]. P. G. is supported by a grant from the State Committee for Scientific Research, KBN no. 6 P04B 01015. G. B. thanks the support of the Hungarian National Fund, OTKA T-029089. The Bruker DRX 500 spectrometer used in these studies was purchased from the following grants: PHARE-ACCORD H-9112-0198, OMFB MEC 93-0098 and OTKA A084.

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