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J. Biol. Chem., Vol. 280, Issue 2, 1209-1216, January 14, 2005

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Calcium-binding Crystallins from Yersinia pestis

CHARACTERIZATION OF TWO SINGLE -CRYSTALLIN DOMAINS OF A PUTATIVE EXPORTED PROTEIN^*

Maroor K. Jobby and Yogendra Sharma

From the Center for Cellular and Molecular Biology, Uppal Road, Hyderabad-500007, India

Received for publication, August 12, 2004 , and in revised form, October 25, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
-Crystallin is a superfamily with diverse members from vertebrate lens to microbes. However, not many members have been identified and studied. Here, we report the identification of a putative exported protein from Yersinia pestis as a member of the -crystallin superfamily. Even though calcium has been known to play an important role in the physiology and virulence of the Yersinia genus, calcium-binding proteins have not yet been identified. We have studied the calcium-binding properties of two of the three crystallin domains present in this putative exported protein designated "Yersinia crystallin." These two domains (D1 and D2) have unique AA and BB types of arrangement of their Greek key motifs unlike the domains of other members of the -crystallin superfamily, which are either AB or BA types. These domains bind two calcium ions with low and high affinity-binding sites. We showed their calcium-binding properties using various probes for calcium and the effect of calcium on their secondary and tertiary structures. Although both domains bind calcium, D1 underwent drastic changes in secondary and tertiary structure and hydrodynamic volume upon calcium binding. Domain D1, which is intrinsically unstructured in the apo form, requires calcium for the typical -crystallin fold. Calcium exerted an extrinsic stabilization effect on domain D1 but not on D2, which is also largely unstructured. We suggest that this protein might be involved in calcium-dependent processes, such as stress response or physiology in the Yersinia genus, similar to its microbial relatives and mammalian lens crystallins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Crystallins are the major structural proteins of the lens. Three major crystallins, -crystallin, -crystallins, and -crystallins, are known to be present ubiquitously in mammalian lenses (1). - and -Crystallins share structural similarities and are classified together as -crystallins (1). Topologically, - and -crystallins are made up of two globular domains; each domain consists of two Greek key motifs arranged as four-stranded antiparallel -sheets. The domain of these proteins is referred to as the crystallin fold or motif. Two types of Greek key motifs are described, "A-type" and "B-type," which are present as either AB or BA combinations.

The , or Greek key, motif was found in several other non-lens proteins, and they were all classified as being within the -crystallin superfamily. Some members of this superfamily are Protein S (2), Spherulin 3a (3, 4), Streptomyces killer toxin-like protein (SKLP) (5), cargo proteins from Tetrahymena thermophila (6), AIM1 (absent in melanoma) (7), epidermis differentiation-specific proteins (8, 9), yeast killer toxin WmKT (10), and Streptomyces metalloproteinase inhibitor (SMPI) (11). These members show structural similarity despite relatively low sequence identity, which reflects the functional diversity found among these proteins.

Two microbial homologues of -crystallins, Protein S and Spherulin 3a, have been studied extensively (for review, see Ref. 12). Protein S is a two-domain protein, whereas Spherulin 3a is one-domain protein present as a dimer. These proteins share the intrinsic thermodynamic stability of -crystallins. Under drastic conditions of starvation, desiccation, or heavy metal exposure, the slime mold Physarum polycephalum sporulates with a high level expression of Spherulin 3a (13). Similarly, upon stress, soil bacterium Myxococcus xanthus differentiates into spores protected by a spore coat containing a high level of Protein S (14). Both proteins are known to bind calcium at the homologous sites (15, 16). The calcium-binding sites are located at the Greek key motif, as depicted in the schematic diagram (Fig. 1). The stability of these proteins is increased severalfold upon the binding of calcium, suggesting a calcium-dependent protective role of these proteins under adverse conditions (12).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Schematic diagram of Greek key crystallin fold showing the calcium-binding site. Location of bound calcium is represented by the solid spheres. Conserved residues are shown at their positions in a typical Greek key motif. a, b, c, and d represent four strands of the motif

Rapid increase in the number of genomes sequenced in the recent past led us to look for more -crystallin members. Because the genome sequence of Yersinia pestis was completed (19), we were interested in knowing whether microbial homologues of -crystallins are present in this pathogen. Y. pestis is the causative agent of the dreaded disease plague. Yersinia genus consists of three species pathogenic to humans and animals, namely Y. pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis. Y. pestis is the most fatal of all three and has caused more numbers of deaths than any other disease (20) and is a threat because of its potential use as a bioweapons agent. It has been known for a long time that Y. pestis is unable to grow at 37 °C in calcium-depleted medium (21), which is known as a low calcium response. Loss of this property makes the bacterium avirulent. Calcium level also affects the physiology and metabolism of Y. pestis during growth at 37 °C (22, 23); still no calcium-binding protein from this pathogen has been identified.

The calcium-binding microbial homologous of crystallins, if any, in Y. pestis, with analogy to microbial -crystallins, could be a possible candidate for stress response encountered during the various phases of the life cycle in animals and humans. We, therefore, searched the genome sequence of Y. pestis CO92 and found a putative exported protein (locus tag YPO2884) and a hypothetical protein (locus tag YPO0466) as microbial relatives of -crystallins, containing three and two domains, respectively.

In this context, we studied two of the three -crystallin domains present in the sequence of a putative exported protein, which we refer to as "Yersinia crystallin." Yersinia crystallin domains show novel Greek key motif combinations (AA and BB types), which have not been found in other crystallin domains. Our data showed that domains of Yersinia crystallin bind calcium with micromolar affinity. We report that calcium was required for the proper fold and stability of one of the domains. We also report the presence of a homologue and a paralogue of Yersinia crystallin that we have identified in this pathogen and another microorganism.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Identification and Selection of -Crystallin Domains from Y. pestis—Using -crystallin and other members of the crystallin superfamily as a template sequence, we performed a BLAST program (24) search against the genome sequence of the Y. pestis strain CO92. The signature sequence YXXXX(F/Y)XG forms the beginning of a crystallin-type Greek key motif about 35 residues in length with a conserved Ser at about 34th position in a stretch of (D/N)XXS. The hits were scanned for the presence of crystallin signature sequence YXXXX(F/Y)XG. We obtained two proteins with genuine Greek key motifs occurring in pairs. These proteins were annotated as the putative exported protein (GenBank^TM accession number NP_406389 [GenBank] , locus tag YPO2884) and the hypothetical protein (GenBank^TM accession number NP_404108 [GenBank] , locus tag YPO0466). Two single domains of Yersinia crystallin, D1 (first) from residue 21 to 112 (92 residues) and D2 (second) from amino acid 110 to 215 (107 residues) were selected.

Molecular Modeling—Molecular modeling was performed either using the web-based Swiss Modeler server (25) or the three-dimensional PSSM fold recognition server (26). The nearest homologue for D1 was the N-terminal domain of Protein S (Protein Data Bank (PDB) code 1nps [PDB] ) (24% identity and 56% similarity) (27), and the nearest homologue for D2 was Spherulin 3a (PDB code 1hdf [PDB] ) (32% identity and 49% similarity) (16). Region YRGNYPERKQS (137–147) of D2 was an insertion and could not be modeled. The initial coordinates generated were energy-minimized using 200 iterations each of steepest descent and conjugate algorithm on an Onyx work station (Silicon Graphics, Inc.) using the Discover module of InsightII software (Accelrys Inc.). The final model of D1 consisted of residues from Ala-2 to Ala-92, and the root mean square deviation at C atoms was 1.873 Å compared with the template structure. Similarly, the final model of D2 had residues from Lys-2 to Phe-104 and the root mean square deviation of 1.959 Å. The final models were evaluated using the PROCHECK program (28), and 95% of the residues were found to be in the allowed region for D2; for D1, this was 94%.

Cloning, Expression, and Purification of D1 and D2 Crystallin Domains—Genomic DNA of Y. pestis was a kind gift from Dr. S. Shivaji, Center for Cellular and Molecular Biology (29). Both domains D1 and D2 were amplified from the genomic DNA using PCR and cloned into an expression vector pET21a (Novagen) under T7 promoter. To facilitate fluorescence studies, we engineered a Trp residue at the penultimate amino acid position at the C-terminal end of D2. The cloned sequences were confirmed by DNA sequencing on an automated sequencer (Applied Biosytems ABI PRISM 3700). The expression constructs were transformed to Escherichia coli strain BL21 (DE3). The transformed E. coli cells were grown to mid-log phase and heterologous protein expression was induced by the addition of isopropyl 1-thio--D-galactopyranoside. The cultures were harvested after 3 h, and the cell pellet was stored at -80 °C.

The cell pellet containing D1 in buffer A (50 mM Tris-Cl, pH 7.0, 50 mM NaCl, 1 mM EDTA, 2 mM DTT,¹ and 5 mM phenylmethylsulfonyl fluoride) was lysed by sonication, and the supernatant was loaded on a Q-Sepharose FF column (Amersham Biosciences) equilibrated in buffer A without DTT and phenylmethylsulfonyl fluoride. The bound protein was eluted using a gradient of 50–200 mM NaCl. Fractions containing the protein were concentrated to 2 ml and loaded on a Sephadex G-75 column (Amersham Biosciences) (1.0 x 150 cm) equilibrated in 50 mM Tris-Cl (pH 7.0) containing 150 mM KCl and 1 mM DTT. The purified protein was decalcified using Chelex-100 (Bio-Rad)-treated buffer and stored in plastic ware at -80 °C.

D2 was expressed as an inclusion body in the E. coli. After cell lysis in 50 mM imidazole-Cl (pH 6.5), 50 mM NaCl, and 1 mM EDTA by sonication, the pellet was washed with the lysis buffer containing 1% Triton X-100 and then subsequently with Triton X-100-free buffer. The washed inclusion body was solubilized in lysis buffer containing 8 M urea and 5 mM DTT for 2 h. The solubilized inclusion body was centrifuged, and the supernatant was loaded on a Q-Sepharose FF column equilibrated in the same buffer. Under these conditions, protein bound to the column. After washing with the start buffer to remove unbound proteins, the column was washed with urea-free start buffer for refolding. The bound protein was then eluted with start buffer containing 200 mM NaCl. The eluted protein was further purified on a Sephadex G-75 (Amersham Biosciences) (1 x 150 cm) equilibrated in 50 mM Tris-Cl (pH 7.0) containing 150 mM KCl and 1 mM DTT. The protein was decalcified with Chelex-100-treated buffer. All the buffers used in the studies were treated with Chelex-100 (Bio-Rad) to remove calcium.

⁴⁵Ca Overlay Assay—The assay was performed as described earlier (30) with minor modifications. 50 µg of protein was spotted on a nitrocellulose paper and washed with buffer containing 20 mM Tris (pH 7.0) and 50 mM KCl. The blot was incubated in this buffer containing 1 µCi ⁴⁵Ca (PerkinElmer Life Sciences) for 10 min at room temperature. The blot was then washed three times with 50% ethanol, air-dried, and scanned in a phosphorimaging device (Fuji FLA-3000).

Determination of Macroscopic Binding Constants—Chromophoric calcium chelator BAPTA tetra potassium salt (Molecular Probes) was used to determine the macroscopic calcium-binding constants for the domains as described earlier (31). Chelex-100-treated buffer (10 mM Tris-Cl (pH 7.5) containing 10 mM KCl) was used at 25 °C in a 1-ml quartz cuvette. Protein concentrations used were 20–30 µM. Aliquots from stock calcium solution were added, and absorbance at 263 nm was read in a Shimadzu UV-visible 1601 spectrophotometer. The titration data were analyzed using the Caligator software (32), which uses a Levenberg-Marquardt non-linear curve-fitting routine. The data were best fit to a two-site model. The macroscopic dissociation constants from the best fit to data were used.

Fluorescence Spectroscopy—Fluorescence emission spectra were recorded on a Hitachi F-4500 spectrofluorometer. The cuvettes were soaked in EDTA solution and were rinsed with Chelex-100-treated MQ-water (Millipore) and dried before use. The spectra were recorded in the correct spectrum mode of the instrument using excitation and emission band passes of 5 nm. All measurements were done at room temperature.

Circular Dichroism (CD) Spectroscopy—Far- and near-UV CD spectra of both domains were recorded at room temperature on a Jasco-715 spectropolarimeter using 0.01 and 1 cm path-length cuvettes, respectively. Secondary structure fractions from far-UV CD spectra were calculated using the CDPro software package (33), which uses multiple algorithms and CDNN based on neural networks (34).

Terbium Binding—Terbium binding to both the domains was carried out on a Hitachi F-4500 spectrofluorometer. The excitation wavelength was 285 nm with band passes of 5 nm for both excitation and emission. The titration buffer consisted of 10 mM BisTris (pH 6.7) and 10 mM KCl. Dissociation constants of the protein for terbium were calculated by non-linear curve fitting to the following equation (35) using the program Microcal Origin 6.0,

(Eq. 1)

where F, F_max, and F_min represent the fluorescence intensity at a given point, at saturation and without terbium, respectively. Tb_f is the free terbium concentration at a given point, which was calculated using the following equation.

(Eq. 2)

P is the total protein concentration.

Hydrodynamic Volume—Hydrodynamic volumes of the domains were determined using a Superdex G-75 (Amersham Biosciences) column, calibrated using standard molecular weight markers. Each domain protein was run with EDTA or with calcium added to a final concentration of 1 and 10 mM respectively. The buffer used was 50 mM Tris-Cl (pH 7.5), 100 mM KCl, 1 mM EDTA and 1 mM DTT. EDTA was replaced with 10 mM CaCl₂ for holoproteins.

Glutaraldehyde Cross-linking—Glutaraldehyde (Sigma) was used for cross-linking studies. 100 µM of protein was exchanged to 50 mM HEPES (pH 7.5) containing 100 mM KCl for cross-linking reaction. Final glutaraldehyde concentrations were 0.1 and 0.2%. The reaction mixture was incubated at 25 °C for 30 min. Unreacted glutaraldehyde was quenched by the addition of 1 M Tris-Cl (pH 7.5) at the end of incubation. SDS-PAGE sample buffer was added and the sample boiled for 5 min at 100 °C. The samples were analyzed on a 15% SDS-polyacrylamide gel, and the gel was stained using Coomassie Brilliant Blue R-250.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Sequence Analysis and Identification of Yersinia Crystallins—We identified a putative exported protein (locus tag YPO2884) from Y. pestis strain CO92 as a member of the -crystallin superfamily. This 806 amino acid protein has a 22 amino acid long secretion signal at the N-terminal, suggesting possible localization in periplasm. We identified six Greek key motifs in their N-terminal ends organized as three -crystallin domains. The last two Greek key motifs are variants. This is the first multidomain microbial crystallin. The long C-terminal end of Yersinia crystallin shows probability for the presence of a coiled-coil motif (residues 533–566) (36).

Paralogue and Homologue of Yersinia Crystallin—We also found a paralogue and a homologue of Yersinia crystallin. Hypothetical protein (GenBank^TM accession number NP_404108 [GenBank] , locus tag YPO0466) from Y. pestis CO92 is a paralogue of this protein. Both proteins show 35.3% identity and 57.5% similarity. This protein also has a secretion signal. The fourth Greek key does not have the usually conserved Gly, which is replaced by Ala-180 (Fig. 2). We have found this pair of crystallin paralogues in the genome of Y. pseudotuberculosis and Y. enterocolitica (37) with minor variations and also in other strains of Y. pestis, namely strain 91001 (38) and Kurdistan Iran Man (KIM) (39). We have also identified a hypothetical protein (GenBank^TM accession number CAG22738 [GenBank] locus tag PBPRB0866) having homology to Yersinia crystallin with two crystallin domains, in the genome of Photobacterium profundum,² a marine bacterium living at high pressure and low temperature (Fig. 2). It is likely that all these proteins had a common ancestor and evolved to their present form, because minor variations are found in the paralogous pair of genes in Y. pseudotuberculosis, from which Y. pestis has recently originated (41).

View larger version (40K): [in this window] [in a new window]   FIG. 2. A- and B-type Greek key motif. The sequence of D1 and D2 is aligned with the Greek key crystallin motif found in the paralogue and homologue. Locus tags YPO2884, YPO0466, and PBPRB0866 represent putative exported protein (Y. pestis), hypothetical protein (Y. pestis), and hypothetical protein (P. profundum) respectively. Underlined sequences represent residues with helical propensity. Putative residues involved in calcium binding are indicated by an asterisk. a, b, c, and d represent the four strands of a Greek key motif.

Unique Combination of Greek Key Motifs in Yersinia Crystallin—Two types of Greek key motifs have been described. They are called A-type and B-type motifs arranged as AB or BA. The arrangement of Greek key motifs is AA in D1 and BB in D2 (Fig. 2), which has not been reported in other crystallin domains. This combination of Greek key motifs is also found in the paralogue and homologue of Yersinia crystallin. All B-type motifs of Yersinia crystallin lack usually conserved Trp in the "a" strand, and the typical GDY patch of sequence is replaced by EDY, DLY, or IKY (Fig. 2).

Molecular Modeling—Our attempts to crystallize these domains for structure determination failed, and therefore we could not pursue solving their three-dimensional structure. We built homology models of both of the domains. Modeled structures of D1 and D2 were typical of -crystallin domain, having four anti-parallel strands (Fig. 3). We calculated the secondary structure composition from our models using the MOLEMAN 2 software program, which indicated the prevalence of sheet (47 and 49%) and loop (46 and 47%) in D1 and D2, respectively, with the remainder constituting a helical region (5.5 and 3.2%, respectively). D2 has a short stretch of -helix in the loop. Spherulin 3a also has a helical segment (GLNDVV from amino acids 43–48) in its first Greek key motif. A homologous sequence PLNDEV from amino acids 152–157 is present in this domain forming an -helical stretch. It is seen that D1 and D2 share structural characteristics with -crystallins from vertebrate eye lens.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Homology models of (a) D1 and (b) D2. The models were generated using Swiss-Modeler and three-dimensional PSSM. Two calcium ions are shown as spheres. The side chains of putative residues involved in calcium coordination are shown. The figures were generated using InsightII. The arrow in b shows the helical region.

View larger version (59K): [in this window] [in a new window]   FIG. 4. Purified D1 and D2 domains. 10 µg of D1 and D2 were loaded onto a 15% SDS-polyacrylamide gel, and the gel was stained using Coomassie Brilliant Blue R-250 dye.

Direct Calcium Binding to D1 and D2 by ⁴⁵Ca Overlay Assay—We investigated calcium binding by dot-blot calcium overlay method (30). Direct assay using radioactive ⁴⁵Ca revealed that both domains, D1 and D2, bind calcium as shown in Fig. 5. Bovine serum albumin was used as a negative control and exhibited the anticipated results. This qualitative and simple assay confirmed that these proteins are calcium-binding crystallins and prompted us to further study their calcium-binding properties.

View larger version (54K): [in this window] [in a new window]   FIG. 5. 45Ca overlay assay. 50 µg of D1, D2, and bovine serum albumin (BSA) were spotted on a nitrocellulose paper and washed with buffer containing 20 mM Tris (pH 7.0) and 50 mM KCl. The blot was incubated with the above buffer containing 1 µCi 45Ca for 10 min. It was washed with 50% ethanol three times, dried, and scanned in a phosphor imaging device.

View larger version (18K): [in this window] [in a new window]   FIG. 6. a, fluorescence emission spectra of D1. 10 µM protein in 20 mM Tris (pH 7.5), 150 mM KCl, and 1 mM DTT was excited at a wavelength of 295 nm. Calcium aliquots from stock solution were added until saturation was reached. The figure shows Trp fluorescence in the presence of 0, 100, 200, 300, 400, 500, 700, 1000, and 2000 µM of calcium. b, fluorescence emission spectra of D2. 6 µM protein in 10 mM Tris (pH 7.5), 20 mM KCl, and 1 mM DTT was excited at a wavelength of 295 nm. Calcium aliquots from stock solution were added to a final concentration of 0, 100, 220, 465, and 953 µM. Direction of arrows follows the increasing order of calcium concentration.

View larger version (16K): [in this window] [in a new window]   FIG. 7. a, far-UV CD spectra of D1. Spectra were recorded on a Jasco J-715 spectropolarimeter. Protein concentration at 1.78 mg/ml in 20 mM Tris (pH 7.5), 150 mM KCl, and 1 mM DTT was used. Calcium chloride was added to a final concentration of 0, 10, 50, 100, 200, 300, 400, 500, 750, and 1000 µM. b, far-UV CD spectra of D2. Spectra were recorded using a protein concentration of 1.35 mg/ml in a buffer containing 15 mM Tris (pH 7.5), 100 mM KCl, and 1 mM DTT. Final calcium concentrations were 0, 100, 200, 500, and 1000 µM. Direction of arrows follows the increasing order of calcium concentration.

View larger version (25K): [in this window] [in a new window]   FIG. 8. Near-UV CD spectra of D1. Protein concentration at 0.86 mg/ml in buffer containing 50 mM Tris (pH 7.5) and 100 mM NaCl was used in a 1-cm path-length cuvette. Calcium chloride was added to a final concentration of 0, 40, 200, 300, 400, 500, and 800 µM.

View larger version (17K): [in this window] [in a new window]   FIG. 9. a, terbium binding to D1. 6.0 µm of D2 in 10 mM BisTris (pH 6.7) and 10 mM KCl was excited at 285 nm and emission spectra recorded from 300 to 580 nm. Final concentration of TbCl3 was 0, 20, 50, 100, and 200 µM. b, terbium binding to D2. 6.0 µM of D2 in 10 mM BisTris (pH 6.7) and 10 mM KCl was excited at 285 nm and emission recorded from 300 to 580 nm. TbCl3 was added to the final concentration of 0, 4, 6, 11, and 14 µM. Direction of arrows follows the increasing order of terbium concentration.

Hydrodynamic Volume and Chemical Cross-linking Studies—To study the effect of calcium on the hydrodynamic volume of Yersinia crystallin domains, we performed analytical gel filtration chromatography. In the apo form, D1 eluted at a volume higher than expected for a monomer (Fig. 10a). The presence of calcium drastically decreased the hydrodynamic volume, and the elution volume corresponds to a monomer (Fig. 10a). Thus, there is significant change in the Stokes radius of the D1 upon calcium binding, which could be attributed to the transition of the protein from an unstructured to a structured state and the resulting compaction (44, 46). There was no significant change in the hydrodynamic volume of D2 upon calcium addition (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 10. a, effect of calcium on the hydrodynamic volume of D1. 250 µg of protein were loaded on a Superdex G-75 column equilibrated in 50 mM Tris (pH 7.5), 100 mM KCl, and 1 mM DTT in the presence of EDTA or CaCl2. Elution volumes of molecular mass standards are indicated by arrows. b, glutaraldehyde cross-linking of D1. 100 µM D1 was incubated with 0.1 and 0.2% of glutaraldehyde for 30 min. Lane 1 is the control with only protein and no cross-linker present. Lanes 2 and 4 are the protein without calcium and 0.1 and 0.2% of cross-linker, respectively. Lanes 3 and 5 are protein with 10 mM CaCl2 and 0.1 and 0.2% of cross-linker, respectively. M is the protein molecular mass marker lane.

Putative Calcium-binding Sites—Based on the similarities in the sequences of D1 and D2 with Protein S and Spherulin 3a, we propose homologous calcium-binding sites in D1 and D2 located in the Greek key motifs (Fig. 2). The first residue that coordinates calcium is from the "a" strand (residue next to the first conserved aromatic residue); the other three residues are from the loop between strands "c" and "d" (near the conserved Ser at the stretch ((D/N)-X-X-S) of the Greek key motif. We strongly suggest that all paralogues and homologues of this protein would bind calcium at the putative sites shown in Fig. 2.

Our studies established that both the domains showed properties not predicted from their modeled structure. From UV, fluorescence, and CD spectral studies, we conclude that the homology model of D1 holds good only for the holoform, whereas biophysical properties of D2 do not match with those expected from the model. This large deviation from the predicted structure and properties might be attributed to the unique Greek key combination and sequence variation.

Calcium Binding, Domain Stability, and Possible Functions—-Crystallins have high intrinsic stability, which these domains lack in the apo form. Removal of calcium from D1 destabilized the protein, suggesting its extrinsic stabilizing effect, whereas D2 showed no such characteristics. Two microbial homologues, Protein S and Spherulin 3a, also show the high extrinsic stabilization effect by calcium ions (42, 47), but they have intrinsic stability.

The life cycle of Y. pestis involves phases in flea, rat, and humans. During the colonization in these hosts, the bacterium is exposed to various drastic conditions before it can establish its niche. Under these circumstances, the bacterium needs to be protected. Based on our results, we suggest the possible functions of Yersinia crystallins in stress protection and ionic homeostasis. Earlier workers have suggested that the presence of high levels of calcium is a type of physiological stress unique to Yersinia, which results in growth inhibition of Yersinia species at 37 °C (48, 49). Some proteins, such as Gsr A (50), Omp R (51), Rpo S (52), and response regulator PhoP (40) have been shown to protect the organism from stresses encountered during colonization of phagocytes. We suggest that, by similarity with other crystallins, Yersinia crystallin might be protecting the bacterium during adverse conditions, because domain D1 is stabilized by calcium, and D2 can act as a calcium buffer. On the basis of genomic differences between virulent and non-virulent strains, Golubov et al. (37) reported the presence of Yersinia crystallin and its paralogue in Y. enterocolitica with possible impact on virulence. Therefore, a possible role of Yersinia crystallin in virulence cannot be ruled out completely.

Conclusions—In summary, we have described Yersinia crystallin from Y. pestis as another microbial relative of -crystallins, possessing some common as well as several distinct properties. To our knowledge, this is the only calcium-binding protein studied from Y. pestis. These domains are intrinsically unstructured. Domain D1 requires calcium for its structural stabilization, whereas domain D2 does not show any such effect by calcium binding. Our results suggest that members of the -crystallin superfamily are calcium-binding proteins. The paralogues of this protein in other Yersinia genera are also calcium-binding proteins and might play important roles in calcium-regulated processes in this pathogen. Our study should lead to the more detailed in vivo study of the roles played by these proteins in the physiology and host-parasite interaction of this deadly pathogen. Such studies would also improve our limited understanding of the crystallin superfamily.

	FOOTNOTES

 
^* This work was supported by a Third World Academy of Science (TWAS, Italy) Research Grant 03-231 RG/BIO/AS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a senior research fellowship from the Council of Scientific and Industrial Research, Government of India.

To whom correspondence should be addressed. Fax: 91-40-27160591; E-mail: yogendra{at}ccmb.res.in.

¹ The abbreviations used are: DTT, dithiothreitol; CD, circular dichroism; BAPTA, 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.

² A. Vezzi, S. Campanaro, M. D'Angelo, F. Simonato, N. Vitulo, F. Lauro, A. Cestaro, G. Malacrida, B. Simionati, N. Cannata, D. Bartlett, and G. Valle, genome analysis of Photobacterium profundum revealing the complexity of high pressure adaptations; direct submission to the EMBL/GenBank^TM/DDBJ data bases, 2004.

	ACKNOWLEDGMENTS

 
We thank Syed Syeed Abdul for technical assistance and anonymous reviewers for their helpful and insightful comments.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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