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J. Biol. Chem., Vol. 277, Issue 12, 10581-10589, March 22, 2002

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Disease-causing Mutations in Cartilage Oligomeric Matrix Protein Cause an Unstructured Ca²⁺ Binding Domain^*

Quinn Kleerekoper^§, Jacqueline T. Hecht^¶, and John A. Putkey^§

From the  Departments of Biochemistry and Molecular Biology and ^¶ Pediatrics, ^§ Structural Biology Research Center, University of Texas, Houston Medical School, Houston, Texas 77030

Received for publication, October 15, 2001, and in revised form, December 11, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chondrocytes from pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) patients display an enlarged rough endoplasmic reticulum that accumulates extracellular matrix proteins, including cartilage oligomeric matrix protein (COMP). Mutations that cause PSACH and EDM1 are restricted to a 27-kDa Ca²⁺ binding domain (type 3 repeat). This domain has 13 Ca²⁺-binding loops with a consensus sequence that conforms to Ca²⁺-binding loops found in EF hands. Most disease-causing mutations are found in the 11-kDa C-terminal region of this domain. We expressed recombinant native and mutant forms of the type 3 repeat domain (T3) and its 11-kDa C-terminal region (T3-Cterm). T3 and T3-Cterm bind ~13 and 8 mol of Ca²⁺/mol of protein, respectively. CD, one-dimensional proton, and two-dimensional ¹H-¹⁵N HSQC spectra of Ca²⁺-bound T3-Cterm indicate a distinct conformation that has little helical secondary structure, despite the presence of 13 EF hand Ca²⁺-binding loops. This conformation is also formed within the context of the intact T3. 19 cross-peaks found between 9.0 and 11.4 ppm are consistent with the presence of strong hydrogen bonding patterns, such as those in -sheets. Removal of Ca²⁺ leads to an apparent loss of structure as evidenced by decreased dispersion and loss of all down field resonances. Deletion of Asp-470 (a mutation found in 22% of all PSACH and EDM1 patients) decreased the Ca²⁺-binding capacity of both T3 and T3-Cterm by about 3 mol of Ca²⁺/mol of protein. Two-dimensional ¹H-¹⁵N HSQC spectra of mutated T3-Cterm showed little evidence of defined structure in the presence or absence of Ca²⁺. The data demonstrate that Ca²⁺ is required to nucleate folding and to maintain defined structure. Mutation results in a partial loss of Ca²⁺-binding capacity and prevents Ca²⁺-dependent folding. Persistence of an unstructured state of the mutated Ca²⁺ binding domain in COMP is the structural basis for retention of COMP in the rough endoplasmic reticulum of differentiated PSACH and EDM1 chondrocytes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pseudoachondroplasia (PSACH)¹ and multiple epiphyseal dysplasia (EDM1) are related autosomal dominant skeletal dysplasias that are characterized by disproportionate short stature, lax joints, and early osteoarthritis (1, 2). These diseases are caused by mutations in cartilage oligomeric matrix protein (COMP). COMP, a member of the thrombospondin family of proteins (3), is found in bone growth plates, tendon bundles, ligaments, and smooth muscles (4-7). High concentrations of the protein are seen in the territorial matrix of chondrocytes (4) of mature cartilage from normal individuals but is depleted in the matrix of cartilage from PSACH and EDM1 patients (7). In these patients, COMP is sequestered in the rough endoplasmic reticulum (rER) (8), which becomes massively enlarged with alternating electron dense and electron lucent material (9, 10). Other extracellular matrix proteins, such as type IX collagen (8), are also retained in the enlarged rER of PSACH chondrocytes. These observations suggest that expression of the PSACH and EDM1 cellular phenotype results, at least in part, from improper protein processing/trafficking in chondrocytes.

COMP is a homopentamer composed of multidomain subunits. Each subunit consists of four domains as follows: an N-terminal association domain, followed by an epidermal growth factor-like type 2 repeat domain, a Ca²⁺-binding type 3 repeat domain, and a C-terminal globular domain. Interestingly, 72 of 76 known disease-causing mutations in COMP are localized to the type 3 repeat Ca²⁺ binding domain, and the majority of these are found in the C-terminal portion of this domain (8, 11, 12). Several general mechanisms could contribute to the observed cellular phenotype of PSACH and EDM1 chondrocytes. First, mutations may compromise the function of COMP. Second, mutations may cause gain of aberrant intracellular function or undesirable protein-protein interactions. A third possibility is that mutations may induce improper folding that leads to aggregation of partially folded intermediates or undesirable nonspecific interactions between mutant COMP and other proteins in the rER. Such a mechanism would be dominant negative and would be independent of the normal function of COMP.

To investigate these potential mechanisms, we have initiated a study to provide detailed information on the structural consequence of mutations in the Ca²⁺-binding type 3 repeat domain of COMP. This domain has 13 Ca²⁺-binding loops that conform to the consensus sequence of an EF hand Ca²⁺-binding loop such as those found in calmodulin and troponin C (13). A typical EF hand, or helix-loop-helix Ca²⁺-binding motif, consists of a calcium-binding loop flanked by helices of 10-12 amino acids. In contrast, 5 pairs of putative Ca²⁺-binding loops in COMP are separated by only 1 or 3 amino acids. Thus, the Ca²⁺ binding domain in COMP and other thrombospondins represent a novel utilization of the EF hand Ca²⁺-binding loop. We report here comparative analysis of the wild type and mutant COMP Ca²⁺ binding domains, with a focus on the C-terminal 11-kDa subdomain that incurs the majority of disease-causing mutations. The recombinant mutant proteins have Asp-470² deleted; a mutation that is found in 22% of all PSACH and EDM1 patients. The data are consistent with a model in which removal of Ca²⁺ or mutation leads to a loss of defined structure in the Ca²⁺ binding domain of COMP.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Expression and Isolation-- Selected regions of the COMP cDNA were amplified by PCR and inserted into the expression plasmid pET23d. All recombinant proteins encoded Ala as the second amino acid to enhance overall expression in Escherichia coli BL21pLys. The COMP derivatives could be easily isolated by standard procedures developed for cardiac troponin C (14). The cells were grown and induced as described previously (15) in either L-broth or M9 media prepared using ¹⁵N-ammonium chloride. Cells from 4 liters of culture were collected by centrifugation and resuspended in 120 ml of 50 mM Tris, pH 7.5, 0.2 mM EGTA, 0.1% BME. Resuspended cells were frozen at 70 °C, thawed, and then sonicated using a Branson sonicator with medium tip 3 times for 5 min at 50% power and 80% duty cycle while kept on ice. The lysed cells are centrifuged at 45,000 rpm in a Beckman Ti45 rotor. All COMP derivatives are found in the soluble fraction.

Soluble proteins were applied to a 2.5 × 30-cm Macro-Q (Bio-Rad) anion exchange column at a flow rate of 5 ml/min. The column was washed with 50 mM Tris, pH 7.5, 100 mM KCl, 0.2 mM EDTA, 0.1% BME until a base-line A₂₈₀ was achieved. Bound proteins were then eluted by a linear 100-500 mM KCl gradient (5 ml/min for 120 min). Fractions containing COMP proteins were pooled and made 1 M in ammonium sulfate, which precipitates most proteins but leave COMP soluble. After centrifugation, the soluble fraction was dialyzed against 50 mM Tris, pH 7.5, 100 mM KCl, 0.1% BME, 2 mM CaCl₂, and then applied to a Water semi-preparative DEAE Protein Pak high performance liquid chromatography column. After washing, the bound proteins were eluted with a 100-500 mM KCl gradient. Fractions containing COMP were pooled, dialyzed against 20 mM, ammonium bicarbonate, pH 8.0, 10 mM DTT, and lyophilized. The lyophilized proteins were refolded in vitro by first resuspending in 50 mM Tris, pH 7.5, 8 M guanidine HCl, 10 mM DTT, 2 mM CaCl₂. This was then diluted 50-100-fold into 50 mM Tris, pH 7.5, 100 mM KCl, 20 mM CaCl₂, 1 mM DTT, 0.1 mM oxidized DTT at room temperature. The refolded protein was concentrated and dialyzed against the buffer of choice for a given experiment. Proteins were analyzed by electrospray mass spectroscopy to confirm sequence and by amino acid analysis to determine protein concentration. The N-terminal Met residue was removed by the bacteria.

Calcium Binding-- Refolded proteins were transferred to a buffer of 50 mM MOPS, pH 7.0, 100 mM KCl, 2 mM CaCl₂, 1 mM DTT, 0.1 mM oxidized DTT and concentrated to a protein concentration of about 4 mg/ml. A 1-ml volume of protein was placed in a Slidylizer (Pierce) dialysis cassette and dialyzed at room temperature against the same buffer containing trace amounts of ⁴⁵CaCl₂. The time necessary to reach equilibrium was determined using a control Slidylizer that did not contain protein. Under these conditions the free Ca²⁺ was set by the total concentration of Ca²⁺ in the buffer reservoir. After equilibrium, 0.1 ml was removed from the buffer reservoir and from each Slidylizer, and the cassettes were transferred to another buffer reservoir containing a lower total Ca²⁺ concentration. This procedure was repeated to collect data points at 2, 1, 0.5, 0.25, and 0.1 mM free Ca²⁺. Protein remaining after the final dialysis was analyzed by SDS-PAGE to assess potential degradation. The concentration of protein at each level of free CaCl₂ was determined using the Pierce BCA assay. COMP proteins, the concentration of which had been determined by amino acid analysis, were used as standards for the protein assays.

Circular Dichroism-- CD spectra were collected on a JASCO J-710 spectrofluorimeter using a cylindrical cuvette with a 0.5-mm path length. Proteins that were refolded in vitro were dialyzed against 10 mM MOPS, pH 7.0, 100 mM KCl, 1 mM reduced DTT, 0.1 mM oxidized DTT, 2 mM CaCl₂, and then diluted to a concentration of 5-20 µM in the same buffer. CD spectra were collected before and after the addition of 3 mM EDTA.

NMR-- NMR spectroscopy was carried out on a Bruker Avance DRX 600, or Varian Unity Plus 600 spectrometers equipped with a inverse triple resonance gradient probes. Proteins used for NMR samples varied in protein concentration from 1 to 2 mM and were exchanged into a NMR buffer consisting of 20 mM Tris, pH 7.0, 100 mM KCl, 20 mM CaCl₂, 10 mM reduced DTT, 1 mM oxidized DTT (presence or absence of oxidized DTT did not greatly affect chemical shifts) in a 9:1 ratio of H₂O to ²H₂O by repeated washing in a Millipore Ultrafree-4 Centrifugal Filter Unit with high flow Biomax membrane. Repeated washing with the CaCl₂-containing buffer allowed the concentration of free CaCl₂ in the NMR sample to remain at 20 mM. Thus the total CaCl₂ in the NMR samples was 20 mM plus the amount of CaCl₂ bound to the protein. Apoproteins were generated by washing into a buffer that contained 1 mM EDTA rather than CaCl₂. Preparation of apoproteins consisted of repeated washing with NMR buffer containing 1 mM EDTA to the NMR sample. All experiments were carried out at 298 K and a pH of 6.8. HSQC spectra were acquired with spectral widths of 11,000 Hz for ¹H and 2000 Hz for ¹⁵N. A total of 2048 data points in t₂ and 128 t₁ points were used for the HSQC experiments. ¹H chemical shifts were referenced to 3-(trimethylsilyl)-1-propanesulfonic acid. ¹⁵N chemical shifts were referenced indirectly by using the gyromagnetic ratio. The NMR data were processed on a PC work station using FELIX2001 (Accelrys, San Diego, CA)

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Selection of Recombinant Proteins-- The domain organization of COMP monomers is shown in Fig. 1A. Epidermal growth factor-like and globular domains flank the type 3 repeat Ca²⁺ binding domain. Fig. 1B illustrates consensus sequences in the Ca²⁺-binding loops and the longer interloop regions of human COMP, as well as the pattern of Cys residues in the type 3 repeat. There are 13 potential Ca²⁺-binding loops that conform to the consensus sequence of an EF hand Ca²⁺-binding loop. The longer interloop sequences are rich in Pro residues, which likely inhibit formation of -helices found in typical EF hands. Five of the interloop spacers consist of only 1 or 3 amino acids.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Domain structure of COMP (A), primary sequence of the type 3 repeat Ca2+ binding domain (B), and recombinant proteins expressed in bacteria (C). The domain structure of a COMP monomer is based on primary sequence analysis of the thrombospondins, as well as electron microscopy (44), which shows a bouquet-like structure, topped with globular heads. Depiction of the various domains in not meant to infer specific structural conformations. The primary sequence of the type three repeat is aligned to highlight similarities within the Ca2+-binding loops and interloop spacers. Consensus sequences are shown at the bottom. The consensus sequence for the interloop spacers is based only on those of 11 amino acids in length. X, Y, Z, Y, X, and Z refer to the coordination positions for Ca2+ in the EF hands of calmodulin and troponin C (see Ref. 13 for review). Cys residues are boxed.

Fig. 1C summarizes the proteins used in this study. The protein called T3 encodes amino acids 302-524 (loops 2-13) and was described previously (16). The N- and C-terminal regions of the type 3 repeat have distinguishing characteristics. This includes a different distribution of short and long interloop spacers, different patterns of Cys residues, and the presence of His residues only in the C-terminal half. These features, together with the fact that 66% of disease-causing mutations are located in the C-terminal half of the type three repeat, led us to construct an expression plasmid for an 11-kDa protein called T3-Cterm which encodes amino acids 420-524 (loops 8-13). A pair of mutated derivatives, T3D and T3-CtermD, was generated in which Asp-470 was deleted to mimic the most common disease-causing mutation. The net effect of this mutation was to remove the single amino acid between loops 10 and 11.

We used a bacterial system to express COMP proteins because it provides high levels of expression and efficient labeling of proteins with stable isotopes required for multidimensional heteronuclear NMR. Tissue-derived human COMP is glycosylated at residues 101, 124, and 721, which are outside the type 3 repeat (17). The native folding environment of COMP in the rER is rich in Ca²⁺ and contains chaperones such as protein-disulfide isomerase, which may aid in the proper folding of the Cys-rich type 3 repeat. In contrast, the intracellular environment of bacteria is low in Ca²⁺ and high in reducing potential. The proteins used in this study have all been refolded in vitro as described under "Experimental Procedures." Fig. 2 shows an SDS gel of the purified native and mutant proteins. Both T3 and T3-Cterm proteins migrate with apparent relative molecular masses that are greater than predicted from primary sequence. The molecular weight of the recombinant proteins was confirmed by electrospray mass spectroscopy.

View larger version (59K): [in this window] [in a new window]   Fig. 2.   SDS-PAGE of isolated recombinant T3, T3D, T3-Cterm, and T3-CtermD. Cardiac troponin C is used for size comparison. The gel was run under reducing conditions with 1% BME in the sample buffer.

Ca²⁺ Binding to Type 3 Repeat Domain and Subdomain-- Typically, the Ca²⁺-binding properties of proteins are determined by sequential addition of Ca²⁺ to the apoprotein. Unlike regulatory EF hand proteins that are designed to cycle between apo- and Ca²⁺-bound forms, COMP may require Ca²⁺ to maintain a proper structure that is not readily regained if Ca²⁺ is removed from the folded protein and then replenished. Thus, we generated Ca²⁺-binding isotherms for recombinant COMP proteins by sequential dilution of Ca²⁺ after the proteins had been refolded in the presence of Ca²⁺. Fig. 3 compares the Ca²⁺-binding curves of T3 and T3-Cterm with T3D and T3-CtermD. T3 binds 12-13 mol of Ca²⁺ per mol of protein with a K_50,Ca of about 0.25 mM. T3-Cterm binds 8-9 mol of Ca²⁺ per mol of protein with a K_50,Ca of about 0.5 mM. The mutant T3D and T3-CtermD bind less Ca²⁺ than the wild type proteins at all concentrations of free Ca²⁺, and with slightly lower affinity. At 2 mM Ca²⁺ both T3 and T3-Cterm bind about 3 more mol of Ca²⁺/mol of protein than the mutated derivatives. This suggests that the effect of deletion of Asp-470 in the C-terminal half of the type 3 repeat domain is restricted to Ca²⁺-binding sites in this subdomain.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Ca2+ binding to recombinant T3, T3D, T3-Cterm, and T3-CtermD. T3 equilibrium dialysis was used to generate the Ca2+-binding curves. Data were collected by sequential dilution of the Ca2+-loaded proteins as described under "Experimental Procedures."

Circular Dichroism-- The predicted secondary structure of T3-Cterm (PSIPRED V2.0) indicates a high content of coiled structures. To confirm this, we performed circular dichroism on all proteins in the presence and absence of Ca²⁺ as an initial screen for differences in protein structure. Preliminary NMR studies indicated that low ionic strength has a significant effect on the structure of T3-Cterm. Because chloride ions interfere with CD measurements in the far UV range, and because fluoride precipitates with Ca²⁺, we performed all measurements in 100 mM KCl and show CD spectra from 195 to 240 nm. Fig. 4 compares the CD spectra of the wild type and mutant T3 proteins (Fig. 4A) and T3-Cterm (Fig. 4B). The large negative ellipticity at 200 nm and small negative values at 220 nm indicate high content of coiled secondary structure and relatively little helical structure for all proteins. The CD data shows several overall trends for T3 and T3-Cterm proteins. The wild type proteins displayed an increase in negative ellipticity at ~200 nm and a shift to higher wavelength upon addition of Ca²⁺ to the apoproteins. Unlike the wild type proteins, mutant D proteins behaved differently. Addition of Ca²⁺ to apoT3D resulted in a decrease in negative ellipticity at ~200 nm, a similar shift to higher wavelength as seen for wild type T3, and less change than T3 in [] at all higher wavelengths. T3-CtermD exhibited similar characteristics as T3D at ~200 nm upon Ca²⁺ addition but to a greater degree. Unlike T3-Cterm, there is a substantial decrease in ellipticity at all wavelengths for T3-CtermD. The data suggest the T3-Cterm proteins undergo different structural transitions in response to Ca²⁺. The changes observed for T3-Cterm could represent an unfolded to folded transition, but for T3-CtermD the results are most consistent with an unfolded to aggregated transition.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   CD spectra for wild type and mutant T3 and T3-Cterm in the presence and absence of Ca2+.

NMR-- CD measures the sum of secondary structures in a protein. It is sensitive to structural changes, but it does not provide great insight into the nature of these changes. We employed heteronuclear NMR to determine the effect of Ca²⁺ and mutation on the structure of the type 3 repeat. The focus of the present study is T3-Cterm because it incurs the majority of disease-causing mutations. All spectra were collected using proteins that had been refolded in vitro in the presence of Ca²⁺ and a 10:1 ratio of reduced and oxidized DTT (1/0.1 mM). Subsequent addition of excess DTT to the refolded protein did not greatly affect the NMR spectra (data not shown). Calcium was removed from the refolded protein to generate the apoform by sequential dilution and concentration with a buffer containing EDTA.

Fig. 5 compares the amide region of the one-dimensional proton spectra for wild type T3-Cterm (Fig. 5A) and T3-CtermD (Fig. 5B) in the presence and absence of Ca²⁺. The spectrum of Ca²⁺-bound T3-Cterm is well dispersed with a number of distinct down field-shifted resonances. Removal of Ca²⁺ from wild type T3-Cterm dramatically reduces or eliminates the down field shifted resonances and generally decreases resonance dispersion. Spectra for the mutant T3-CtermD show little evidence of the down field shifted resonances, and the resonances are much less dispersed than for wild type T3-Cterm in the presence or absence of Ca²⁺. The two groups of chemical shifts at about 6.8 and 7.6 ppm correspond to the side chain amides of Gln and Asn residues. Thus, chemical shifts of the backbone H^N for apoT3-Cterm and T3-D in the presence or absence of Ca²⁺ are found in a narrow spectral window between 7.8 and 8.8 ppm. This lack of dispersion is characteristic of unstructured protein (18).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Amide region of the proton NMR spectrum for T3-Cterm and T3-CtermD in the presence (red) and absence (black) of Ca2+. The samples contained 2 mM protein, 20 mM Tris D11, pH 7.0, 100 mM KCl, 5 or 1 mM DTT, with 35 mM CaCl2 or without Ca2+ as described under "Experimental Procedures."

Fig. 6, A and B, shows the ¹H-¹⁵N HSQC spectrum of T3-Cterm in the presence and absence of Ca²⁺, respectively. The spectrum of Ca²⁺-bound T3-Cterm is well dispersed and characteristic of a protein with defined structure. 93 cross-peaks (excluding side chain amides) resolved at this contour level, which agrees well with the 96 backbone amide protons predicted from the primary sequence. The spectrum shows 19 cross-peaks down field of 9.0 ppm in the proton dimension. These down field resonances likely correspond to protons that participate in strong hydrogen-bonded structures, such as -sheets. Four of the down field cross-peaks are identified as the H^N protons of the four glycine residues at the 6th position of Ca²⁺-binding loops 9-12.³ The H^N chemical shifts of Gly residues in the 6th position of EF hand Ca²⁺-binding loops of calmodulin and troponin C shift down field in the presence of Ca²⁺ (19-22). Removal of Ca²⁺ causes a significant spectral change that is dominated by a substantial decrease of dispersion in the proton dimension (Fig. 6B). This is consistent with the one-dimensional spectra in Fig. 5. Most notably, all of the down field resonances seen in the presence of Ca²⁺ are absent upon removal of Ca²⁺. There is little overlap of resonances between the spectra for T3-Cterm in the presence and absence of Ca²⁺.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   1H-15N HSQC of T3-Cterm and T3-CtermD in the presence and absence of Ca2+. A and B show the entire amide region for T3-Cterm in the presence and absence of Ca2+, respectively. C and D show the amide region of T3-CtermD in the presence and absence of Ca2+, respectively. Buffer conditions are described in Fig. 5.

Fig. 6, C and D, shows the ¹H-¹⁵N HSQC spectra for T3-CtermD in the presence and absence of Ca²⁺, respectively. In contrast to wild type T3-Cterm, the mutant protein shows little resonance dispersion in the presence of Ca²⁺. The 19 down field cross-peaks seen for the wild type protein in the presence of Ca²⁺ are absent at this contour level in T3-Cterm D. Removal of Ca²⁺ from T3-CtermD has little effect on the ¹H-¹⁵N HSQC spectra. Fig. 6 suggests that T3-Cterm assumes an unstructured or multiply structured state. A similar conclusion can be drawn in the absence or presence of Ca²⁺ for the Asp-470 deletion mutant.

Fig. 7 compares the down field region of the ¹H-¹⁵N HSQC spectrum for Ca²⁺-bound T3-Cterm (black) and the 27-kDa T3 (red). It is clear that all resonances in T3-Cterm are present in the spectrum for T3 with identical chemical shifts. Coincidence of cross-peaks is also seen in other regions of the spectra. Only three resonances in the spectra for Ca²⁺ bound T3-Cterm do not overlap with corresponding resonances in T3 (data not shown). Thus, we conclude that Ca²⁺-bound T3-Cterm assumes a single dominant conformation in solution and that T3-Cterm is an independently folding subdomain of the larger type 3 repeat Ca²⁺ binding domain in COMP.

View larger version (9K): [in this window] [in a new window]   Fig. 7.   2+H-15N HSQC of T3-Cterm and T3. The down field amide region of Ca2+-bound T3 (red) and T3-Cterm (black) are overlaid to highlight similarities and differences. Buffer conditions are described in the Fig. 5.

We wished to collect ¹H-¹⁵N HSQC spectra for T3-Cterm at sub-saturating levels of Ca²⁺. Rather than add Ca²⁺ to the apoprotein, which may not readily refold under conditions used to collect NMR spectra, we elected to dilute Ca²⁺ in a sample that had been refolded in the presence of calcium. The total Ca²⁺ in a 2 mM sample of T3-Cterm was diluted to a final predicted total Ca²⁺ concentration of 0.1 mM by sequential rounds of dilution and concentration. Fig. 8A compares the spectra of the same protein at high (black) or low (red) concentrations of Ca²⁺. All resonances observed at saturating Ca²⁺ are also present at the lower Ca²⁺ concentration, but the relative intensities were about 30% those observed for the Ca²⁺-saturated protein. At the lower concentration of Ca²⁺, additional resonances are observed between 7.8 and 8.8 ppm that are characteristic of the apoprotein. Fig. 8B expands the boxed region in A. The cross-peaks in B are the sum of resonances observed in spectra of the Ca²⁺-saturated (C) and apoproteins (D). The presence of distinct structured and unstructured populations at sub-saturating levels of Ca²⁺ is typical of a slow exchange process associated with high affinity ligand binding. The equilibrium Ca²⁺ binding constant of COMP is on the order of 10⁴ M¹. Thus we were surprised to see evidence of slow exchange for COMP. Additional studies will be necessary to characterize this phenomenon.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   1H-15N HSQC spectra of T3-Cterm at different levels of Ca2+. A compares HSQC spectra of T3-Cterm at high (black) and low (red) levels of Ca2+. Protein was refolded in the presence of 20 mM Ca2+ and concentrated to about 2 mM in a buffer that contained 20 mM Ca2+ as described under "Experimental Procedures." The total Ca2+ was about 35 mM. After collection of an HSQC spectrum, total Ca2+ in the sample was effectively diluted to about 0.1 mM by successive rounds of dilution of the protein into a Ca2+-free buffer followed by concentration to the original volume. The relative intensities of the down field cross-peaks at low Ca2+ levels were about 30% of the Ca2+-saturated protein. Contour levels were adjusted optimally to observe cross-peaks in both spectra. B shows and an expanded view of the indicated region from A. The black contours correspond to resonances found in the Ca2+-saturated protein (C), and the green contours correspond to those found in the fully apoprotein (D).

The results in Fig. 8 suggest that very low affinity binding of Ca²⁺ to mutated T3-CtermD could be detected as a minor population of protein with a dispersed HSQC spectrum. Thus, ¹H-¹⁵N HSQC spectra of T3-CtermD were analyzed at a very low contour level. Fig. 9 shows this analysis in red with the corresponding spectra of Ca²⁺-saturated T3-Cterm shown in black. Only the down field region of the spectral window is shown because the very intense signals at this low contour level obscure comparisons in other spectral regions. At a low contour level, dispersed cross-peaks are observed for T3-CtermD. The relative intensities of the cross-peaks seen for T3-CtermD are less than 2% those of wild type protein, and the chemical shifts are not identical between the two proteins. These data suggest that a small population of the mutant protein exists in a more structured state in the presence of 20 mM free Ca²⁺. This likely reflects a dramatic decrease in Ca²⁺ affinity, rather than complete inhibition of Ca²⁺ binding due to deletion of Asp-470. Nevertheless, deletion of Asp-470 causes an unstructured or scrambled state in the bulk of the population of protein in the presence or absence of biologically relevant concentrations of Ca²⁺.

View larger version (9K): [in this window] [in a new window]   Fig. 9.   Comparison of the down field cross-peaks in the 1H-15N HSQC spectra of Ca2+-bound T3-Cterm (black) and T3-CtermD (red). The observed cross-peaks in the spectra of T3-CtermD are observed only at a very low contour level and are not observed at the contour level used in Fig. 6.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Dominant negative mutations in COMP cause the rER in chondrocytes of PSACH and EDM1 patients to become grossly enlarged and to retain COMP and other extracellular matrix proteins. 72 of 76 known disease-causing mutations are located in the type 3 repeat Ca²⁺ binding domain of COMP. 70% of these mutations are in the C-terminal portion of the type 3 repeat, and 22% involve deletion of 1 of 5 Asp codons (Asp-469-473). This essentially deletes the single Asp-470 spacer between Ca²⁺-binding loops 10 and 11 (see Fig. 3). The goal of the current study was to apply NMR to gain insights into the structural consequence of this deletion. We report the first high resolution NMR data for a member of the thrombospondin family of proteins. The results have general implications with respect to the effect of Ca²⁺ on the structure of Ca²⁺-binding proteins in normal and disease states.

Primary sequence analysis suggests that the N- and C-terminal portions of this domain have distinct structures. Our data show that T3-Cterm is indeed a structurally distinct subdomain that can fold independent of the intact type 3 repeat domain. We hypothesize that the N-terminal portion of the type 3 repeat is also a structurally distinct subdomain. Independence of the N and C subdomains of the type 3 repeat may insulate them from global effects of mutations. For example, deletion of Asp-470 in the C-terminal subdomain may minimally affect the structure of the N-terminal subdomain. This would be analogous to what is observed for a dimer of LDL-A Ca²⁺/ligand binding domains from the LDL receptor, which are structurally independent, and are insulated from the effects of mutation in the paired domain (23). Structurally distinct subdomains in the type 3 repeat may confer important functional characteristics. For example, the adjacent globular domain is known to bind collagen (24), and mutations in the type 3 repeat affect collagen binding (25). Although the biological significance of these observations is unknown, the structural integrity of both type 3 repeat subdomains may be necessary to orient properly the globular domain for optimal ligand binding.

The data presented here argue that the Ca²⁺-binding loops in COMP provide a coordination sphere that is similar if not identical to that of a canonical EF hand. Four of the Ca²⁺-binding loops in T3-Cterm (loops 9-12) have Gly at the 6th position (Gly-440, -463, -476, and -499). Glycine residues at this relative position in the canonical EF hands of CaM and troponin C allow a tight ~90° turn of the Ca²⁺-binding loops (13). In the presence of Ca²⁺, the amide proton of this Gly residue forms a strong hydrogen bond with the carboxyl oxygen of the acidic residue at loop position 1. This confers an unusually large down field chemical shift (8.9-11.0 ppm) on the H^N of this Gly in the EF hands of cardiac troponin C (19, 20), skeletal troponin C (21), and calmodulin (22) that can be diagnostic for Ca²⁺ binding. The circled cross-peaks centered on 10 ppm in Fig. 6 correspond to the H_N of Gly-440, -463, -476, and -499 in Ca²⁺-binding loops 9-12, which we attribute to a similar structural feature associated with Ca²⁺ binding. Thus we propose that loops 9-12 provide a coordination geometry and pentagonal bipyramidal distribution of ligands that is similar to prototypical EF hands. These four Ca²⁺-binding loops may constitute a stable core structure for T3-Cterm that is hypersensitive to mutations.

Although Ca²⁺-binding loops in T3-Cterm conform to those found in a canonical EF hand, it is unlikely that the Ca²⁺ binding domain of COMP consists of multiple copies of true helix-loop-helix EF hand motifs. These observations are consistent with the primary sequences of T3 and T3-Cterm, which are dominated by Ca²⁺-binding loops. The first 9 amino acids in an EF hand Ca²⁺-binding loop have a coiled or -strand secondary structure, whereas the last three amino acids form the first portion of the F helix. Four pairs of Ca²⁺-binding loops in COMP are separated by only a single amino acid, and most of the longer (11 amino acids) interloop spacers have two Pro residues. It is likely that these features inhibit extensive helix formation between Ca²⁺-binding loops. In addition, the 19 amide protons with chemical shifts between 9.0 and 11.4 ppm in the spectra for Ca²⁺-bound T3-Cterm are consistent with the presence of strong hydrogen bonding patterns such as those found in -sheets (26), but the structural context remains in question.

There is precedent for the use of EF hand-like Ca²⁺-binding loops outside the context of -helices. Vyas et al. (27) reported a variant EF hand in bacterial galactose-binding protein that has a partial EF loop bordered by helix and -strand secondary structures. The Ca²⁺-binding motif found in bacterial alkaline protease (28, 29) and the -propeller domain of integrin _V3 (30) have a short Ca²⁺-binding loop flanked by -strands. However, the Ca²⁺-binding loops in these proteins differ significantly from the 12-amino acid loop of an EF hand in that the Z Ca²⁺ coordination site is not provided by position 12 of the loop. We predict that the type 3 repeat domain in COMP has multiple copies of a motif that uses canonical EF hand Ca²⁺-binding loops in a novel context.

The NMR spectra reported here show that removal of Ca²⁺ from T3-Cterm results in a general loss of proton resonance dispersion. All of the down field amide proton resonances seen for Ca²⁺ bound T3-Cterm are absent from the one-dimensional ¹H and two-dimensional ¹H-¹⁵N HSQC spectra of the apoprotein. This is consistent with a fully unstructured state or an ensemble of rapidly converting structures (18) for T3-Cterm in the absence of Ca²⁺. We hypothesize that Ca²⁺ binding nucleates folding and is required to maintain a folded state with defined tertiary topology stabilized by hydrogen bonding patterns. This defined topology is lost in the absence of Ca²⁺. Under appropriate oxidizing conditions, removal of Ca²⁺ may induce scrambling of disulfide bonds. However, we do not feel that potential disulfide scrambling is the primary cause of loss of defined structure, because high concentrations of DTT fail to promote resonance dispersion in the apoprotein (data not shown).

Whereas a number of EF hand containing proteins (e.g. CaM and skeletal troponin C, calbindin D_9k, calcyclin, S100B) are well structured in the absence of Ca²⁺. The removal of Ca²⁺ can have severe global structural consequences for proteins with Ca²⁺-binding motifs. The cysteine-rich LDL-A modules of the LDL receptor and the Tva cellular receptor both require Ca²⁺ for proper folding (31, 32). Removal of Ca²⁺ from these motifs results in an unstructured or scrambled ensemble. The overall effect of removal of Ca²⁺ on the NMR spectra of the LDL-A motif is very similar to that reported here for T3-Cterm. Dramatic structural effects of removal of Ca²⁺ have also been observed in EF hand proteins from invertebrates. The EF hand protein calerythrin from Saccharopolyspora erythraea is in equilibrium between a structured and less structured form in the absence of Ca²⁺ (33), and the sarcoplasmic EF hand protein NSCP from the invertebrate Nereis diversicolor appears unstructured in the absence of Ca²⁺ (34).

Deletion of Asp-470 causes a loss of about 3 mol of Ca²⁺/mol of protein for the intact type 3 repeat domain as well as T3-Cterm. This is consistent with the results of Chen et al. (35) who observed that intact pentameric 470 COMP bound 2-4 mol less Ca²⁺ per subunit than the wild type protein. The NMR spectra show that T3-CtermD is largely unstructured in the presence or absence of 20 mM free Ca²⁺. Because the wild type protein is unstructured in the absence of Ca²⁺, we conclude that inhibition of Ca²⁺ binding is the primary reason the mutated protein fails to adopt defined structure. These data are consistent with electron micrographs of native and mutant COMP pentamers (35). In the presence of Ca²⁺ the stalk region of native COMP homopentamers appears compact, and in the absence of Ca²⁺ they appear more extended. The stalk region in mutant COMP homopentamers appears more extended in the presence or absence of Ca²⁺. The data presented here suggest that the more extended conformation is not the result of a misfolded yet defined structure but rather to an unfolded or unstructured Ca²⁺ binding domain that may have a high content of extended and flexible random coil.

It is not presently clear by what mechanisms the unstructured mutated type 3 repeat domain in COMP promotes intracellular protein retention resulting in a grossly enlarged rER in PSACH and EDM1 chondrocytes. Retention of COMP is gradual and requires or is at least enhanced by expression of other extracellular matrix proteins in the rER of differentiated chondrocytes (36, 37). This suggests the possibility of specific interactions between mutated COMP and other extracellular proteins that are being processed in the rER. But the unstructured mutated Ca²⁺ binding domain of COMP raises the potential for nonspecific interactions as well. Unstructured or partially folded proteins can associate in a concentration-dependent manner with other partially folded proteins, primarily via hydrophobic interactions. Intracellular protein crowding would enhance such interactions (38) and possibly promote intracellular deposition of long lived partially folded intermediates that result from pathological conditions (39). Differentiated chondrocytes may provide a crowded protein environment that favors heterologous interactions between mutated COMP and a variety of other large extracellular proteins that are being processed in the rER.

The potential role of chaperone proteins must also be considered as suggested previously (16, 35). Creemers et al. (40) demonstrated that binding of BiP to lymphoma proprotein convertase prevents aggregation but slows protein maturation. Thrombospondin transiently associates with BiP, Grp94, and Erp72 (41, 42), and native and mutant COMP are in close proximity to these proteins as well (43). Prolonged association of chaperonins with an unstructured COMP Ca²⁺ binding domain may inhibit protein secretion and/or disrupt the equilibrium distribution of chaperonins between other proteins in the crowded chondrocyte rER, thus leading to global trafficking problems in the active rER of differentiated chondrocytes.

In summary, we have shown that the type three repeat Ca²⁺ binding domain of COMP is composed of distinct subdomains. Removal of Ca²⁺, and a disease-causing mutation, both led to a dramatic loss of structure in the C-term subdomain. This is likely central to the pathogenesis of PSACH and MED and is a striking example of a disease-causing mutations that cause a loss of structure in a human Ca²⁺-binding protein.

	ACKNOWLEDGEMENTS

We thank Dr. Ed Nikonowicz at Rice University and Dr. David Gorenstein and the Sealy Center for Structure Biology at the University of Texas Medical Branch, Galveston, TX, for the use of their respective NMR facilities. We also thank Wen Liu and Natasha Bogdanov for their expert technical assistance.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant RO1HL45724 (to J. P.) and Robert A. Welch Foundation Grant AU-1144 (to J. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Structural Biology Research Center, University of Texas, Houston Medical School, Houston, TX 77030. Tel.: 713-500-6061; Fax: 713-500-0652; E-mail: John.Putkey@uth.tmc.edu.

Published, JBC Papers in Press, January 8, 2002, DOI 10.1074/jbc.M109944200

² The literature typically refers to this mutation as deletion of Asp-469, which is the first of a cluster of five Asp-(469-473) residues encoded by GAC codons. We refer to the mutation here as deletion of Asp-470 because the net effect of deletion of any one of these 5 Asp residues is removal of the single Asp residue (Asp-470) that separates calcium-binding loops 10 and 11.

³ Identification of the Gly residues was made by assigning the neighboring spin systems of each Gly in Ca²⁺-bound ¹⁵N,¹³C-enriched T3 C-term using triple resonance experiments HNCACB, CBCA(CO)NH, C(CO)NH, and H(CCO)NH.

	ABBREVIATIONS

The abbreviations used are: PSACH, pseudoachondroplasia; EDM1, multiple epiphyseal dysplasia; COMP, cartilage oligomeric matrix protein; T3, type 3 repeat domain; T3-Cterm, T3 and its 11-kDa C-terminal region; rER, rough endoplasmic reticulum; BME, 2-mercaptoethanol; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; LDL, low density lipoprotein.

	REFERENCES

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