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J. Biol. Chem., Vol. 277, Issue 12, 9989-9996, March 22, 2002

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Comparative Analysis of Cobalamin Binding Kinetics and Ligand Protection for Intrinsic Factor, Transcobalamin, and Haptocorrin^*

Sergey N. Fedosov^§, Lars Berglund, Natalya U. Fedosova^¶, Ebba Nexø, and Torben E. Petersen

From the  Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark, the ^¶ Department of Biophysics, University of Aarhus, Ole Worms Alle 185, 8000 Aarhus C, Denmark, and the  Department of Clinical Biochemistry, AKH Aarhus University Hospital, Nørrebrogade 44, 8000 Aarhus C, Denmark

Received for publication, November 29, 2001, and in revised form, January 7, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Changes in the absorbance spectrum of aquo-cobalamin (Cbl·OH₂) revealed that its binding to transcobalamin (TC) is followed by slow conformational reorganization of the protein-ligand complex (Fedosov, S. N., Fedosova, N. U., Nexø, E., and Petersen, T. E. (2000) J. Biol. Chem. 275, 11791-11798). Two phases were also observed for TC when interacting with a Cbl-analogue cobinamide (Cbi), but not with other cobalamins. The slow phase had no relation to the ligand recognition, since both Cbl and Cbi bound rapidly and in one step to intrinsic factor (IF) and haptocorrin (HC), namely the proteins with different Cbl specificity. Spectral transformations observed for TC in the slow phase were similar to those upon histidine complexation with Cbl·OH₂ and Cbi. In contrast to a closed structure of TC·Cbl·OH₂, the analogous IF and HC complexes revealed accessibility of Cbl's upper face to the external reagents. The binders decreased sensitivity of adenosyl-Cbl (Cbl·Ado) to light in the range: free ligand, IF·, HC·, TC·Cbl·Ado. The spectrum of TC·Cbl·Ado differed from those of IF and HC and mimicked Cbl·Ado participating in catalysis. The above data suggest presence of a histidine-containing cap shielding the Cbl-binding site in TC. The cap coordinates to certain corrinoids and, possibly, produces an incapsulated Ado-radical when Cbl·Ado is bound.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intrinsic factor (IF),¹ transcobalamin (TC) and haptocorrin (HC) are three proteins involved in assimilation and transport of cobalamin (Cbl) in an organism (1). They all have extraordinary affinity to the physiologically active forms of Cbl with K_d < 1 pM (1-4) but exhibit different selectivity toward the nonfunctional Cbl analogues. IF and, to some extent, TC are sensitive to variations in the structure of the ligand, which helps these proteins to discriminate between the physiologically active and inactive corrinoids (2, 5). On the contrary, HC can successfully bind many defective corrinoids lacking even the whole nucleotide moiety (2, 5). Binding to the carriers shields the lower part of the Cbl molecule (also called -site), which contains the nucleotide. On the contrary, the upper surface of Cbl (-site) with the active group is thought to be open, as judged from its reactivity with the external compounds in the case of holo-IF and holo-HC (6-8).

Low amounts of the Cbl-binding proteins available from the natural sources (1, 6, 9, 10) hampered their investigations until several binders were successfully expressed in the recombinant organisms (11-15). The sufficient amounts of both bovine and human transcobalamin were obtained from the recombinant yeast Pichia pastoris. It allowed to establish the structure of the disulfide bridges in bovine TC (14) and investigate in detail Cbl·OH₂ binding to human TC by a stopped-flow technique (15). It was shown that the association between TC and Cbl·OH₂ occurred in two steps, when the initial attachment to an open conformation of the protein was followed by transition to a closed conformation with the shielded upper face of Cbl. As a result of this transition, cobalt-coordinated water in Cbl·OH₂ was thought to be displaced by a protein residue. The suggestion was supported by the fact that the external compounds coordinated to the -position of TC·Cbl·OH₂ at exceedingly slow rates. The described features, however, appeared to be characteristic only for Cbl·OH₂ interacting with TC, whereas binding of Cbl·OH₂, for instance, to HC occurred in one step (15). Cobalamins with the tightly associated -groups (Cbl·CN and Cbl·N₃) bound both to TC and HC in one step as well (15).

The experiments on Cbl·OH₂ interaction with TC and HC suggested a correlation between high specificity of the carrier for Cbl and the biphasic nature of the binding reaction. In this paper we, therefore, investigated the rapid kinetics of Cbl·OH₂ binding to the most Cbl-specific protein IF. Interaction of two other ligands (Cbl·Ado and an analogue Cbi) with IF, TC, and HC was also characterized. We found no correlation between the ligand specificity and the biphasic kinetics of binding. Slow spectral transformations were observed only for two ligands, Cbi (this paper) and Cbl·OH₂ (15), when interacting with TC. The character of these changes was identical to those induced by coordination of external histidine to Cbi or Cbl·OH₂. This fact supports the hypothesis of a cobalt-coordinated histidine residue within the complexes between TC and certain corrinoids. We also addressed the accessibility of bound Cbl to the external cobalt-specific reagents in different protein complexes. The results suggest that the -surface of Cbl associated with IF or HC is moderately open, in contrast to practically closed complex with TC. Binding of Cbl·Ado to the proteins protected to some extent this ligand from light-induced decomposition. In addition, the absorbance spectrum of TC·Cbl·Ado alluded to homolytic cleavage of the carbon-cobalt bond in 10-20% of the associated ligand molecules.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

All salts and media components were purchased from Merck, Roche Molecular Biochemicals, Sigma, and Beckton Dickinson. The enzymes and kits for DNA handling were obtained from New England Biolabs, Stratagene, and Roche Molecular Biochemicals, the kit for the PCR reaction was from HT Biotechnology Ltd. Oligonucleotides were synthesized by DNA technology. The employed yeast expression system was purchased from Invitrogen. The fermentor Biostat B from B. Braun Biotech International was employed during expression of the recombinant proteins. Sephacryl S-200 and CM Sepharose were obtained from Amersham Biosciences, Inc. The anti-IF serum was raised by DACO.

Methods

Preparation of the DNA Material for Expression of Human IF-- IF-encoding fragment of DNA was produced from a gastric RNA by the reverse transcriptase and polymerase chain reactions employing IF-specific primers with adaptors for XhoI and NotI endonuclease sites. The obtained product was purified and ligated to the corresponding sites in the expression plasmid pGAPZ. The designed sequence of the fusion protein contained the following components counting from the N terminus: a yeast secretion signal (-factor), the site for yeast protease Kex2, and the mature human IF. This construction ensured cleavage of the N-terminal peptides from the recombinant protein during its secretion:  ... LEKRSTQTQ ... , IF residues are underlined.

Expression and Purification of Human IF-- The recombinant IF was expressed according to recommendations of the manufacturer (Invitrogen) in yeast P. pastoris (strain SMD 1168). The constitutive promoter of glyceradehyde-3-phosphate dehydrogenase induced the expression. The fermentation of the recombinant yeast was carried out at 30 °C for 2 days in 1 liter of YPD medium (containing 0.5 µM Cbl·OH₂) with the constant supply of glucose. The level of oxygen and pH in the medium were maintained at 25% and 6.0, respectively. The cell-free supernatant was saturated with ammonium sulfate (520 g/liter) and centrifuged at 4,000 × g for 40 min. The pellet was dissolved in 50 ml of 0.05 M P_i buffer, pH 7.5, whereupon centrifuged once more at 12,000 × g for 10 min. The solution was concentrated by ultrafiltration to the volume 10-15 ml and applied to a 250-ml Sephacryl S-200 column equilibrated with 0.1 M Tris, 1 M NaCl, pH 7.5. The fractions with red protein were pooled, concentrated to 5-8 ml, and subjected to repeated gel filtration under analogous conditions. The red fractions with IF were collected, concentrated, and stored frozen. SDS electrophoresis, staining of the gel by Coomassie, staining of the glycoproteins by PAS method, and Western blotting were performed according to the standard procedures.

Expression and Purification of Human TC-- The recombinant TC was produced as described in our previous publication (15).

Isolation of Human HC-- The protein was purified from human plasma as described elsewhere (17).

Preparation of the Apo Forms of Cbl Binders-- Holo forms of IF, TC, and HC were dialyzed against 5 M GdnHCl (IF and TC) or 8 M GdnHCl (HC) at 30 °C for 2 days with one change. Liberation of Cbl was monitored visually. The proteins were renaturated by overnight dialysis against 0.2 M P_i buffer, pH 7.5, 5 °C.

Spectral Measurements-- The spectra were recorded on M350 Double Beam UV Visible Spectrophotometer (Camspec) or on the stopped-flow equipment, see the next paragraph.

Stopped-flow Experiments on Cbl Binding-- Binding of different corrinoids to the specific apo-proteins was followed on DX.17MV stopped-flow spectrofluorometer (Applied Photophysics) using difference in the absorbance spectra of the ligands in their free and bound state, see Ref. 15. The reactions were performed in 0.2 M P_i, pH 7.5, at 20 °C.

Dissociation of the Protein-Ligand Complexes-- When dissociation of Cbl·OH₂ from its protein complexes was investigated, the holo-protein (20 µM) was mixed with Cbl·CN (100 µM) and incubated at room temperature for 4 days. The samples of 0.15 ml were collected at different time intervals, suspended for 1 min with charcoal (pellet from 0.3 ml of 1% solution), and centrifuged for 1 min at 15,000 × g. Supernatants were centrifuged once more for 5 min. The loss of the protein due to adsorption on charcoal did not exceed 15%. Spectra of the protein-associated Cbls were recorded, whereupon displacement of Cbl·OH₂ by Cbl·CN was measured according to the ratios A₃₆₁/A₃₃₀, A₃₆₅/A₃₃₅, and A₃₆₃/A₃₃₀ for IF, TC, and HC, respectively. The transition spectra were compared with those of protein·Cbl·OH₂ and protein·Cbl·CN complexes to establish completeness of the reaction.

Dissociation of protein-Cbi complexes (20 µM) was initiated by adding 20 µM Cbl·OH₂. The measurements were carried out as described above except for the registration wavelengths: A₅₀₀/A₅₈₀, A₅₁₅/A₅₈₀, and A₅₀₀/A₅₉₀ for IF, TC, and HC, respectively.

Exchange of the Cobalt-coordinated Groups in the Corrinoids-- The displacement of the cobalt-coordinated groups in Cbi, Cbl·CN, IF·Cbl·OH₂, and HC·Cbl·OH₂ (20-25 µM) by the external ligands (CN, N and histidine) was followed spectroscopically in 0.2 M P_i buffer, pH 7.5, at 20 or 37 °C. Transformation of Cbl·Ado to Cbl·OH₂ (either free or bound to IF, TC, and HC) was induced by light when the sample in a quartz cuvette was placed in front of a 30 W daylight lamp at the distance of 20 cm. The changes in absorbance were measured at 352 nm (free Cbl) and 359 nm (protein-bound Cbl) with 1-min intervals.

Mathematical Analysis-- Fitting of the curves was performed by a computer program for nonlinear regression analysis² or a program Gepasi (18). The presented data were obtained from two to four parallel experiments and are shown as the mean values.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of the Cbl-binding Proteins-- Details of the isolation procedures for human HC and human recombinant TC were described elsewhere (15, 17). Human recombinant IF was expressed in yeast P. pastoris and purified as described under "Methods." The gel filtration profile of the purified recombinant IF contained one protein peak of 70 kDa saturated with Cbl·OH₂. SDS electrophoresis in the presence of a reducing agent revealed the major protein pool of 50-55 kDa (Fig. 1, lane 2), which was reactive toward IF-specific antibodies (Fig. 1, lane 4). The determined N-terminal sequence was identical to human gastric IF (STQTQS ... ). The 50-55 kDa band was not sharp, probably because of variation in the composition of carbohydrates coupled to the protein core of IF. The presence of carbohydrates on recombinant IF was confirmed by PAS staining of the gel (Fig. 1, lane 3).

View larger version (92K): [in this window] [in a new window]   Fig. 1.   SDS electrophoresis of recombinant IF treated with dithiothreitol. Lane 1, supernatant after fermentation concentrated by ammonium sulfate precipitation (Coomassie stained). Lane 2, purified IF (Coomassie stained). Lane 3, purified IF (PAS stained). Lane 4, Western blot of purified IF (polyclonal antibodies from rabbit were generated to human gastric IF and used as the primary antibodies).

All three Cbl binders were purified in complex with Cbl·OH₂ and preparation of apo-proteins required treatment with GdnHCl followed by a renaturing step. The regained binding capacity corresponded to 80-90% (TC), 60-70% (HC), and 30-40% (IF), when compared with the initial amounts of the bound Cbl.

Changes in Cbl Absorbance Upon Its Binding to the Cbl-specific Proteins-- Association of Cbls with IF, HC, and TC caused typical changes in the ligand spectrum (Fig. 2) (6, 8, 14, 15). The extinction coefficients of Cbl·OH₂ in complex with the proteins investigated are shown in Table I. These data were obtained on the originally purified holo forms as well as on the GdnHCl-treated, renatured and resaturated proteins. GdnHCl treatment had certain effects on the extinction coefficients. It was particularly evident for IF where all peaks increased by 15-20% (Table I) mainly due to intensified absorbance of the apo-protein (Fig. 2, B and C). The corresponding changes were insignificant for TC and practically absent for HC (Table I). The spectra of the free ligands are given for comparison in Fig. 2D.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Spectra of different corrinoids when free or bound to the specific proteins. The data were recorded at 20 °C, pH 7.5, with 0.5-nm steps as lines with long dashes for IF, short dashes for HC, and solid lines for TC. Panel A, spectra of the purified binders (20 µM) in complex with Cbl·OH2. Panel B, apo forms (22 µM) saturated with 20 µM Cbl·Ado. Panel C, apo forms (22 µM) saturated with 20 µM Cbi. Panel D, spectra of the free ligands (20 µM): Cbi (dashed line), Cbl·OH2 (line with dash-dot pattern), Cbl·Ado (line with one dash, two dots pattern).

View this table: [in this window] [in a new window]   Table I Extinction coefficients for Cbl·OH2 and its protein complexes at pH 7.5, 37 °C All extinction coefficients (M1 cm1) were determined with S.D. between ±200 and ±600.

The most significant shifts in the absorbance spectra of all ligands took place after their association with TC (Fig. 2, solid lines): (i) for Cbl·OH₂ one can see a noticeable red shift for all peaks (Fig. 2A); (ii) for Cbl·Ado this was a distortion of the shape at 350-380 and 480-550 nm (Fig. 2B); (iii) for Cbi there occurred an unusual redistribution of intensities from A₅₄₀/A₅₈₀ <1 to >1 (Fig. 2C). Curiously enough, the spectrum of TC·Cbl·Ado reminded very much of those for Cbl·Ado acting as a cofactor in glutamate mutase (19) and methylmalonyl-CoA mutase (20) under steady-state conditions. On the contrary, the spectra of analogous complexes with IF and TC were similar to glutamate mutase·Cbl·Ado in rest (19).

Development of Slow Spectral Distortions-- All spectral changes induced by the binding of Cbl·OH₂, Cbl·Ado, and Cbi to the specific proteins were accomplished in less than 1 s, except for the pairs TC + Cbl·OH₂ and TC + Cbi. Those cases attracted our special attention.

During the binding of Cbl·OH₂ to TC, the initial jump of the -peak was followed by continual spectral changes during the next 3 min (Fig. 3A). These slow perturbations significantly contributed to initially moderate red shift and amplification of the -peak. The process developed exponentially in time with the rate constant of 2.5 × 10 ² s¹, which did not differ from k₊₂ obtained earlier in the stopped-flow experiments at a single wavelength (15). Increase of the temperature essentially accelerated the slow phase but had no affect on its amplitude (at least between 20 and 37 °C).

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Slow spectral changes upon the binding of Cbl·OH2 to TC and during coordination of histidine to IF·Cbl·OH2, HC·Cbl·OH2, and Cbl·OH2. Apo-proteins (22 µM) were mixed with Cbl·OH2 (20 µM), and the spectra were recorded at 20 °C, pH 7.5, with 5-nm steps. Spectra at 1 s were reconstructed from the stopped-flow measurements at different wavelengths. Panel A, interaction of apo-TC with Cbl·OH2. Panel B, reaction of IF·Cbl·H2 (upper solid line) with 5 mM histidine. The spectral changes were recorded as dashed lines. Panel C, reaction of HC·Cbl·OH2 (upper solid line) with 5 mM histidine. The spectral changes were recorded as dashed lines. Panel D, spectra of free Cbl·OH2 (20 µM) after incubation with 5 mM aspartic acid, lysine, serine (dashed lines) and histidine (solid line). Incubation was carried out for 2 h at 65 °C. The spectra were recorded at 20 °C with 0.5-nm steps.

The slow decrease of absorbance for the -peak, induced by attachment of Cbi to TC, was not very noticeable due to the originally high absorbance of Cbi at 350-370 nm (not shown). The spectral transition was more evident for the smaller - and -peaks (Fig. 4A), and the effect was expressed better at 37 °C than at 20 °C. The transition was exponential with the rate constants of 4.3 × 10³ s¹ (20 °C) and 1.1 × 10² s¹ (37 °C).

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Spectral changes during the binding of Cbi to TC, and simulation of this process by mixing free Cbi with histidine or free Cbl with cyanide. The data were collected at 37 °C, pH 7.5, with 5-nm steps. Panel A, interaction of apo-TC (22 µM) with Cbi (20 µM). Panel B, reaction of 20 µM Cbi with 15 mM histidine. Positions of the spectra between 0 s and 60 s were reconstructed from time dependence at different wavelengths. The spectrum at histidine   was deduced from the saturation curve at 0-100 mM histidine. Panel C, transition of Cbl·CN (20 µM) to CN·Cbl·CN in the presence of 20 mM KCN. Positions of the spectra between 0 and 48 s were reconstructed from time dependence at different wavelengths.

Imitation of the Slow Phases by Coordination of Histidine to Cbl·OH2 and Cbi-- We have suggested earlier that the unusual spectral behavior of Cbl·OH₂ during binding to TC may have been caused by coordination of a protein residue to cobalt (15). The control experiment with several amino acids and free Cbl·OH₂ showed that only incubation with histidine caused noticeable spectral response (Fig. 3D, solid line), at least under the shown conditions. This is not surprising since imidazol is a known ligand with intermediate affinity to Cbl (21). The reaction between histidine and Cbl·OH₂ was reversible and characterized by the rate constants k_+His = 0.92 M¹ s¹ and k_His = 2.2 × 10⁴ s¹ (K_His= 0.24 mM) at pH 7.5 and 20 °C. At higher temperature (37 °C) the rate coefficients increased 2.0-2.2-fold without significant change in the equilibrium constant K_His.

Addition of 5 mM histidine to either IF or HC complex with Cbl·OH₂ caused gradual shift of the -peak (Fig. 3, B and C, respectively) analogous to the reaction between histidine and free Cbl·OH₂ (Fig. 3D). All above processes were similar in their manifestation to the second phase observed for TC + Cbl·OH₂ interaction (compare Fig. 3, B and C, dashed lines, with A). The rate coefficients of the forward reaction determined for IF·Cbl·OH₂ and HC·Cbl·OH₂ were equal to 0.44 M¹ s¹ and 0.05 M¹ s¹, respectively. The complex TC·Cbl·OH₂ did not react with histidine for at least 2 h (not shown).

Addition of histidine to Cbi also evoked spectral changes (Fig. 4B), which testified for coordinatioin of the imidazol group to either  or  surface of the corrinoid (21). The recorded spectra reversibly mirrored those during displacement of the dimethyl-benzimidazol base by cyanide: Cbl·CN + CN  CN·Cbl·CN (Fig. 4C). This may suggest attachment of histidine to the lower axial site of Cbi. The half-maximal optical response was reached at His = 20 mM (not shown). The apparent rate coefficient of the process k_+app = 0.021 s¹ (20 °C), k_+app = 0.077 s¹ (37 °C), was, however, practically independent on histidine concentrations at His = 5-100 mM. This means that the velocity of conversion CN·Cbi·CN  His·Cbi·CN is not limited by attachment of histidine to cobalt, although, the details of kinetics are not quite understood. Coordination of 15 mM histidine to Cbi (Fig. 4B) caused the same type of the spectral response as the binding TC + Cbi (Fig. 4A). Protection of the TC-associated Cbi was not as good as for Cbl·OH₂, and addition of 15 mM histidine caused further spectral transition with the velocity 14 times slower, than for free Cbi (not shown).

Binding Kinetics of Different Corrinoids-- The change in the absorbance of Cbls and Cbi upon their attachment to the proteins was used to monitor these processes on stopped-flow equipment. The data depicted in Fig. 5, A-C, represent the rapid phase of the binding. The reactions were fitted to a bimolecular mechanism A + B  C with the rate constants shown in Table II. Ligand binding to IF was characterized by the lowest rate constants. Association of Cbi (corrinoid with incomplete structure) with all proteins was almost as quick as for Cbl. No slow phase was found for IF and HC in the time region 1-100 s (not shown). The values of k_+CblCN from the literature: 236 µM¹ s¹ for chicken HC (3) and 10 µM¹ s¹ for immobilized bovine TC (4), are in good agreement with our results (Table II).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Binding of different corrinoids to the specific proteins. The ligand and the protein (20 °C, pH 7.5) were mixed in equimolar concentrations: 12 µM for panels A-C or 40 µM for panel D. Panel A, interaction of IF with the ligands. The reactions were followed spectroscopically at 356 nm for Cbl·OH2, 360 nm for Cbl·CN, 368 nm for Cbi, and 342 nm for Cbl·Ado. Panel B, interaction of HC with the ligands. The reactions were monitored at 345 nm for Cbl·Ado and 368 nm for Cbi (the optical response at this wavelength is not maximal). Panel C, interaction of TC with the ligands. The reactions were monitored at 361 nm for Cbl·Ado and 369 nm for Cbi. Panel D, slow phase of interaction between TC and Cbi. The reaction was performed at 3-fold increased concentrations and at another wavelength (580 nm) when compared with the analogous processes shown in panel C.

As was already mentioned, the initial attachment of Cbi to TC was followed by a slow monomolecular reaction C  D. The detected decrease of the -peak was difficult to follow at 12 µM TC due to low response on the background of a relatively high absorbance. Therefore, the time course of this second phase was recorded at increased concentrations of TC and Cbi (both 40 µM) and at another wavelength corresponding to -peak (Fig. 5D). The rate coefficients, determined from continuous measurements, were 5.6 × 10³ s¹ (20 °C) and 1.4 × 10² s¹ (37 °C). They did not differ from the data in Fig. 4A. Thorough investigation of Cbl·Ado binding to TC at different concentrations and wavelengths did not reveal any additional phase in this process besides the spectral changes during the first 10 ms induced by attachment of the ligand to the protein.

Dissociation of the Ligand-Protein Complexes-- High velocity of association between Cbi and the recombinant IF or TC raised a question about their affinity to this analogue, since Cbi is known to be a poor substrate for IF and TC from the natural sources (1, 2, 5, 16). We have, therefore, characterized dissociation of the protein·Cbl·OH₂ or protein·Cbi complexes by gradual replacement of the original ligand with added Cbl·CN or Cbl·OH₂, respectively (Fig. 6, A and B). The process was followed in time by the spectral changes of the protein fraction after charcoal treatment.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Liberation of Cbl·OH2 and Cbi from the protein-ligand complexes induced by the presence of another ligand. Panel A, displacement of Cbl·OH2 from its complex with the binders (20 µM) in the presence of Cbl·CN (100 µM), 20 °C, pH 7.5. Samples were taken at different time intervals and the unbound ligands were removed by adsorption on charcoal. Spectra of the protein-associated corrinoids were recorded and used to establish completeness of the reaction. The calculated rate constants of dissociation were: kCblOH = 4.2 × 106 s1 and kCblCN = 9.2 × 106 s1 for IF; kCblOH = 1 × 107 s1 for TC; kCblOH = 6 × 107 s1 for HC (see the main text for details). Panel B, displacement of Cbi from its complex with the binders (20 µM) in the presence of Cbl·OH2 (20 µM), 20 °C, pH 7.5. The rate constants of dissociation were estimated as kCbi > 5 × 102 s1 (IF and TC) and kCbi < 1 × 105 s1 (HC).

The data in Fig. 6A show the reaction, where a 4-fold excess of Cbl·CN was added to the holo-proteins saturated with Cbl·OH₂. Computer simulation of the curve obtained for IF allowed to calculate the dissociation rate constants both for Cbl·OH₂ (k_CblOH = 4.2 × 10⁶ s¹) and Cbl·CN (k_CblCN = 9.2 × 10⁶ s¹), using the known values of k_+Cbl from Table II. The values of k_CblOH for dissociation of the corresponding TC and HC complexes were estimated from the initial slopes (v = k_CblOH [complex]) as 1 × 10⁷ s¹ and 6 × 10⁷ s¹, respectively. Our previous measurements of k_CblCN for bovine and human TCs at higher temperature (37 °C) were in the range of 1 × 10⁶ to 3 × 10⁶ s¹ (4, 15).

When the apo forms of recombinant IF and TC were saturated with Cbi and exposed to equal concentration of external Cbl·OH₂, the complete substitution occurred in less than 1 min (Fig. 6B, upper curves). No detectable dissociation of HC·Cbi was found under the same conditions (Fig. 6B, lower curve). The rate constants of Cbi liberation were estimated as k_Cbi > 5 × 10² s¹ (IF, TC) and k_Cbi < 1 × 10⁵ s¹ (HC).

Exchange of the -Group in Cbl·OH₂ Associated with IF or HC-- It has already been shown that accessibility to the upper face of the ligand in the TC·Cbl·OH₂ complex is hindered (15). In this assay we exposed IF (Fig. 7, A and B) and HC (Fig. 7, C and D), saturated with Cbl·OH₂, to different concentrations of CN or N and then followed replacement of the original -group by changes in the absorbance. The observed reactions were practically irreversible in the case of CN and reversible for N. The calculated rate coefficients are shown in Table III, where the previous results for TC·Cbl·OH₂ and free Cbl·OH₂ (15) are given for comparison. As one can see, neither IF nor HC rendered significant protection against CN. At the same time, coordination of N to cobalt was somewhat decelerated in both directions when compared with free Cbl·OH₂.

View larger version (42K): [in this window] [in a new window]   Fig. 7.   Displacement of -coordinated water in IF·Cbl·OH2 or HC·Cbl·OH2 by the external reagents. The reaction was initiated by the exposure of the holo-protein (25 µM) to different concentrations of the substituting compound (37 °C, pH 7.5). The calculated values of the rate constants are presented in Table III. Panel A, reaction of IF·Cbl·OH2 with cyanide: 0.1, 0.25, 0.5, 1, 2.5, 5, 10 mM CN (curves 1-7, respectively). Panel B, reaction of IF·Cbl·OH2 with azide: 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5 mM N (curves 1-8, respectively). Panel C, reaction of HC·Cbl·OH2 with cyanide: 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 10 mM CN (curves 1-7, respectively). Panel D, reaction of HC·Cbl·OH2 with azide: 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5 mM N (curves 1-7, respectively).

View this table: [in this window] [in a new window]   Table III The rate constants of -exchange in Cbl·OH2 when free or bound to the specific proteins (37 °C)

Specific Proteins Protect Cbl·Ado against Light-induced Decomposition-- When Cbl binders saturated with Cbl·Ado were exposed to light, a gradual transformation of Cbl·Ado to Cbl·OH₂ was observed (Fig. 8). The time course of these photoactivated reactions was monitored spectroscopically and compared with decomposition of free Cbl·Ado under analogous conditions. The performed measurements showed a 7-, 15-, and 17-fold deceleration of Cbl·Ado decay when the ligand was bound to IF, HC, and TC, respectively.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Time course of the light-induced decomposition of Cbl·Ado. From left to right: 1, free ligand in solution, k = 0.54 min1; 2, IF·Cbl·Ado, k = 0.078 min1; 3, HC·Cbl·Ado, k = 0.036 min1; 4, TC·Cbl·Ado, k = 0.032 min1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Binding of the Cbl molecule to the specific proteins affects its absorbance spectrum, which turns spectroscopy to an easy and convenient method for monitoring the protein-ligand interactions. The advantages of the method were used for the investigation of Cbl binding to three transporting proteins: IF, TC, and HC. Two first binders were expressed in recombinant yeast, and HC was purified from human plasma. All proteins were isolated as holo forms with bound Cbl·OH₂ and their absorbance spectra (Fig. 2A, Table I) were typical for the binders from other sources. GdnHCl treatment, necessary for production of the apo-proteins, had practically no effect on the spectra of HC and TC. At the same time, the treatment influenced IF, and the increased absorbance of the apo-protein (Fig. 2, B and C) resulted in artificially high extinction coefficients of the newly bound ligand (Table I). The earlier determined extinction parameters of gastric IF (8) were, nonetheless, closer to the overrated absorbance of recombinant holo-IF after GdnHCl than to the coefficients of "fresh" recombinant holo-IF (Table I). This observation stresses importance of IF's history for its spectral features.

Comparison of the data in Fig. 2, A-C, with Fig. 2D showed that the most pronounced alterations in the ligand spectra took place after binding to TC (solid lines). Thus, the record for TC·Cbl·OH₂ at pH 7.5 (Fig. 2A) demonstrated a remarkable red shift of the -peak (362 nm) and strong expression of the -peak (546 nm). This pattern mimicked better Cbl·CN or Cbl·imidazol than Cbl·OH₂ at neutral pH (21). The spectrum of another complex TC·Cbl·Ado (Fig. 2B) was characterized by increased optical density at 350-380 and 400-450 nm as well as by decreased absorbance at 500-550 nm accompanied by separation of the individual peaks  and . Similar spectra were observed for enzyme-bound Cbl·Ado during catalysis (19, 20), which may suggest partial homolysis of the carbon-cobalt bond also in TC·Cbl·Ado, not trivial for a transporting protein like TC. The complex of TC with the third ligand Cbi (Fig. 2C) likewise revealed some redistribution of intensities between the peaks, i.e. decrease of the -peak (578 nm) and increase of the -peak (544 nm). Analogous spectra can be observed, for instance, for Cbl during transition (base on)·Cbl·CN  (base off)_CN·Cbl·CN, when cyanide and the nucleotide base compete for the lower coordination position at cobalt (Fig. 4C).

The peculiar spectra of the above protein-ligand complexes prompted us to thorough investigation of the binding kinetics. Change in the absorbance of the -peak during the ligand binding was followed by a stopped-flow technique (Fig. 5, A-C). All ligands, including the analogue with the missing nucleotide moiety, attached rapidly and in one step to two proteins with widely different Cbl specificity, IF and HC (Fig. 5, A and B). There was no visible indication of any second phase during 100 s of the binding as well, unlike the interaction between TC and Cbl·OH₂ examined earlier (15). This fact implies that the slow phase is not an attribute of the selective recognition of Cbl but rather a specific characteristic of TC, when interacting with certain ligands. We doubt that presence of carbohydrates on IF and HC (1, 6) and their absence on TC (1, 6, 15) has anything to do with the described effects, because glycosylation does not seem to interfere with the binding of Cbl to IF (22).

There was an interesting observation, concerning the high velocity of association between Cbi and IF or TC. The incomplete ligand bound to these two proteins, known to be Cbl selective (1, 2, 4, 5), almost as quick as the ligand with the correct structure, i.e. Cbl. We compared the association rate constants from Table II with the collision rate constant k_coll = 5 × 10⁹ M¹ s¹ at 20 °C estimated for a corrinoid and a binding site of appropriate geometry (23). The calculations showed that the number of the efficient impacts varied from 2 to 20 per 1000 collisions without particular correlation between k_+Cbl and the ligand structure. Similar rate constants found for Cbi and Cbl mean that the Cbl-specific site is not originally tuned to any particular ligand and can accommodate for a time being even some defective molecule. The sensitivity of IF and TC to the substrate's geometry, and its absence in the case of HC, was revealed only in the dissociation experiments (Fig. 6, A and B).

Calculated values of the rate constants allowed us to make the following estimates of K_d for Cbl·OH₂: 1 pM (IF), 0.01 pM (HC), and 0.005 pM (TC). The values for Cbi were: K_d < 0.1 pM (HC), K_d > 1 nM (TC and IF). The earlier published K_d for Cbl and the specific binders varied in the range 10¹⁶-10⁹ M (see, for review, Refs. 1-4, 8, 9, 15, and 22), which could hardly be explained by real fluctuations of the affinity. Such a broad dispersion was rather caused by inappropriate mathematic approach to the case when the total concentrations of the binding site E_T and the ligand L_T are close to each other (complicated by K_d E_T, L_T). Under these conditions, half-saturation would be reached at L_T(0.5) = K_d + 0.5 E_T, which may represent rather concentration of the binding site than the dissociation constant. More accurate presentation of the results as EL versus L_free may also lead to an erroneous evaluation of K_d if the reaction is almost irreversible. Under these circumstances even a small but reproducible overestimate of L_free (e.g. L_app = L_free + 0.05 L_T) inevitably causes great overestimate of K_d (e.g. half-saturation at L_app(0.5) = 1.05 K_d + 0.025 E_T  0.025 E_T). In such a difficult case, determination of K_d from the ratio of the rate constants k_L/k_+L may be advantageous. This statement can be illustrated by comparison of K_d measured for chicken HC in an equilibrium assay (10¹³ M) and from k_L/k_+L (10¹⁶ M) by the same authors (3).

Investigation of the rapid kinetics contributed to our understanding of the substrate binding, although, it did not give us a clue to the anomalous appearance of the holo-TC spectra. Therefore, the stopped-flow experiments were repeated at different wavelength and higher concentration of TC. They did not exhibit any second phase for the reaction TC + Cbl·Ado but revealed it for TC + Cbi (Fig. 5D). This result demonstrated that Cbl·OH₂ was not the only corrinoid characterized by biphasic binding to TC (15). Two atypical ligands (Cbl·OH₂ and Cbi) were subjected to thorough analysis. We have recorded deformations of the -peak for Cbl·OH₂ (Fig. 3A) and ,-peaks for Cbi (Fig. 4A) at 20 and 37 °C in an attempt to get the best response for each corrinoid. In both cases the initial attachment of the ligands caused slight increase and red shift of the peaks without significant distortions of their shape (see the 1-s records in Figs. 3A and 4A). Continuation of the reactions was, although, accompanied by more pronounced changes in the spectra, similar to those observed during exchange of the cobalt-coordinated groups in Cbl·OH₂ and Cbi. This observation raised, in its turn, a question about the nature of cobalt-coordinated groups in the ligands associated with the transporting proteins.

When Cbl binds to a transporter, its lower part becomes buried inside the protein molecule, whereas the upper part is thought to be open in all carriers under study (7, 8, 24). At the same time, our analysis of the -group exchange in free and bound Cbl·OH₂ confirmed this statement only for IF and HC. The mechanism of the exchange reaction for these two binders was generally the same as for Cbl·OH₂ in solution except for somewhat reduced reaction velocities (Fig. 7, Table III). Protection of the upper surface of Cbl·OH₂ in holo-TC was much more evident. For instance, coordination of 1 mM CN or N to holo-TC (15) was 2 orders of magnitude slower than the same reactions with holo-IF/-HC (Fig. 7). The accessibility of Cbl's upper plane was suggested to be hindered due to coordination of a protein residue to cobalt at upper axial position (15). Several amino acids have been tested on Cbl·OH₂ for their ability to cobalt coordination (Fig. 3D), but only histidine appeared to be the sufficiently potent compound. This result was quite anticipated, because adsorption of Cbl·OH₂ on the proteins with His residues during extraction of Cbl from biological sources is a well known phenomenon (25). We made an attempt to imitate the slow phase by adding histidine to holo-IF and -HC, i.e. the proteins with the open upper surface of Cblc·OH₂. The following spectral changes reflected transition to IF·/HC·Cbl·His (Fig. 2, B and C) and bore an essential resemblance to the second phase of Cbl·OH₂ binding to TC (Fig. 2A).

The interpretation of the spectral changes during association of Cbi and TC seems to be more complex. It is clear that displacement of cyanide from either lower or upper position by a histidine residue of TC is quite feasible (compare Fig. 4, A and B). We have also suggested that histidine coordinates to the -site of Cbi (Fig. 4, A and B) because of resemblance with the reaction (base on)·Cbl·CN  (base off)_CN·Cbl·CN (Fig. 4C). Some aspects remain, however, unclear. Thus, the observed substitution of (?)-cyanide in TC·Cbi was incomplete as followed from comparison with the Cbi spectrum at saturating histidine (Fig. 4B). This result did not match the accomplished replacement of -water in the slow phase of the binding reaction TC + Cbl·OH₂ (15). The activation energies of the second phases for TC + Cbi (43 kJ/mol) and TC + Cbl·OH₂ (120 kJ/mol) also differed quite significantly. In other words, there may be different histidine residues involved in -substitution on Cbl·OH₂ and (?)-substitution on Cbi. Still, we cannot exclude that a disc-shaped Cbi molecule binds to TC upside down with the -site exposed to the same His-containing domain as the -site of Cbl·OH₂.

Another still unraveled issue is the binding of Cbl·Ado to TC. The spectrum of the produced complex TC·Cbl·Ado was stable in time and the associated ligand was well protected against light (Fig. 8). At the same time, appearance of the ,-peaks (Fig. 2B) mimicked Cbl·Ado-dependent enzymes under catalysis (19, 20) when ~20% of Cbl molecules contain detached Ado· radical involved in the substrate transformation. Nonetheless, we did not find any additional phase in the binding reaction, TC + Cbl·Ado, which could be potentially ascribed to homolytic cleavage of the carbon-cobalt bond. The apparent absence of the second phase might be caused by high velocity of cleavage estimated, for instance, in the case of methylmalonyl-CoA mutase as >600 s¹ (20). The ability of TC to induce formation of the Ado· radical may be not as absurd as it seems to be at first sight. Thus, alignment of the pairs (IF, TC, or HC):(methylmalonyl-CoA mutase or glutamate mutase) showed 15-19% of homology in all cases, although, at different regions. Anyway, the unusual spectral properties of the TC·Cbl·Ado complex require additional analysis.

The presented data throw some light upon the structure of the binding sites of Cbl transporting proteins. One can imagine that all three carriers are supplemented with a lid- or a cap-like structure at the entrance to the site cavity. At the same time, development of this structure in the Cbl transporters appears to be different. The cap in IF does not seem to cover any appreciable part of the upper face of Cbl and, therefore, holo-IF demonstrates quite rapid exchange of the -groups as well as a relatively moderate protection of Cbl·Ado. The kindred protein HC is rigged somewhat better. Its cap shields to a certain extent the upper plane of Cbl and hinders the inwards-outwards movements of the -coordinated groups, at least for bulky complexons. Analogous cap in TC renders much better protection against all substituents, and it might even produce and stabilize the Ado· radical above the upper plain of Cbl. In addition, the protective shield of TC is, presumably, supplemented by an active His residue, which can coordinate to cobalt and dislodge -water in Cbl·OH₂ or (?)-cyanide in Cbi (the latter case requires, although, additional clarification). Coordination to cobalt at the -position locks the lid above the binding site and Cbl (but not Cbi) becomes encapsulated inside holo-TC, with only occasional and short-time openings to occur.

The performed investigation strengthens the view on TC as the best protector of the associated Cbl. It also raises a question about the role of the protective cap in stabilization and destabilization of the cobalt-coordinated groups in TC-bound corrinoids.

	FOOTNOTES

^* 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. Tel.: 45-89-42-50-92; Fax: 45-86-13-65-97; E-mail: snf@imsb.au.dk.

Published, JBC Papers in Press, January 11, 2002, DOI 10.1074/jbc.M111399200

² S. N. Fedosov, unpublished data.

	ABBREVIATIONS

The abbreviations used are: IF, intrinsic factor; Cbl, cobalamin; Cbl·OH₂/·CN/·N₃/·Ado, aquo/cyano/azido/5'-deoxyadenosyl cobalamin; GdnHCl, guanidine hydrochloride; HC, haptocorrin; TC, transcobalamin; PAS, periodic acid-Shiff reagent.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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