Probing the H-protein-induced Conformational Change and the Function of the N-terminal Region of Escherichia coli T-protein of the Glycine Cleavage System by Limited Proteolysis*

Kazuko Okamura-IkedaDagger, Naomi Kameoka, Kazuko Fujiwara, and Yutaro Motokawa

From the Institute for Enzyme Research, the University of Tokushima, Tokushima 770-8503, Japan

Received for publication, October 23, 2002, and in revised form, December 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

T-protein, a component of the glycine cleavage system, catalyzes a tetrahydrofolate-dependent reaction. Previously, we reported a conformational change of Escherichia coli T-protein upon interacting with E. coli H-protein (EH), showing an important role for the N-terminal region of the T-protein in the interaction. To further investigate the T-protein catalysis, the wild type (ET) and mutants were subjected to limited proteolysis. ET was favorably cleaved at Lys81, Lys154, Lys288, and Lys360 by lysylendopeptidase and the cleavages at Lys81 and Lys288 were strongly prevented by EH. Although ET was highly resistant to trypsinolysis, the mutant with an N-terminal 7-residue deletion (ETDelta 7) was quite susceptible and instantly cleaved at Arg16 accompanied by the rapid degradation of the resulting C-terminal fragment, indicating that the cleavage at Arg16 is the trigger for the C-terminal fragmentation. EH showed no protection from the N-terminal cleavage, although substantial protection from the C-terminal fragmentation was observed. The replacement of Leu6 of ET with alanine resulted in a similar sensitivity to trypsin as ETDelta 7. These results suggest that the N-terminal region of ET functions as a molecular "hasp" to hold ET in the compact form required for the proper association with EH. Leu6 seems to play a central role in the hasp function. Interestingly, Lys360 of ET was susceptible to proteolysis even after the stabilization of the entire molecule of ET by EH, indicating its location at the surface of the ET-EH complex. Together with the buried position of Lys81 in the complex and previous results on folate binding sites, these results suggest the formation of a folate-binding cavity via the interaction of ET with EH. The polyglutamyl tail of the folate substrate may be inserted into the bosom of the cavity leaving the pteridine ring near the entrance of the cavity in the context of the catalytic reaction.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The glycine cleavage system is a multienzyme complex composed of four proteins, P-, H-, T-, and L-protein, which catalyzes the reversible oxidation of glycine yielding carbon dioxide, ammonia, 5,10-CH2-H4folate,1 and a reduced pyridine nucleotide. T-protein is a folate-dependent enzyme that catalyzes the release of ammonia and the transfer of the methylene carbon unit to H4folate from the intermediate attached to H-protein after the decarboxylation of glycine catalyzed by P-protein (reviewed in Ref. 1). The kinetic properties of the forward (2) and the reverse (3) reaction catalyzed by T-protein have been studied extensively, and the primary structures of T-proteins from eight species have been determined (4-10) as well as many putative T-protein sequences derived from genome analyses.

We found previously that lysine residues of Escherichia coli T-protein (ET) at positions 78, 81, and 352 are involved in the interaction with the polyglutamate moiety of 5,10-CH2-H4PteGlu4 (11). From the results of a kinetic analysis with single and multiple substitution mutants for these lysine residues, it was postulated that Lys352 interacts with the alpha -carboxyl group of the first glutamate residue nearest the p-aminobenzoic acid ring of 5,10-CH2-H4PteGlu4 and that Lys81 interacts with the alpha -carboxyl group of the second glutamate residue. Lys78 seems to participate in the binding of the peripheral glutamate residues. Lys352 is conserved among the T-proteins studied thus far, whereas Lys81 and Lys78 are not. Therefore, other lysine residues must be responsible for the binding of the second and third glutamate residue of 5,10-CH2-H4PteGlu4 in T-proteins from other species. Furthermore, a cross-linking study (12) revealed that the interaction of E. coli H-protein (EH) with ET causes a conformational change of ET and results in intramolecular cross-linking between Asp34 and Lys216 of ET. Intermolecular cross-linking between Lys288 of ET and Asp43 of EH was also identified, indicating the participation of the region in the interaction between ET and EH. The extreme N-terminal region of ET is essential for the interaction with EH because such intramolecular and intermolecular cross-linking was not observed in a complex composed of the N-terminal 7-residue deletion mutant of ET (ETDelta 7) and EH. The marked decrease in the kcat/Km value of ETDelta 7 for EH compared with the wild type enzyme exhibited a good correlation with the cross-linking results.

In the present study, we used limited proteolysis to further examine the function of the N-terminal region as well as the mobile nature of the T-protein catalysis. Two amino acid residues, Thr4 and Leu6, that are conserved in all eight T-proteins were mutated separately to alanine. The resulting mutants (ETT4A and ETL6A), ET, and ETDelta 7 were overexpressed as His6-tagged proteins at the C-terminal end, purified, and subjected to proteolysis. The profile of protease digestion of these proteins in the absence or presence of substrates suggested that the functional lysine residues of ET identified previously are protease-accessible and that some of them are buried in the ET-EH complex. Moreover, it is revealed that the N-terminal region serves as a molecular "hasp" to hold the ET molecule in the proper folding critical for association with EH. Leu6 plays a pivotal role in the hasp function. The formation of the folate binding cavity by the interaction of ET with EH was also probed by limited proteolysis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [14C]Sodium bicarbonate was obtained from Amersham Biosciences. Restriction endonucleases and other DNA modifying enzymes were purchased from New England Biolabs, Roche Molecular Biochemicals, Toyobo (Tokyo, Japan), or Takara Shuzo (Shiga, Japan). Oligonucleotides were from Hokkaido System Science (Sapporo, Japan). L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated bovine pancreatic trypsin (TPCK-trypsin) was purchased from Worthington, and soybean trypsin inhibitor was from Sigma. Lysylendopeptidase (Lys-C) and folic acid were obtained from Wako Pure Chemicals (Osaka, Japan). Pteroyltetraglutamate from Schircks Laboratory (Jona, Switzerland) and folic acid were reduced by the method of Kisliuk (13) and used for the synthesis of 5,10-CH2-H4PteGlu4 and 5,10-CH2-H4folate, respectively, as described (14). Recombinant E. coli P-protein and H-protein were prepared as described previously (11).

Expression and Purification of Wild Type and Mutant E. coli T-protein with a His6 Tag at the C-terminal End-- DNA manipulations were accomplished using standard techniques (15). To construct plasmids for the expression of ET and ETDelta 7 with a His6 tag at the C-terminal end, pET3a with an insertion of ET DNA (pET/ET) (11) or ETDelta 7 DNA (12) was cut with EcoRI and employed in a polymerase chain reaction with oligonucleotides 5'-TAATACGACTCACTATAGGG-3' (T7 promoter primer) as 5'-primer and 5'-AAAAGTCTCGAGCGCGACGGCTTTGCCG-3' (the XhoI site is underlined) as 3'-primer. The products were purified with a QIAEX II kit (Qiagen) after agarose gel electrophoresis, digested with NdeI and XhoI, and ligated into pET23b. The nucleotide sequences of the resultant plasmids, pET23b/ET and pET23b/ETDelta 7, were confirmed with a 373 DNA sequencing system (Applied Biosystems). For the introduction of two mutations, ETT4A and ETL6A, polymerase chain reaction was carried out using EcoRI-digested pET/ET as a template with the respective 5'-primers, 5'-AGATATACATATGGCACAACAGGCTCCTTTG-3' and 5'- AGATATACATATGGCACAACAGACTCCTGCGTACGAAC-3' (modified bases are shown in boldface letters), and the common 3'-primer, 5'-AAAAGTCTCGAGCGCGACGGCTTTGCCG-3' (the XhoI site is underlined). The products were subcloned into pET23b as described above, and nucleotide sequences of the resulting plasmids, pET23b/ETT4A and pET23b/ETL6A, were verified by DNA sequencing.

E. coli BL21(DE3)pLysS cells (16) transformed with the constructed expression plasmids were grown in 100 ml of LB medium containing 20 µg/ml ampicillin and 25 µg/ml chloramphenicol at 30 °C for 24 h. Expression was induced by 25 µM isopropyl-beta -D-thiogalactopyranoside added at the start of the incubation. Cell-free extracts were prepared as described previously (7) with buffer A (20 mM potassium phosphate, pH 7.4, 0.3 M NaCl, and 0.2 mM dithiothreitol) containing 20 mM imidazole and subjected to affinity chromatography in a Ni2+ column (1.5 × 2.2 cm, Qiagen). The samples were eluted with buffer A containing 50 mM imidazole, concentrated with a Centriplus-30 (Millipore), and subjected to size exclusion chromatography on a Superdex 200 pg 16/60 column equilibrated with 50 mM potassium phosphate, pH 7.0, containing 0.2 M NaCl and 1 mM dithiothreitol. The final preparations were dialyzed against 20 mM potassium phosphate, pH 7.0, containing 1 mM dithiothreitol, concentrated with a Centricon-10 (Millipore), and stored at -80 °C.

Protease Digestion and Analysis of the Fragments-- ETs (1.8 mg/ml) were digested in 50 mM HEPES, pH 7.0, at 25 °C with TPCK-trypsin at an ETs/trypsin (w/w) ratio of 100:1. Aliquots containing 18 µg of ETs were removed at various times, and the digestion was terminated with a 2-fold excess (w/w) of soybean trypsin inhibitor. When the trypsinolysis was carried out in the presence of substrates, ETs were preincubated with a 2-fold molar excess of EH or with 0.2 mM 5,10-CH2-H4PteGlu4 at 25 °C for 30 min in 50 mM HEPES, pH 7.0. Then TPCK-trypsin was added at a ratio of 10:1 (w/w) and digested as above. In all cases, an equal aliquot was withdrawn just before the addition of TPCK-trypsin and used as a zero time control. Samples were subjected to an assay of T-protein activity (12) and SDS-PAGE according to the method of Schägger and von Jagow (17). Digestion by Lys-C was also carried out as above in 20 mM Tris-HCl, pH 8.0, at an ET/Lys-C ratio (w/w) of 50:1. In this case, the reaction was terminated by the addition of an equal volume of 2× SDS-PAGE sample buffer to aliquots and immediate treatment in boiling water.

The proteolytic fragments were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Immobilon-PSQ, Millipore), and subjected to Edman sequencing on a Hewlett Packard G100A protein sequencer. When necessary, the digestion products were separated by reversed-phase high performance liquid chromatography as described previously (11), and appropriate peak fractions were subjected to N-terminal sequencing.

Assay of T-protein Activity and Kinetic Analysis-- The routine assay and kinetic analysis of T-protein were conducted as described previously (12). For the kinetic analysis of ET, ETT4A, and ETL6A, concentrations of varying substrates were: reduced EH, 0.4-3.6 µM; L-(±)-5,10-CH2-H4folate, 40-360 µM; L-(±)-5,10-CH2-H4PteGlu4, 4-36 µM; NH4Cl, 20-180 mM; and the concentrations of constant substrates were: reduced EH, 6 µM; L-(±)-5,10-CH2-H4folate, 0.6 mM; NH4Cl, 200 mM. In the experiments employing ETDelta 7, concentrations of varying substrates were: reduced EH, 2.67-24 µM; L-(±)-5,10-CH2-H4folate, 40-360 µM; L-(±)-5,10-CH2-H4PteGlu4, 20-180 µM; NH4Cl, 30-300 mM; and the concentrations of constant substrates were: reduced EH, 36 µM; L-(±)-5,10-CH2-H4folate, 0.6 mM; NH4Cl, 300 mM. The experiments determining the Km values for reduced EH of ET, ETT4A, and ETL6A were conducted in 0.5 ml of reaction mixture. Appropriate amounts of ETs within the following ranges were used: ET, 10-20 ng; ETDelta 7, 80-250 ng; ETT4A, 20-40 ng; ETL6A, 20 ng.

Mass Spectrometry-- The tryptic fragments were analyzed by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. ET and ET·His6 (each 360 µg) digested with TPCK-trypsin (36 µg) in the presence of a 2 molar excess of EH at 25 °C for 90 min were loaded onto a Superdex 200 HR 10/30 column (Amersham Biosciences) equilibrated with 50 mM Tris-HCl, pH 8.0, 0.2 M NaCl, and 1 mM dithiothreitol. Two main peaks corresponding to molecular masses of 54 and 26 kDa were obtained. The former peak, containing a 39-kDa band verified by SDS-PAGE, was concentrated using Microcon-3 (Millipore), and the solvent was replaced with 50% acetonitrile containing 0.1% trifluoroacetic acid by repeated concentration. The sample solution was mixed with a saturated solution of sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) in 30% acetonitrile and 0.1% trifluoroacetic acid. The spectra of positive ions were recorded in the linear mode on a Voyager-DE STR mass spectrometer (Perseptive Biosystems) equipped with a delayed extraction device. External calibration was performed with Sequazyme Peptide Mass Standard Kit Mix 3 (Perseptive Biosystems) containing insulin (bovine), thioredoxin (E. coli), and apomyoglobin (horse) with a mass (M + H+1) of 5734.59, 11,674.48, and 16,952.56 Da, respectively.

Other Methods-- Protein concentrations were routinely determined by the method of Bradford (18) with bovine serum albumin as a standard. Concentrations of purified wild type and mutant ETs with a His6 tag at the C terminus and EH were estimated as described previously (11).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of Wild Type and Mutant E. coli T-protein with a His6 Tag at the C-terminal End-- To facilitate purification and to obtain mutant ETs without contamination by wild type ET originating from the host cell gene, we introduced a His6 tag at the C-terminal end of the ETs with two extra amino acids (-LEHHHHHH). Four proteins, termed ET·His6, ETDelta 7·His6, ETT4A·His6, and ETL6A·His6, were highly expressed in E. coli as soluble forms and purified by Ni2+ affinity column chromatography and subsequent gel filtration. The final preparation of each protein gave a single band on SDS-PAGE (not shown). Purified ET·His6 showed comparable specific activity (16,885 units/mg) to ET (15,127 units/mg) (12), indicating no significant effect of the C-terminal extension on the activity. The specific activity of purified ETDelta 7·His6 (906 units/mg) was about 5% relative to ET·His6, again giving a result comparable to ETDelta 7 (764 units/mg) (12). T4A and L6A mutations led to a reduction in the specific activity to 43% (7221 units/mg) and 79% (13,364 units/mg), respectively, relative to wtET·His6.

Kinetic Analysis-- Steady state kinetic studies were carried out by varying the concentration of one substrate and keeping the concentrations of the other two substrates constant (Table I). ET·His6 and ETDelta 7·His6 gave parameters comparable to those of ET and ETDelta 7, respectively (12), showing a notable decrease (165-fold) in the kcat/Km value of ETDelta 7·His6 for reduced EH. ETT4A·His6 and ETL6A·His6 also exhibited a significant decrease (4- and 5-fold, respectively) in the kcat/Km value for reduced EH compared with ET·His6. These results suggest that both residues play an important role in the function of the N-terminal region of ET.


                              
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Table I
Kinetic constants of wild type and mutant E. coli T-protein

Protease Digestion-- To investigate the effect of the N-terminal mutation, the purified proteins were subjected to limited proteolysis by TPCK-trypsin (an ETs/trypsin ratio of 100:1), and the resulting peptides were analyzed by SDS-PAGE, Edman sequencing, assay of the activity, and/or mass spectrometry. ET·His6 was resistant to trypsin digestion as revealed by the presence of about half of the intact ET band (Fig. 1A, lane 7) and the retention of about half of the original activity (Fig. 3A) even after a 4-h digestion. However, ETDelta 7·His6 was quite susceptible to trypsinolysis and converted to a 37-kDa fragment (F1) within the first 5 min of digestion (Fig. 1B, lane 2). F1 gave the N-terminal sequence starting from Met17 (Table II) and retained the His6 tag at the C-terminal end as probed by the antibody against the His6 tag sequence in Western blotting (not shown). Loss of the N-terminal residues up to Arg16 resulted in a complete loss of activity (Fig. 3A). F1 was rapidly degraded to small pieces through intermediary fragments of 20, 14, and 7 kDa (named F2, F3, and F4, respectively) (Fig. 1B, lanes 2-5). Consequently, the cleavage at Arg16 seems to trigger the subsequent C-terminal fragmentation. The intermediate peptides were subjected to Edman sequencing, and the five N-terminal residues were determined as Met17-His21, Ala82-Gly86, and Met17-His21 for F2, F3, and F4, respectively (Table II), predicting a split of F2 to F4 and F3. F2 and F3 probably have a common C-terminal residue, which can be assumed as Lys203 or Arg208 judging from the molecular mass on SDS-PAGE. As several peptide bands found between F1 and F2 showed the same N-terminal sequence starting from Met17, it is conceivable that random cleavage occurs at several positions in the C-terminal one-third of ETDelta 7·His6 after the removal of 16 residues from the N-terminal. ETL6A·His6 behaved similar to ETDelta 7·His6 (Fig. 1D) in trypsinolysis, indicating a central role for the leucine residue in the function of the N-terminal region. Arg16 of ETT4A·His6 was somewhat resistant to trypsinolysis (Fig. 1C).


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Fig. 1.   Time course of tryptic digestion of E. coli T-proteins. E. coli T-proteins were digested with trypsin at an ET/trypsin (w/w) ratio of 100:1 as described under "Experimental Procedures." Digests containing 3.3 µg of ET were loaded for SDS-PAGE (16.5% T and 3% C acrylamide gels) (17) and stained with Coomassie Blue. A, ET·His6; B, ETDelta 7·His6; C, ETT4A·His6; D, ETL6A·His6. The digestion times in minutes are indicated below the lanes, and the standard protein markers (lanes M1 and M2) are on the left. The fragments derived from ETDelta 7·His6 are termed as indicated on the right.


                              
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Table II
Proteorytic fragments of E. coli T-protein

The effects of substrate binding on susceptibility to trypsinolysis were examined in the presence of EH (a 2-fold molar excess over ETs) or 5,10-CH2-H4PteGlu4. In these experiments, TPCK-trypsin was employed at an ETs/trypsin (w/w) ratio of 10:1 to ensure the thorough digestion of ET·His6. As shown in Fig. 2, both substrates exhibited negligible protection against the cleavage at Arg16 of the mutants except for the partial protection of ETT4A·His6 by EH. However, both substrates substantially prevented the subsequent C-terminal fragmentation, although to somewhat different extents. Digestion of ET·His6 by large amounts of trypsin proceeded gradually without a remarkable accumulation of the intermediate fragments except for the small amount of 30-kDa fragment, which gave the N-terminal sequence starting from Ala1. However, digestion of ET·His6 in the presence of EH resulted in the accumulation of a 39-kDa fragment (F5) (Fig. 2B, lane 12). The fragment gave an intact N-terminal amino acid sequence starting from Ala1 but did not cross-react with the antibody against His6 tag (data not shown). Furthermore, gel-purified F5 gave a [M + H+1] value of 39,767.52 Da, in agreement with the calculated value for the peptide Ala1-Lys360 (39,773.13 Da). The equivalent peptide obtained from the parallel experiment with ET also gave an intact N-terminal sequence and a [M + H+1] value of 39,771.85 Da, although it is impossible to distinguish the mobility of the peptide band from that of intact ET on SDS-PAGE (Fig. 2A, lane 12). In contrast to the cleavage at Arg16, the cleavage at Lys360 affected the activity only slightly (Fig. 3B). To further characterize the cleavage at Lys360, the tryptic digests of ET·His6 in the absence or presence of EH were subjected to reversed-phase HPLC. The peptide from Ala361 to the end of the His6 tag (AVALEHHHHHH) was eluted at 8% acetonitrile as an isolated peak with intensive absorbance at 220 nm. The intensity was compared with that of the peptide Met17-Arg40, a peptide reflecting the fragmentation of the entire ET molecule into small pieces. The results demonstrated that the release of the C-terminal peptide was significantly stimulated in the presence of EH despite the marked decrease of that of the peptide Met17-Arg40 (Fig. 4). 5,10-CH2-H4PteGlu4 also stimulated the cleavage at Lys360 while suppressing the fragmentation of the entire ET·His6, and EH potentiated the event (Fig. 4).


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Fig. 2.   The effects of substrates on the trypsinolysis of E. coli T-proteins. E. coli T-proteins were digested with TPCK-trypsin at an ETs/trypsin (w/w) ratio of 10:1 in the absence (lanes 1-6) or presence (lanes 7-12) of a 2-fold molar excess of EH or 0.2 mM 5,10-CH2-H4PteGlu4 (lanes 13-18) as described under "Experimental Procedures." The digests were analyzed as described in the legend for Fig. 1. A, ET; B, ET·His6; C, ETDelta 7·His6; D, ETT4A·His6; E, ETL4A·His6. The digestion times in minutes are indicated below the lanes, and each intact protein and the fragments derived from ET and ET·His6 are indicated.


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Fig. 3.   T-protein activity during the tryptic digestion. A, T-protein activity in the tryptic digests analyzed in Fig. 1 was assayed as described under "Experimental Procedures." B, ET·His6 digested with TPCK-trypsin at an ET/trypsin (w/w) ratio of 10:1 in the absence or presence of a 2-fold excess of EH was assayed.


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Fig. 4.   Detection of the cleavage at Lys360 during trypsin digestion of ET·His6. ET·His6 digests treated with TPCK-trypsin in the absence or presence of EH and CH2-H4PteGlu4 as described in Fig. 2 were subjected to reversed-phase HPLC. The intensities of the peaks containing the C-terminal fragment (Ala361-His6) or the fragment Met17-Arg40 were compared after the identification by Edman sequencing.

In contrast to trypsin, Lys-C easily digested ET·His6 at an ET·His6/Lys-C (w/w) ratio of 50:1, in which several fragments accumulated as shown in Fig. 5. Edman sequencing of up to 5 residues of these fragments was carried out, and the C-terminal ends were predicted from the molecular masses determined by SDS-PAGE (Table II). The results revealed that at least four lysine residues at positions of 81, 154, 288, and 360 are favorable cleavage sites. Cleavage at Lys81 and Lys288 was strongly prohibited in the presence of EH, although that at Lys360 was again stimulated by the interaction with EH. About half of the EH that was added, i.e. ET-bound EH, remained intact in contrast with the immediate degradation of free EH.


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Fig. 5.   Time course of digestion of E. coli T-proteins by lysylendopeptidase. ET·His6 was digested with Lys-C at an ET/Lys-C (w/w) ratio of 50:1 in the absence (lanes 1-6) or presence (lanes 7-12) of a 2-fold molar excess of EH as described under "Experimental Procedures." The digestion times in minutes are indicated below the lanes, and the standard protein markers (lanes M1 and M2) are indicated on the left. The fragments derived from ET·His6 (termed F5-F12) and EH (termed EH2 and EH3) are indicated on the right.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we used limited proteolysis and mutation analysis to further elucidate the structural nature of ET in the catalytic reaction as well as the function of its N-terminal region in the interaction with EH. The mutant proteins were constructed by replacing two invariant residues among the 7 N-terminal residues, Thr4 and Leu6, with alanine as well as the deletion of up to 7 residues. The introduction of a His6 tag to the C-terminal end not only facilitated the purification of proteins having activity comparable with that of proteins without His6 but also provided a convenient tool for detecting local conformational change at the C terminus, as mentioned below. A single mutation, T4A or L6A, resulted in a 4- or 5-fold decrease in the kcat/Km value for reduced EH, respectively, compared with ET, indicating the importance of these residues in the T-protein activity.

The purified wild type and mutant proteins were subjected to limited proteolysis. The difference in the profile of digestion of ET·His6 and ETDelta 7·His6 by a small amount of TPCK-trypsin demonstrated that Arg16 in ETDelta 7·His6 is oriented toward a trypsin-accessible location. After cleavage at Arg16 and loss of 16 N-terminal residues, the remaining C-terminal polypeptide chain would be unfolded and attacked by trypsin. Because EH and folate substrate substantially prevented the latter event, it is conceivable that they facilitate the intramolecular interaction between the relatively stable N-terminal two-thirds and the extended C-terminal one-third of ET. These findings are consistent with our previous suggestion that Lys352 serves as the binding site for the first glutamate residue nearest the p-aminobenzoic acid ring of 5,10-CH2-H4PteGlu4 and that Lys81 and Lys78 participate in the binding with the second and third glutamate residue, respectively (11). Consequently, the N-terminal region might serve as the molecular hasp locking its own molecule in a compact fold. The point mutations, L6A and T4A, also resulted in an extended N terminus, probably because of the loss of interaction between these residues and some others. The contribution of Leu6 is likely to be essential to the event. Furthermore, the marked reduction (165-fold) in the kcat/Km value of ETDelta 7·His6 for reduced EH and the absence of cross-linking between Lys288 of ETDelta 7 and Asp34 of EH (12) may reflect the disordered conformation in the stabilized C-terminal polypeptide. In this connection, it is worth noting that two point mutations in the N-terminal region of human T-protein, H42R (19) and G47R (20), cause the clinical disorder nonketotic hyperglycinemia. His42 and Gly47 of the human precursor T-protein correspond to His10 and Ala15, respectively, of ET. His42 is conserved in all eight T-proteins reported thus far, and Gly47 is present in all but E. coli T-protein (11).

Extensive sequencing of the Lys-C digests of ET·His6 demonstrated 4 sensitive lysine residues, Lys81, Lys154, Lys288, and Lys360. It is well known that residues sensitive to proteolytic cleavage are localized to regions that are solvent-accessible, unstructured, or flexible. The results are quite reasonable because functional lysine residues such as Lys81 and Lys288 are situated in a solvent-accessible location. Lys288 might be masked by the interaction with Asp34 of EH. The significant protection by EH of the cleavage at Lys81 indicates a buried position in the ET-EH complex of the binding site for the second glutamate residue of folate polyglutamate. In contrast, the promoted cleavage at Lys360 in the stabilized ET-EH complex suggests that the region containing Lys360 and probably Lys352 is located on the surface of the complex. These results lead us to speculate that the folate binding cavity is formed by the interaction of ET with EH, in which the binding site for the second glutamate residue of folate substrate is localized deeper in the cavity than that for the first glutamate residue. Consequently, the polyglutamate tail of the folate substrate may be inserted into the bosom of the cavity leaving the pteridine ring near the entrance of the cavity. This configuration is compatible with the previously revealed Ordered Ter Bi mechanism of the reverse reaction catalyzed by T-protein, in which reduced EH is the first substrate that binds ET followed by folate substrate and ammonia (3). In the study with ET, we could not distinguish the polypeptide Ala1-Lys360 from the intact polypeptide on SDS-PAGE nor detect the small peptide that was released, Ala361-Val-Ala363. However, detection of polypeptide Ala1-Lys360 in the tryptic digest of ET in the presence of EH ruled out the possibility that the positioning of Lys360 near the surface of the ET-EH complex may be due to the addition of a His6 tag at the C terminus.

The orientation of folate in the cavity is compatible with the conformation of the lipoyl moiety of H-protein reported by Cohen-Addad et al. (21). The lipoate arms of pea H-protein in oxidized or reduced forms, i.e. carrying no methylamine, are located at the surface of the H-protein in a rather flexible conformation, and following the transfer of methylamine, the cofactor pivots around the lysine linkage and is locked into the binding cleft of the H-protein. Therefore, the presence of the pteridine ring harboring a C1 unit near the surface of the ET-EH complex might facilitate the transfer of C1 to the lipoyl cofactor of EH.

Recently, Guilhaudis et al. (22) reported an NMR analysis probing the structural change of pea H-protein bearing two forms of lipoate cofactor, oxidized and methylamine-loaded, by the addition of T-protein. They showed that the interaction of both proteins occurs in a relatively restricted area surrounding the lipoate arm binding site and Glu42 corresponding to Asp43 of ET. Consequently, the functionally concentrated area was required on T-protein involving the H-protein binding site, folate substrate binding site, and other related regions. These results are again consistent with our conclusions. Guilhaudis et al. (22) proposed that the role of T-protein is not only to locate the tetrahydrofolate cofactor in a position favorable for a nucleophilic attack on the methylene carbon but also to destabilize the H-protein in order to facilitate the unlocking of the arm and initiate the reaction. Its structural nature might be elucidated by crystallographic analysis, a project that is now in progress.

    ACKNOWLEDGEMENTS

We thank Drs. Hisaaki Taniguchi and Emiko Yamauchi of the University of Tokushima for help with mass spectrometry and Ryoichi Kunai of the University of Tokushima for help with amino acid sequence analysis.

    FOOTNOTES

* This investigation was supported in part by grants from the Ministry of Education, Science, and Culture of Japan.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.

Dagger To whom correspondence should be addressed: Institute for Enzyme Research, the University of Tokushima, Kuramotocho 3-chome, Tokushima 770-8503, Japan. Tel.: 81-88-633-9254; Fax: 81-88-633-7428; E-mail: ikeda@ier.tokushima-u.ac.jp.

Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M210853200

    ABBREVIATIONS

The abbreviations used are: 5, 10-CH2-H4folate, methylenetetrahydrofolate; 5, 10-CH2-H4PteGlu4, methylenetetrahydropteroyltetraglutamate; ET, E. coli T-protein; EH, E. coli H-protein; Lys-C, lysylendopeptidase; TPCK-trypsin, L-1-tosylamino-2-phenylethyl chloromethyl ketone-treated trypsin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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