1 Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8501, 2 Institute of Physical and Chemical Research (RIKEN), Wako 351-0198 and 3 Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji 192-0392, Japan
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Abstract |
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Keywords: closed conformation/3-isopropylmalate dehydrogenase/minor groove/modified C-terminus/paperclip motion
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Introduction |
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3-Isopropylmalate dehydrogenase (IPMDH, EC 1.1.1.85), encoded by a leuB gene of an extreme thermophile, Thermus thermophilus, is a thermostable enzyme catalyzing the oxidative decarboxylation of (2R,3S)-3-isopropylmalate (IPM) to 2-oxoisocaproate in leucine biosynthesis. The catalytic properties and thermal stability of the enzyme have been studied (Yamada et al., 1990) and its three-dimensional structure has also been determined at high resolution (Imada et al., 1991
). The subunit structure of IPMDH is composed of two domains, domain 1 and domain 2. The first domain includes both termini and consists of residues 199 and 252345, and the remaining amino acid residues from 100 to 251 form the second domain.
Numerous efforts to improve the thermal stability of IPMDH from T.thermophilus particularly emphasized the role of Ala residue at position 172 in the conformational stability (Kotsuka et al., 1996, Akanuma et al., 1997
). The thermostability of IPMDH A172L mutant (A172L hereafter), in which Ala was replaced with Leu at position 172, is 3°C higher than that of the wild-type enzyme. The thermostability enhancement of A172L is caused by the fact that the larger side chain of Leu in the minor groove gives extra interactions at the interface between domains in the active-site cleft (Qu et al., 1997
). In a sense, the enzyme is more stabilized when the active-site cleft is closed (closed form).
In a further study of A172L, the last five amino acid residues at the C-terminus (LeuArgHisLeuAla) were replaced with a dipeptide GlyIle. It results in a temperature-sensitive IPMDH mutant, called CD071. The melting temperature of CD071 dropped sharply from that of A172L by 14°C (Akanuma et al., 1996). Furthermore, insertion of tandemly duplicated sequences (Table I
) to the C-terminus of CD071 produces novel mutants that partly restore the thermal stability. The catalytic activities of these mutants are not significantly different from that of the wild-type IPMDH or A172L.
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Materials and methods |
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The mutants of T.thermophilus IPMDH were constructed and overexpressed in Escherichia coli as described previously (Akanuma et al., 1996). Purification of the IPMDHs was performed according to Kotsuka et al. (1996) with 10 min heat treatment at 72, 70 and 70°C for HD711, HD708 and HD177, respectively. The purified enzymes were analyzed with SDSPAGE.
Crystallization and data collection
The enzyme was dissolved in 20 mM potassium phosphate buffer (pH 6.5) containing 0.5 mM EDTA. Crystals of IPMDH HD711 were grown at 25°C by the hanging drop vapor diffusion method. The drops contained 0.05 M sodium acetate buffer (pH 4.8), 10% (v/v) PEG400 and 57.5 mg/ml protein and the reservoir solution was 0.1 M sodium acetate buffer (pH 4.8) containing 20% (v/v) PEG400. Rod-like crystals with the largest size of 0.3x0.5x0.3 mm were observed after overnight incubation. Crystals of IPMDH HD708 and HD177 were grown under the conditions similar to those for HD711 except for a twofold higher concentration of PEG400.
Data sets for HD711 and HD177 were collected at room temperature and 100 K, respectively, with use of the Weissenberg camera for macromolecular crystallography installed at the BL6A and BL18B of Photon Factory (Tsukuba, Japan). The intensity data were indexed, integrated, merged and scaled using the DENZO packages (Otwinowski and Minor, 1997). The crystal HD711 belongs to the monoclinic space group P21, with cell dimensions a = 55.4 Å, b = 87.6 Å, c = 70.9 Å and ß = 100.5°. HD177 crystallizes also in the monoclinic space group P21 with unit cell dimensions a = 69.9 Å, b = 86.0 Å, c = 54.4 Å and ß = 100.3°. Assuming a dimer per asymmetric unit, the Vm values of HD711 and HD177 are 2.30 and 2.20 Å3/Da, respectively (Matthews, 1968
). Detailed data collection statistics are listed in Table II
.
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Structure determination and refinement
HD711.
HD711 was solved by the molecular replacement method using the program X-PLOR (Brünger, 1992). The dimer A172L structure (PDB, accession code 1OSJ) was utilized as a search model. A rotation search using data between 10 and 4 Å resulted in the unique solution that the molecule corresponds to the search model, which is rotated by 10.2° about the axis of the direction cosine of (0.3, 0.9, 0.2). A translation search of the oriented molecule followed by rigid body refinements also yielded a unique solution with an R factor of 27.4%. After the molecule was located in the unit cell, manual-rebuilding structure was performed with the program TURBO-FRODO (Jones, 1985
). Again, rigid body and positional refinements using data between 6.88 and 2.8 Å were carried out with the program X-PLOR 98.1. Solvent positions were automatically assigned by WATERPEAK standard protocol with the hydrogen bond distance set between 2.5 and 4 Å and they were also checked with 2Fo Fc map using the program TURBO-FRODO. Further molecular dynamics stimulated-annealing refinements were achieved with 10% of data left out randomly for Rfree factor calculation. In the last refinements, non-crystallographic symmetry (NCS) restraint was applied to the domain backbones to obtain a good stereochemical quality and a bulk solvent correction was also made. Residues 346349 in subunit A and residues 345349 in subunit B were not included in the structural model because of their uninterpretable electron densities. The final model has an R factor of 17.7% and an Rfree factor of 26.7%. Detailed refinements statistics are given in Table III
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Results |
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IPMDH is a homodimer (subunits A and B hereafter) and each subunit consists of two domains; domain 1 contains five ß-strands (AE) and seven helices (ad and ik) and domain 2 contains seven ß-strands (FL) and four helices (eh) (Figure 1a) (Imada et al., 1991
). Ten ß-strands from A to J are important pillars for the IPMDH structure while the surrounding
-helices and loops cover the pillar from the solvent. The active-site groove is composed of helix h, strand F, strand E and helix d (Figure 1b
). The minor groove between helices e and j has no specific function. Table IV
summarizes the rotation angles of HD711, HD708 and HD177. The proper arrangement of the subunits may be essential for rigidity of IPMDH conformation. The dimer fitting angles of HD711 and HD177 are close to the twofold symmetry angle but that of HD708 deviates slightly from the symmetry angles.
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To detect the conformational changes in the mutants, 64 C-atoms in ß-strands forming the pillar structure (Imada et al., 1991
) (residues 27, 3540, 6669, 100110, 126133, 179184, 210215, 232236, 258263 and 266271) of each mutant were superimposed on those of A172L and the root mean square (r.m.s.) differences between the two coordinate sets were calculated for all mutants. The pillars of the mutants were fitted to the A172L pillar by rotation of 0.3° about the axis of (0.4, 0.7, 0.6) for HD711, 2.9° about the axis of (1.0, 0.5, 0.4) for HD708 and 83.8° about the axis of (0.2, 1.0, 0.2) for HD177, then by shifting oriented pillars in the direction of the orthogonal vectors of (28.1, 4.1, 0.5), (26.5, 5.0, 1.4) and (21.1, 82.2, 79.4) in units of ångstroms for HD711, HD708 and HD177, respectively. The r.m.s. differences of overall molecule and subunits are listed in Table V
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Dimer interface interactions
The dimer interface involves twofold symmetry interactions of four helices, g, h (in subunit A), g' and h' (in subunit B), two sticking out arm-like peptide chains hooking both subunits and two loops (Figure 2). The dimer interface interactions were evaluated by measuring the cavity volumes with radius probe of 1.4 Å. The largest cavity in the dimer interface was found in the space between the sticking out arm-like peptide chains and the mouth of a four-helix-bundle structure. The cavity volumes for A172L, HD711, HD708 and HD177 are 49.6, 54.0, 42.4 and 53.7 Å3, respectively.
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Active-site groove
The active-site groove is sandwiched between helix d and h and is in a closed conformation for all mutants used in this study. The width of the substrate binding cleft is defined by the distance between C atoms of Glu87 and Asp241 and that of the coenzyme binding cleft can be measured by estimating the distance between C
atoms of Gly255 and Ile279. As shown in Table VI
, the average widths of the substrate binding clefts of HD711 and HD177 are similar to that of A172L. These results are consistent with the result of functional analyses that the kinetic constants of these IPMDHs are similar to each other (Akanuma et al., 1996
). In contrast, the average width of the substrate-binding cleft of HD708 is closer than the others. The average widths of the coenzyme binding clefts of these IPMDHs remain unchanged (in a range 13.213.6 Å).
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The minor groove in IPMDH is located at the opposite side from the active-site groove. The function of this groove is not known. It may play a role in `open and close' movement of the active-site enzyme. The active site is opened upon binding of the substrate (Kadono et al., 1995). In the wild-type enzyme or A172L, the structure around the C-terminus was stabilized by a hydrophobic pocket surrounded by two residues on helix a (Val22, Leu26), one on helix j (Leu307) and three on helix k (Val340, Leu341, Leu344) and ionic interactions among His343, Arg344 (helix k), Lys317 and Glu321 (helix j) (Figure 3a
).
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In HD177, the ionic interactions in the C-terminal region are lost owing to mutations of Arg342Met and His343
Gly (Figure 3d
). In addition, mutation of Leu344
Ile may increase the hydrophobic force that pushes the minor groove closer (Table VII
).
To detect conformational changes due to the mutations in the C-terminal region, the cavity volumes of the region surrounded by strand B, helices a, i and j in the first domain, are also measured. In the case of A172L, the total accessible volume of the cavity is 1.6 Å3. The volumes of HD708 and HD177 are smaller than the volume of A172L, indicating that the strengthened hydrophobic pocket shifts not only the minor groove but also the pillar structure. In contrast, the volume in the corresponding location of HD711 is larger than that of A172L.
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Discussion |
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At least three major intramolecular interactions can be pointed out as an essential interaction for the folding of T.thermophilus IPMDH. First, the IPMDH pillar structure is built with a series of ß-strands from A to J (Figure 1b). The active-site groove and the minor groove are sandwiched between the first and second domains with a hinge pivot consisting of strands E and F. Second, the subunit interface interactions are composed of four-helix bundle structures with a hydrophobic well in the center, hydrogen bonds between two symmetric arm-like stretches from each subunit and interactions between two symmetric loops from each subunit (residues P117L118, Figure 2
). Finally, helices and loops prevent the pillar from making contact with the solvent. In the present study, mutant IPMDHs with different C-terminal sequences and different denaturation temperatures were analyzed using X-ray crystallographic techniques in connection with the change in thermal stability.
Although the experimental temperatures were either room temperature or 100 K, the IPMDH structure did not change as much as reported by Nagata et al. (1996). In fact, the catalytic activity and the major internal interactions of each domain of HD711, HD708 and HD177 are similar to those of A172L, hence the change in thermostability can be attributed to a result of `mechanical movement' alterations of the active-site groove and/or the minor groove.
IPMDH is a paperclip-like enzyme and the mouth of the active-site groove is closed when the minor groove is expanded. The inner space of the minor groove is mainly built by hydrophobic interactions except the space at its mouth segment. This segment is composed of two loops: a loop with hydrophobic residues (residues 301AFGLV305) in the first domain and a positively charged loop (residues 174RLRRL178) in the second domain. The antagonistic movements of the mouth of the minor groove may regulate the active-site groove, which is located at the opposite side of the enzyme molecule. Although it is not easy to define a simple criterion for the openclose movement of the active site because of the small angle between the substrate binding cleft and the hinge pivot, it is possible to define the movement of the minor groove by measuring the distance between two C atoms at the mouth segment.
With respect to the C-terminal region of the wild-type IPMDH or A172L, the width of the minor groove may depend on the inflexibility of helices a, j and k (Figure 3a). The top side of helix j, which is close to the hydrophobic loop of the minor groove, is stabilized by hydrophobic interactions among the side chains of Leu307 (helix j), Leu26 and Leu32 (helix a). The middle part of helix j is also supported by hydrophobic interactions among the side chains of Ala314 (helix j), Val22, L26 (helix a) and L344 (helix k), among the side chains of Ala318 (helix j), Val340 and Phe336 (helix k). The bottom side of helix j, which is exposed to the solvent, is stabilized by ionic interactions (Rhode and Martin, 1999
) among the side chains of Lys317, Glu321 (helix j), Arg342 and His343 (helix k). In addition, hydrophobic interactions between the side chains of Leu341 (helix k) and Ala25 (helix a) make helices a, j and k more compact structures.
HD711
The cleavage of ionic interactions at the bottom of helix j due to mutation of Arg342Ala and His343
Phe brings helix k close to helix j. On the other hand, the side chain of Glu341 located at the hydrophobic pocket tends to push helix k away (Figure 3b
). Although destabilization of the structure occurs particularly in the C-terminal region of helix k, the width of the minor groove remains constant. As shown in Table VII
, the width of the minor groove of HD711 is similar to that of A172L. In accordance with this movement, the width of the active-site cleft is also similar to that of the untruncated enzyme (Table VI
). The results may explain the partial restoration of the thermal stability of HD711 and also provide the reason why this enzyme is the most thermostable among the mutants. However, the slight constraints in the pillar structure (Table V
) and the expansion of the cavity at the interface between the domains are induced by the mutations and destabilize the structure of HD711, giving rise to a lower denaturation temperature than that of A172L.
HD708
Unlike HD711, the mutations in HD708 improve the strength of the hydrophobic pocket in the C-terminal region (Figure 3c). The lack of ionic interactions at the bottom of helix j and the exposed side chain of the hydrophobic residue (Ala342) to the solvent may support the strengthened hydrophobic pocket, by shifting helix k close to helix j. In addition, the side chains of Thr341 and Val344 located in the hydrophobic pocket drive helix k close to helix a. This makes the cavity in the first domain smaller and the minor groove closer (Table VII
). The closing of the mouth of the minor groove may explain the thermal instability of HD708 compared with that of HD711 or A172L. At the same time, the increase in the hydrophobic forces in the C-terminal region pushes the second domain away from the first domain. This gives rise to distortion of the interface angle between the subunits (Table IV
) and reduces the cavity in the dimer interface. The distorted dimer interface makes the active-site groove closer (Table VI
), suggesting the reason why HD708 restores thermal stability higher than that of HD177.
HD177
As the case in HD708, the mutations in HD177 also improve the hydrophobic interactions in the C-terminal region but the improvement is not as great as that in HD708. The loss of ionic interactions at the bottom of helix j makes helix k closer (Figure 3d). The side chain of Ile344 undergoes hydrophobic interactions with the side chains of Val22, Val36 (helix a) and Ala 314 (helix j). However, the residue of Gly341 seems not to contribute to the enhancement of the hydrophobic interactions because of its too small size. The mutations in HD177 only make the minor groove closer (Table VII
) and compress the cavity in the first domain. The closing of the mouth of the minor groove and the expansion of the cavity in the dimer interface may explain the thermal instability of HD177 and may also be the reason why HD177 has the lowest thermal stability among the mutants.
Hence it is obvious that the molecular mechanisms for the restored thermostability of the three mutants are different from one another. Although different mechanisms restored the thermal stability of three IPMDH mutants in this study, the results suggest that maintenance of the pillar structure and the interactions at the dimer interface is important for keeping the enzyme structure rigid. In addition, it is predictable from the present results that the thermostability of IPMDH can be engineered by point mutations (e.g. replacing Val305 with a larger hydrophobic residue), by inserting ions or a specific ligand in the mouth of the minor groove to expand the width of the groove and eventually close the active-site groove located at the opposite side of the subunit through a paperclip-like movement of the ß-strand pillar structure.
The mechanical movement mechanisms that are deduced from the present study are useful for the protein thermostability arrangement without any drastic change in the catalytic properties by way of the adjusted minor groove.
The atomic coordinates and structure factors have been deposited at the Protein Data Bank with the accession codes 1DPZ, 1DR0 and 1DR8 for HD711, HD708 and HD177, respectively.
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Notes |
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Acknowledgments |
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References |
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Received January 11, 2000; revised February 9, 2000; accepted February 14, 2000.