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1 Department of Chemistry, Knox College, Galesburg, Illinois 61401, USA
2 Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA
Reprint requests to: Andrew F. Mehl, Department of Chemistry, Knox College, Galesburg, IL 61401 USA; e-mail:
amehl{at}knox.edu; fax: (309) 341-7718.
(RECEIVED January 8, 2003; FINAL REVISION March 4, 2003; ACCEPTED March 5, 2003)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0300803.
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Abstract |
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-helices at the NH2-terminal end from each monomer (forming a "tail"), which lead into a small four-helix bundle from which each monomer contributes two short sequential -helices in an antiparallel topological arrangement. We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four-helix bundle formation. Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long -helical tail portion (GrpE1–88) is unable to form a dimer, most likely due to a decrease in -helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region. Mutants containing both of the short -helices (GrpE1–138 and GrpE88–197) are able to form a dimer and presumably the four-helix bundle at the dimer interface. These two mutants have equilibrium constants for the monomer–dimer equilibrium that are very similar to the full-length protein suggesting that the tail region does not contribute significantly to the stability of the dimer. Interestingly, one mutant that contains just one of the short -helices (GrpE1–112) exists as a tetrameric species, which presumably is forming a four-helix bundle structure. A proposed model is discussed for this mutant and its relevance for factors influencing four-helix bundle formation. Keywords: GrpE; heat shock protein; dimerization; four-helix bundle; protein stability
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Introduction |
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GrpE is a co-chaperone that helps regulate, along with a second co-chaperone DnaJ, the ATPase activity of the molecular chaperone DnaK (Jordan and McMacken 1995; Packschies et al. 1997; Pierpaoli et al. 1997, 1998). GrpE from E. coli has 197 amino acid residues, and structural information for GrpE is derived from a crystal structure of a GrpE mutant missing 33 residues from the NH2-terminal end that is also bound to the ATPase domain of DnaK (Harrison et al. 1997). An additional unique feature to the structure is the presence of a noncoiled-coil tail where each monomer contributes a long -helix that pair together in the dimer. There is also a small ß-sheet domain at the COOH-terminal end that does not contribute to the dimer interface.
To investigate the mechanism of dimerization and thus four-helix bundle formation within GrpE, we have used a deletion mutation analysis approach. Because the dimer interface is contained within the -helical domain of GrpE, we have focused on creating mutants that vary in their extent of -helical structure within the -helical domain to probe structural requirements for dimerization. Here we report the construction and initial characterization of four deletion mutants of GrpE and their comparison to full-length GrpE in their ability to form dimers and thus four-helix bundles.
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Results |
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) in a complex with DnaK, we designed four deletion mutant proteins of GrpE (Table 1). One (GrpE1–88) was constructed to encompass only the long -helical tail plus a presumably flexible portion that has an extended conformation at the extreme NH2-terminal end. Another mutant (GrpE1–112) was designed to include the long tail region, plus a portion of the four-helix bundle that contains only one of the short -helices that contribute to the bundle. GrpE1–138 was designed to have both the long tail portion and both short -helices that contribute to the formation of the four-helix bundle in the dimer structure. The last mutant (GrpE88–197) constructed is missing the unique NH2-terminal tail structure and thus contains the two short -helices (amino acids 88–138) that contribute to the four-helix bundle in the dimer, and a ß-sheet domain (amino acids 138–197). We were unsuccessful in creating a clone for GrpE88–138, presumably due to the small size of the insert in the cloning process. All the mutant proteins were purified by similar means based on the purification of full-length GrpE (Mehl et al. 2001). The purity level was sufficient for the physical characterization experiments described in this work.
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are the predicted molecular masses and those determined by mass spectrometry (using the MALDI technique). Note that the predicted mass accounts for the fact that those mutants having the "native" NH2 terminus are missing the initial methionine residue (Zylicz et al. 1987). The predicted masses agree with the masses determined by mass spectrometry and thus the constructs are unmodified during overexpression and are as originally designed and confirmed by DNA sequencing.
Circular dichroism spectroscopy of GrpE and the GrpE deletion mutants
Protein secondary structural content of full-length GrpE and the four deletion mutants was probed for by circular dichroism (CD) spectroscopy. Note that CD analysis for full-length GrpE, GrpE1–88, and GrpE88–197 have been reported (Mehl et al. 2001) and are reproduced here for comparison purposes. As shown in Figure 2, full-length GrpE gives a spectrum that is characteristic of a protein with significant -helical secondary structure, showing negative dips in the spectrum at 208 and 222 nm. GrpE1–112, GrpE1–138, and GrpE88–197 also give spectra that show significant amounts of -helical secondary structure. The spectrum for the GrpE1–88 mutant looks different from the full-length and the other mutants, and clearly there is less -helical content with this mutant. The spectrum for the GrpE1–88 does have some -helical content, however, and does not indicate that the mutant is completely unfolded and in a random-coil conformation. When analyzed for amount of -helical content using SELCON3 software (Sreerama et al. 1999), the GrpE1–88 showed 22% -helical content (Table 1). The percentage of -helical content reported previously (Mehl et al. 2001) was done incorrectly. Clearly, with the GrpE1–88 mutant the "native" structure for the tail region is lost.
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and summarized in Table 2 as to the predominant state for each protein after the 2-h incubation with the crosslinker. All of the mutants were able to form a dimeric species except for the GrpE 1–88 mutant. The GrpE 1–112 mutant showed a dimeric species and a higher order species that is a tetramer as determined by analytical ultracentrifugation experiments (see below). Note that under these conditions for crosslinking, some amount of protein, in all cases is degraded due to the crosslinker and this is seen most dramatically with the GrpE 1–88 mutant. Also, those proteins with the tail region, the crosslinked species tends to run faster and become more spread out during the electrophoresis. Both of these observations do not influence the conclusions from the crosslinking study.
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summarizes the monomer molecular mass as determined by AUC equilibrium experiments. These results agree with recent studies (Gelinas et al. 2002) carried out with GrpE1–197 and other similar deletion mutants, namely GrpE84–197 and GrpE1–140. Also shown are the association constants for the monomer–dimer equilibrium or in the case of the GrpE1–112 the dimer–tetramer equilibrium. For GrpE1–197 and GrpE88–197, the fit overlays and residuals, as well as the Monte Carlo distributions for the molecular mass and association constants are shown in Figures 4 and 5, respectively.
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shows an example of the results of the native polyacrylamide gel electrophoresis (PAGE) for the GrpE1–88 mutant. When various proteins are analyzed for mobility versus concentration of polyacrylamide, one can construct a Ferguson plot (Ferguson 1964) to get an estimate for the molecular mass for an unknown protein. When GrpE1–88 was analyzed in this way a molecular mass of 
15 kD was obtained, a value less than that for a dimeric species. This result taken together with the crosslinking result for GrpE1–88 suggests that this protein is unable to form a dimer.
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shows the results of the gel filtration studies. Full-length GrpE (Fig. 7A) eluted at a single peak at about an apparent molecular mass of 230 kD. This result is in general agreement with previous gel filtration studies with GrpE, where a value of 190 kD was reported (Schonfeld et al. 1995). Wu and co-workers (1996) reported a value of 135 kD from gel filtration studies with GrpE. As noted previously (Schonfeld et al. 1995), this technique is problematic for GrpE in obtaining an accurate size; we only present the size exclusion chromatography results for comparison purposes between full-length and the deletion mutants and to note the behavior of the mutants as related to structure of the mutants (and not to determine molecular mass). Both GrpE1–88 and GrpE88–197 eluted at similar positions (Fig. 7B,C). The GrpE1–88 mutant includes the NH2-terminal portion that contains the presumably flexible polypeptide end of 33 amino acids and the long -helical region, again contributing to the elution position for this protein being higher than expected, as was the case for full-length GrpE. The GrpE88–197 does not contain the unique NH2-terminal end and thus elutes at a position that is fairly close to that predicted for a dimer species. The elution pointed to an approximately molecular mass of 35 kD, and the predicted molecular mass would be 24 kD. GrpE1–112 eluted at an apparent molecular mass that is higher than GrpE1–197 (Fig. 7D). This result agrees with the crosslinking studies and AUC results that show a higher ordered oligomer being the predominant species. Lastly, the GrpE1–138 mutant eluted at a position slightly smaller than full-length GrpE (Fig. 7E), again in agreement with this mutant having the long extended tail in its structure, but missing the ß-sheet domain.
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Discussion |
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We have begun to investigate the mechanisms by which the dimeric GrpE protein from E. coli oligomerizes into its dimeric structure. Our initial approach has been to use a deletion mutation strategy to identify specific structural requirements that are potentially needed for proper oligomerization. We have found that for GrpE to oligomerize into a dimer, one of two short -helices that each monomer contributes to the dimer must be present within the mutant protein. Carrodeguas and co-workers (2001) have used this same approach to show that the polymerase 
B subunit requires a unique four-helix bundle for dimerization. The GrpE1–88 mutant that is missing any contribution to the four-helix bundle was unable to form a higher order species that could be crosslinked together with the EDC crosslinking agent, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride. In addition, native PAGE analysis suggests a monomeric species. The CD spectrum for the GrpE1–88 shows a significant amount of nonhelical structure (although not 100% random coil); therefore, the possibility exists that the mutant is not adopting the native structure that is found within the tail region of GrpE. It could be postulated that for the long -helical tail region to form, as is seen in the crystal structure, the four-helix bundle region must be present. These results and conclusion with the GrpE1–88 mutant are consistent with those recently reported by Gelinas and co-workers (2002). Their study used a synthetic GrpE44–88 construct to investigate this region of the protein. The tail region is thought to play an important role in the interaction with DnaK (Harrison et al. 1997; Mehl et al. 2001) and thus the dimer structure with the four-helix bundle at the interface is clearly needed to support the unique tail portion of the protein.
The GrpE1–138 and GrpE88–197 deletion mutants contain the full complement of the four-helix bundle and both form dimeric species presumably in the same manner as the full-length protein with the four-helix bundle at the dimer interface. The positions of equilibrium for the monomer–dimer equilibrium are very similar for all three proteins (GrpE1–197, GrpE1–138, and GrpE88–197). These results indicate that the long tail region does not contribute, to any great extent, to the stability of the dimer.
The most interesting mutant, GrpE1–112, contains only one of the two short -helices and was found to form a higher ordered oligomer, which is a tetramer. Five other examples of homotetramers that formed four-helix bundles were identified in the literature: the H3 region of syntaxin 1A (Misura et al. 2001), the zipper domain of hnRNP C (Shahied et al. 2001), glycogen phosphorylase (Lin et al. 1995), human relaxin (Lin et al. 1995), and acetylcholinesterase (Bourne et al. 1999). The AUC results revealed a dimer–tetramer equilibrium as opposed to a monomer–tetramer system and we propose that the dimer would be similar to that found in the full-length protein with two monomers forming a long tail with the helices parallel to one another in the region between residues 33 and 88. The formation of the four-helix bundle in the full-length protein is achieved by the two helices in the tail moving apart and then becoming diagonally parallel in the bundle; this is followed by a loop to the adjacent helix (residues 116–138) that is oriented antiparallel to the first helix on the same strand. The GrpE1–112 mutant could form the bundle in a similar fashion as depicted in Figure 8, model A, where the monomers in the dimer separate slightly to accommodate the other dimer that would provide the other two helices in the bundle. The dipoles for the helices provided by each dimer would again be diagonally parallel in the bundle. Comparison of the residue polarity/nonpolarity for residues 88–112 with that of 116–138 reveals very similar amphipathic sequences, as one would expect for helices found in four-helix bundles and thus in model A the helical segment formed by residues 88–112 is replacing that formed by residues 116–138, as is found in the full-length protein.
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as model B. In this model a dimer is created by bringing two monomers together to form a tail as in model A, but the formation of the four-helix bundle is accomplished by bringing the dimers together to form a bundle where all helices are parallel to one another. All five of the homotetramers cited above form four-helix bundles with an antiparallel topology, with the H3 region of syntaxin having the unusual arrangement of the two parallel helices being adjacent as opposed to the more commonly observed parallel helices located across the diagonal (Misura et al. 2001). Generally, the antiparallel arrangement is more common in any four-helix bundle as opposed to having all four parallel (Lin et al. 1995). Thus, model B is probably not likely to represent a dominant species for GrpE1–112. The dimers in model B show each helix remaining adjacent, even in the bundle region, because to create a situation where the helices from each dimer are located across the diagonal did not seem structurally feasible due to steric constraints. In both models proposed, we restricted the region for four-helix bundle formation to that observed in the full-length protein because the helices formed using residues 33–88 have many more polar and charged groupings dispersed throughout (nonamphiphatic), rendering it less likely to be involved in bundle formation where a hydrophobic core is necessary. Future work will involve structural characterization of the GrpE1–112 mutant or a mutant that has similar properties.
Conclusions
As was predicted from studying the X-ray crystal structure of the GrpE dimer complexed to the ATPase domain of DnaK, the formation of the dimer by a four-helix bundle is necessary to support formation of the unique NH2-terminal tail region of GrpE. The tail cannot exist by itself in the same type of conformation found in the full-length protein, and additionally, the tail region does not contribute significantly to the stability of the dimer. A GrpE deletion mutant having the first 112 amino acids (GrpE1–112) at the NH2-terminal contains the tail region and one of the two -helical regions that participate in the formation of a four-helix bundle was found to exist as a homotetramer. The structural model proposed for this mutant involves the formation of an antiparallel four-helix bundle where each monomer contributes one helix in a similar manner as is found in the full-length protein. The proposed model supports the notion that helical dipole alignment in the four-helix bundle is an important stabilizing factor.
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-globulin as a standard. The molar protein concentration of full-length GrpE and all the mutants described in this paper are based on the molar mass of the monomeric form of the protein determined from the known amino acid sequence (Table 1).
Strains and plasmids
Strain RLM 988 (C600dnaK103) was used for the expression of full-length GrpE. Strain RLM 569 (C600, recA, hsdR, tonA, lac-, pro-, leu-, thr+, dnaJ+) was used for the expression of all of the GrpE deletion mutants. Plasmid pRLM 156 was used as the expression vector for all proteins described in this paper. This plasmid is the same as pRLM76 (Karzai and McMacken 1996), only it has a more versatile polylinker region for cloning purposes.
Plasmids carrying the wild-type grpE gene and the appropriate grpE deletion mutant genes for GrpE1–88 and GrpE88–197 have been previously described (Mehl et al. 2001). Note there is a correction in this reference in that the GrpE1–89 is in fact GrpE1–88. Plasmids carrying the grpE deletion mutant genes for GrpE1–112 and GrpE1–138 were constructed in a similar manner as described for grpE mutants GrpE1–88 and GrpE88–197 (Mehl et al. 2001); E. coli genomic DNA was used for the template in the PCR reaction and new forward or reverse primers were used as needed. A new reverse oligonucleotide (D 5'-CCACGGCGCCCTGCAG GTATACCTATTAAGCTTTATCAGCCACTTC-3') was used with the construction of GrpE1–112, and a new reverse oligonucleotide (E 5'-CCACGGCGCCCTGCAGGTATACCTATTAGCCAAAC TTACGCACAAC-3') was used with the construction of GrpE1–138. The new reverse primers contained a PstI restriction site, two tandem translation stop codons, and the complement of grpE coding sequence (underlined above). In each mutant the amplified DNA fragment was digested to completion with SalI and PstI, and ligated to pRLM156 that had been similarly digested. DNA from the ligation reaction was transformed into RLM 156, and ampicillin-resistant clones were screened for the ability to overproduce a protein in the appropriate size range when grown at 42°C. A resulting plasmid was named pAFM7 for the GrpE1–112 mutant and the strain was named AFM16. A resulting plasmid was named pAFM9 for the GrpE1–138 mutant and the strain was named AFM12.
DNA sequencing
The grpE region coding for amino acid sequence GrpE1–112 of AFM16 and the grpE region coding for amino acid sequence GrpE1–138 of AFM12 were sequenced on both strands using the dideoxynucleotide chain termination method with modified T7 DNA polymerase (Sequenase) as described by the manufacturer (U.S. Biochemical). All were found to contain the correct nucleotide sequences.
Expression and purification of GrpE and the various deletion mutants
Expression and purification of GrpE1–197, GrpE1–88, and GrpE88–197 have previously been described (Mehl et al. 2001). For the purification and overexpression of GrpE1–112 and GrpE1–138, the same procedure was used as for GrpE1–197.
CD spectroscopy
The CD analysis was carried out at the University of Illinois Champaign-Urbana’s Laboratory for Fluorescence Dynamics. The CD spectra were recorded at 20°C on a Jasco model 720 instrument using a 2.0-mm pathlength cell. Proteins in 20 mM potassium phosphate buffer (pH 7.0), 1 mM DTT, 5.0 mM MgCl2, 25.0 mM KCl, and 10% (v/v) glycerol were used at concentrations of 30 µM. Data were collected with a scanning rate of 50 nm/min with 0.5 nm intervals at a spectral band width of 1.0 nm. Mean residue molar ellipticity was calculated as described (Schmid 1989).
EDC crosslinking reactions
All crosslinking reactions were carried out at 25°C. Samples (usually 50 µL total volume) contained 25 mM HEPES/KOH (pH 7.6), 0.5 mM EDTA, 33 mM EDC, and 10 mM Sulfo-NHS. Reactions were initiated by the addition of protein (45 µg) and incubated for the times indicated. Reactions were stopped by the addition of 50 µL of gel-loading buffer and immediate incubation in boiling water for 5–10 min. Samples were analyzed by SDS-PAGE (usually a 12.5% resolving gel and a 4.5% stacking) and the crosslinked and uncrosslinked proteins were identified by staining with Coomassie Brilliant Blue R-250.
Analytical ultracentrifugation equilibrium experiments
All sedimentation equilibrium experiments were performed with a Beckman Optima XL-A at the Keck Biophysical Facilty at Northwestern University. Equilibrium and Monte Carlo analyses were performed with UltraScan version 5.0 (developed by one of the authors, Demeler 2001). Hydrodynamic corrections for buffer conditions were made according to data published by Laue et al. 1992, and as implemented in UltraScan. The partial specific volume of each protein was estimated from the primary protein sequence according to the method of Cohn and Edsall (1943) and as implemented in UltraScan (GrpE1–112: 0.73041 mL/g, GrpE1–138: 0.73364 mL/g, GrpE1–197: 0.73654 mL/g, and GrpE88–197: 0.74905 mL/g). Monte Carlo analyses were calculated on a 40-processor Linux Beowulf cluster running Slackware Linux version 7.0. All samples were analyzed in a buffer containing 25 mM sodium phosphate at pH 7.2, with 100 mM NaCl and 5% (v/v) glycerol (density: 1.017 g/mL). All experiments were performed at 4°C. The samples were centrifuged to equilibrium at speeds ranging between 15,000 and 50,000 rpm with 5000-rpm increments. Equilibrium times were estimated using UltraScan as follows: 15 krpm, 31 h; 20 krpm, 19 h; 25 krpm, 13 h; 30 krpm, 9 h; 35 krpm, 8 h; 40 krpm, 6 h; 45 krpm, 5 h; and 50 krpm, 4 h. The samples were centrifuged in six-channel epon-filled centerpieces in the AN-50-TI rotor. For each sample, three loading concentrations were measured (0.3, 0.5, and 0.7 OD 230 nm). Because the proteins had very little absorbance at 280 nm, all scans were collected at 230 nm in radial step mode with 0.001-cm step size setting and 20 replicates. Twenty-four scans of three concentrations and eight speeds for each sample were collected, with all samples providing data from a 2.5- to 2.8-mm column length. Data exceeding 0.9 OD were excluded from the fit. Extinction coefficients at 230 nm were estimated by determining the protein concentration for each sample using the Bradford protein assay as described above.
Size exclusion chromoatography
The FPLC chromatography was carried out at 4°C on a Superdex 200 column. A 50-µL aliquot containing GrpE protein or any of the deletion mutants at 100 µM in 100 mM sodium phosphate at pH 7.6 and 10% (v/v) glycerol was loaded onto the column previously equilibrated with the same buffer. Elution of the protein was obtained using the same buffer at a flow rate of 0.4 mL/min.
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References |
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