Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M513721200 on July 25, 2006

J. Biol. Chem., Vol. 281, Issue 43, 32508-32515, October 27, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/43/32508    most recent
M513721200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Márquez, JoseA.
Right arrow Articles by Scheffzek, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Márquez, JoseA.
Right arrow Articles by Scheffzek, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Structure of the Full-length Enzyme I of the Phosphoenolpyruvate-dependent Sugar Phosphotransferase System*Formula

JoséA. Márquez{ddagger}§1, Stefan Reinelt{ddagger}12, Brigitte Koch, Roswitha Engelmann, Wolfgang Hengstenberg, and Klaus Scheffzek{ddagger}3

From the {ddagger}European Molecular Biology Laboratory, Structural and Computational Biology Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany, the §European Molecular Biology Laboratory, Grenoble Outstation, 6 rue Jules Horowitz, B. P. 181, 38042 Grenoble Cedex 9, France, and the AG Physiology of Microorganisms, Ruhr-University-Bochum, D-44780 Bochum, Germany

Received for publication, December 23, 2005 , and in revised form, July 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Enzyme I (EI) is the phosphoenolpyruvate (PEP)-protein phosphotransferase at the entry point of the PEP-dependent sugar phosphotransferase system, which catalyzes carbohydrate uptake into bacterial cells. In the first step of this pathway EI phosphorylates the heat-stable phospho carrier protein at His-15 using PEP as a phosphoryl donor in a reaction that requires EI dimerization and autophosphorylation at His-190. The structure of the full-length protein from Staphylococcus carnosus at 2.5Å reveals an extensive interaction surface between two molecules in adjacent asymmetric units. Structural comparison with related domains indicates that this surface represents the biochemically relevant contact area of dimeric EI. Each monomer has an extended configuration with the phosphohistidine and heat-stable phospho carrier protein-binding domains clearly separated from the C-terminal dimerization and PEP-binding region. The large distance of more than 35Å between the active site His-190 and the PEP binding site suggests that large conformational changes must occur during the process of autophosphorylation, as has been proposed for the structurally related enzyme pyruvate phosphate dikinase. Our structure for the first time offers a framework to analyze a large amount of research in the context of the full-length model.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Group translocation is the membrane transport mechanism by which a substrate is chemically modified to an impermeable derivative as it crosses the cell membrane. This energy-efficient transport strategy is used by bacteria for the uptake of rapidly metabolizable sugars, and it is achieved through a highly conserved three component phospho-relay system called the phosphoenolpyruvate:sugar phosphotransferase system (PTS)4 (1-3). The PTS catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to a sugar while it is being transported across the membrane. It consists of two universal components, Enzyme I (molecular mass, 63 kDa) (4, 5), hereafter referred to as EI, and the heat-stable histidine phospho carrier protein (HPr) (molecular mass, 9 kDa), and in addition several membrane-associated components, which are sugar-specific and are collectively designated as Enzyme II (EII) complexes (3). The PTS cascade starts with the autophosphorylation of EI on a conserved histidine (His-190 in the Staphylococcus carnosus EI studied in this report) in a reaction that uses PEP as phosphoryl donor (4). Subsequently, the phosphoryl group is transferred to His-15 of the HPr protein, and ultimately to the imported hexose, in a series of transphosphorylation reactions mediated by the components of the sugar-specific EII complex. The PTS is not only responsible for sugar uptake; it also represents a major sensing and signaling system in the bacterial cell. The phosphorylation state of the components of the PTS pathway is directly coupled to the regulation of carbohydrate metabolism, chemotaxis toward carbon sources (6), carbon catabolite repression (7, 8), and nitrogen metabolism (9). Because EI catalyzes the first step in the pathway and because its activity levels will determine the phosphorylation state of the downstream PTS components, it may play a key regulatory role in the control of PTS and its downstream metabolic effects (10-12).

EI is highly conserved throughout bacteria, displaying a high degree of sequence similarity among different species (13). It consists of an N-terminal protease-resistant portion (EIN, residues 1-264) and a protease-sensitive C-terminal domain (EIC, residues 265-573) (4). The N-terminal region is responsible for HPr binding, whereas the C-terminal region binds PEP (14). The EIN domain consists of two subdomains: an {alpha}-helical domain and an {alpha}/beta domain, called the phospho-histidine (P-His) domain, that contains the intermediate phosphoacceptor His-190 (in S. carnosus) (15, 16). The EIN and the HPr protein form a stable complex that has been studied by NMR, showing that interactions between HPr and EI occur through the helical domain (from now on termed the HPr-binding domain), which is responsible for substrate specificity. This interaction places the phosphoacceptor His-15 of HPr and the His-190 of EI at optimal separation for efficient phosphotransfer (17).

The C-terminal domain of EI displays sequence similarity with the PEP-binding domain of the pyruvate phosphate dikinase (PPDK) enzyme (18, 19). This enzyme catalyzes the reversible conversion of ATP, pyruvate, and inorganic phosphate (Pi) into PEP, AMP, and pyrophosphate through a phosphoryl-enzyme intermediate, and it has a phosphohistidine acceptor domain structurally equivalent to that of EI. Erni and coworkers (14) have recently obtained the crystal structure of the C-terminal domain of Thermoanaerobacter tengcongensis EI showing that the overall fold is very similar, and the configuration of the active site is almost identical to that of the PEP-binding domain from PPDK. Both PPDK and the PEP-binding region of T. tengcongensis crystallized as dimers, and in both cases the dimerization interface involves equivalent regions of the PEP-binding domains. On the other hand, biochemical studies have shown that EI exists in a monomer-dimer equilibrium (20, 21) where only the dimeric form is competent for autophosphorylation (22). Those studies showed that dimer formation is stimulated by PEP and magnesium ions but that the interconversion between monomer and dimer is very slow, suggesting that oligomerization may be the rate-limiting step for the activation of EI and as a consequence may determine the activity of the PTS pathway and its downstream effects (21, 22). More recently Roseman and coworkers (11, 12) using ultracentrifugation experiments demonstrated that the presence of PEP and magnesium induce conformational changes in both the monomeric and dimeric forms of EI. Nevertheless, it remains unclear how dimerization activates the autocatalytic potential of EI and more generally how the different catalytic centers distributed in different domains of the protein interact during the reaction cycle.

We present here the first crystal structure of the full-length EI from S. carnosus at a resolution of 2.5 Å, which for the first time reveals the spatial arrangement of the three protein domains. Aspects of dimer formation and stabilization as well as implications for catalysis will be discussed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Expression and Purification—EI from S. carnosus was overexpressed in Escherichia coli (strain DH5{alpha}) harboring the plasmid pUC-ptsO2.6X (23). Cells were grown in the presence of isopropyl-beta-D-thiogalactoside in TBY broth (10 g of Tryptone, 5 g of NaCl, 5 g of yeast extract/liter). Cells (13 g of wet weight from 6 liters of culture) were suspended in 25 ml of standard buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride), disrupted by sonication, and centrifuged at 25,000 x g. The supernatant was applied to a Q-Sepharose column (120 ml) using a gradient of 600 ml (0-0.6 M NaCl in standard buffer). EI was detected by SDS-PAGE (10% acrylamide) at 0.5 M NaCl. The enzyme pool was adjusted to 20% ammonium sulfate, applied to a butyl-TSK column (420 ml, Tosohaas, Montgomeryville, PA) and eluted with a linear gradient of 2 liters of 20-0% ammonium sulfate in standard buffer. EI eluted at 5% ammonium sulfate and was pure according to native (pH 9) and denaturing (SDS) PAGE. Standard yields were 40-50 mg of protein. Prior to crystallization experiments the protein was passed through a Sephadex G-25 column equilibrated with 50 mM HEPES, pH 6.5, and concentrated in Centricon tubes (Millipore) to a concentration of 20 mg/ml. The storage solution was supplemented with PEP and MgCl2, both to a final concentration of 5 mM.

Crystallization—Crystals of full-length EI were obtained by the vapor diffusion method. Initially hanging drops made with 1 µl of protein, 1 µl of crystallization buffer (30% polyethylene glycol 4000, 0.2 M Li2SO4, and 0.1 M Tris-HCl, pH 8.5), and 0.2 µl of additive solution (solution 11 of the Hampton Crystal Screen, Hampton Research: 0.1 M trisodium citrate dehydrate, pH 5.6, 1 M ammonium dihydrogen phosphate) were equilibrated against 500 µl of crystallization buffer in standard Linbro crystallization plates. Hexagonal crystals appeared after 1-2 days and diffracted typically to 7.0 Å in a synchrotron beam. After 5-7 days new monoclinic crystals appeared in the drops while the hexagonal crystals tended to disappear. These crystals diffracted typically to 2.5 Å with a synchrotron x-ray source. Initially the presence of solution 11 of the Hampton Crystal Screen in the crystallization mixture was the result of an accidental contamination, however this proved to be essential for the reproducibility of the crystals.

A second crystallization condition was obtained using sitting drops and 30% (w/v) polyethylene glycol 4000, 0.2 sodium malonate, and 0.1 M Tris-HCl, pH 8.5, as crystallization buffer. This second condition did not require additives and produced only monoclinic crystals with similar symmetry properties and diffraction power. For data collection the crystals were soaked in crystallization buffer containing 7.5% (v/v) polyethylene glycol 400 as cryoprotectant and flash-cooled by immersion in liquid nitrogen.

Data Collection, Structure Determination, and Refinement—All diffraction data were collected under a cryogenic stream at 100 K. Preliminary characterization was performed at beamlines ID14-1 and ID29 of the European Synchrotron Radiation Facility and beamline BW7a of the Deutsches Elektronen-Synchroton. EI crystals obtained in the presence of sodium malonate were soaked for 3 h in crystallization buffer supplemented with 0.1 M of the Gadolinium complex Gd·DTPA-BMA (24) and used in a multiple-wavelength anomalous dispersion (MAD) experiment conducted at beamline BM14 of the European Synchrotron Radiation Facility (Table 1). Data processing and scaling were done with the program suite XDS (25).


View this table:
[in this window]
[in a new window]
  		TABLE 1
Summary of the crystallographic analysis

 
The coordinates of the heavy atoms and the initial set of phases were obtained with the program SOLVE (26). These phases and the atomic model of the {alpha}-helical subdomain of the N-terminal region of EI (amino acids 20-142, PDB code 1ZYM [PDB] ) were used for phased molecular replacement using the program CNS (27), which produced a solution for the {alpha}-helical N-terminal domain that agreed with the density map. Phase combination and density modification with the CNS program resulted in an improved electron density map, which allowed model extension. The structure of the PPDK (1DIK) (18) was superimposed on a partial model of the C-terminal portion and subsequently adjusted in the experimental electron density map. The structural model was completed based on the revised sequence of the S. carnosus enzyme,5 in alternate rounds of model building and refinement using CNS (27) and O (28). In the later stages of model building, structure completion was continued with data collected from another crystal grown under the original conditions for the monoclinic form. This crystal was soaked in (NH4)2Os4Br6, which improved diffraction data but did not lead to a heavy atom derivative and was thus considered as a native crystal (Table 1). A large electron density peak in the PEP-binding region of the C-terminal domain, showing interactions with the side chains of two arginine and one asparagine residue compatible with a tetrahedral coordination sphere, indicated the presence of a phosphate or sulfate ion. Given that LiSO4 (0.2 M) is a component of the crystallization mixture, this peak was modeled as a sulfate ion. A summary of the crystallographic analysis is given in Table 1. Structural visualizations were done with the programs MOLSCRIPT along with Raster3D (Figs. 1B, 2, and 3) (29, 30) and PYMOL (Figs. 1A, 1C, and 4A).6 The atomic coordinates have been deposited with the Protein Data Bank (www.rcsb.org), under accession code 2HRO.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Structural Organization of EI—EI from S. carnosus (23) was overexpressed and purified as described under "Experimental Procedures." Initial hexagonal crystals appearing in Hampton Standard screens diffracted poorly, whereas monoclinic crystals, appearing 3-5 days later, diffracted up to 2.5 Å. For phase determination a novel gadolinium compound (24) was particularly useful, because it allowed straightforward recording of a full data set using MAD that was used for structure determination as described under "Experimental Procedures." The resulting electron density map showed one molecule per asymmetric unit and was easy to interpret in the regions corresponding to the HPr-binding and the PEP-binding domains. However, the region containing the P-His domain (residues 4-22 and 156-229) showed weak electron density even at the later stages of refinement. Despite this, 70 of the 102 C{alpha} atoms in this region were assigned, which is enough to identify the fold and determine its orientation relative to the other two domains; however, many of the side chains of this domain, including His-190, were not visible and were modeled as alanines. A stereo view of an electron density map calculated with the final model but omitting the P-His domain (to exclude any possibility of phase bias in this region) is presented in Fig. 1A. The electron density corresponding to the backbone of the P-His domain is clearly visible. The final model (Rwork/Rfree: 22/27%, see Table 1) consists of 551 residues, 1 sulfate ion, and 172 water molecules. 22 of 573 residues in the full-length protein were not visible in the electron density: 3 and 2 residues at the N- and C-termini, respectively, and 17 in the P-His domain.

The full-length EI monomer is composed of three structurally independent domains separated by long linkers (Fig. 1B). It shows an extended conformation, ~110 Å in length with a maximum width of ~60 Å. The N-terminal region (EIN) and the PEP-binding C-terminal domain (EIC) are separated by a long (40 Å) and easily accessible linker helix (residues 233-260, in gray in Fig. 1, B and C), which explains the proteolytic sensitivity of EI (32, 33). The HPr-binding and the P-His domain (depicted in red and blue, respectively, in Fig. 1) are also separated by two extended linker regions (depicted in gray) with only few interactions between them.

The P-His domain (residues 4-22 and 156-229) contains the His-190, the initial acceptor of the phosphoryl group from PEP (15). This region of the structure showed weak electron density and high temperature factors (see above), suggesting that it is flexible in our crystals, presumably due to a lack of interactions with the other domains that could stabilize its position. This conformational flexibility may have functional implications, as discussed below. The HPr-binding domain (red in Fig. 1) is inserted in the beta1-beta2 loop region of the P-His domain. It spans amino acids 37-144 and is composed of two helical hairpins forming a V-shaped structure with an angle of ~60 degrees. The HPr-binding region, as shown by Garrett and co-workers (17), is located at the open end of the "V." A surface representation of the HPr protein is included in Fig. 1C to indicate the approximate region of binding. A superposition of the P-His and HPr-binding domains with that determined from the N-terminal region of E. coli EI (15) indicates that the two domains in the respective proteins have a very similar structure, as would be expected from the degree of sequence conservation. This superposition allowed us to determine the approximate position of the side chain of His-190 involved in the phosphotransfer reaction, which is not visible in our model. The HPr-binding and the P-His domains are connected by two long linkers that adopt an extended conformation (Fig. 1B). In the structure of the E. coli EIN·HPr complex these linker regions are rather straight, which results in an optimal positioning of the phosphoacceptor histidine of the HPr protein with respect to the donor histidine of EI (17). In the present structure, however, the major axis of the same linker regions are inclined by ~35 degrees with respect to that of the E. coli enzyme, which results in an outward rotation of the HPr-binding domain relative to the P-His domain. Assuming that in S. carnosus EI HPr would bind in a similar way, as would be expected from the degree of sequence conservation, the two histidines would be separated by a distance of ~20 Å. This indicates that significant rearrangements must occur before phosphotransfer from the P-His domain to HPr can take place.

The C-terminal Domain—The C-terminal PEP-binding domain of EI consists of an eight-stranded beta-barrel surrounded by ten helical segments in a topology indicative of a TIM barrel fold. Its structure is very similar to that of the PEP-binding domain from T. tengcongensis EI (14) (300 C{alpha} atoms superimposed with a root mean square deviation of 1.1 Å), as would be expected given the high sequence identity (59%) in this region. As previously reported (14), this domain also shows significant structural similarity with the PEP-binding domain of the PPDK enzyme (18, 19, 34) (300 C{alpha} atoms superimposed with a root mean square deviation of 2.9 Å), although in this case the sequence identity was only 28%. The major structural differences between PPDK and EIC have been described (14). A large positive electron density peak was found in the PEP-binding region of the C-terminal domain. It was stabilized through interactions with Arg-297, Arg-333, Asn-455, and Arg-466. Given its size and coordination pattern we interpret this electron density peak as a sulfate ion (one of the components of the crystallization buffer). A detailed view of the PEP binding site is presented in Fig. 2.


Figure 1
View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 1.
Structural organization of the EI monomer. A, stereo view of an electron density omit map around the P-His region. The atoms in a sphere centered at residue 179 of the P-His domain and with a radius of 17 Å (which contains the P-His domain and part of the HPr-binding domain) are omitted from the calculation. The refined model (C{alpha}-trace) is displayed along with the electron density. The HPr-binding helical domain is depicted in red, the P-His-segment in blue, and the C-terminal PEP-binding domain in green. Symmetry-related molecules are displayed in orange. The electron density corresponding to the backbone of the P-His domain is clearly visible. B, ribbon diagram of the final EI model with N-terminal (EIN) and PEP-binding (EIC) regions indicated as in A. Interdomain linkers are represented in gray. The sulfate ion bound at the C-terminal domain is depicted as a CPK model. C, surface view oriented as in B with the same coloring schemes. A model of the HPr protein (pink) has been included based on a superposition with the model for EIN and HPr (17). Red dots indicate the approximate position of His-190 in the P-His domain and the sulfate ion at the C-terminal domain. Arrows indicate the direction of phosphoryl group transfer.

 
In the crystal, two monomers from adjacent asymmetric units show an extensive contact interface between the C-terminal domains (Fig. 3A), burying 3790 Å2 of solvent-accessible surface area. Residues involved in interface stabilization derive primarily from regions 434-439, 352-358, and 459-464. The same interaction surface is found in the dimeric PEP-binding domain from T. tengcongensis EI and in the PPDK (14) (Fig. 3B). This finding strongly suggests that in the present structure two symmetry-related molecules represent the biochemically relevant dimer (see below).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The PEP Binding Site—PPDK has a phosphohistidine acceptor domain structurally equivalent to that of EI. In PPDK the phosphoryl group of PEP, as inferred from substrate analogue binding studies (19), is exposed to the solvent, and its strong negative charge is stabilized through interactions with conserved residues (Arg-617, Arg-561, and Asn-768). A magnesium ion binds in a deep region of the pocket and contributes to PEP binding by forming a network of interactions that involves the carboxyl groups of two acidic residues (Glu-745 and Asp-769) and two oxygen atoms of the substrate. The side chain of a presumed catalytic cysteine (Cys-831) is located deeply in the pocket and directly under the substrate. Erni and co-workers (14) found a similar constellation in the dimeric PEP-binding domain of T. tengcongensis EI, although in this case the catalytic site was empty. In the present structure we find a very similar configuration of the active site as compared with either PPDK (Fig. 2) or T. tengcongensis EIC. In our case, however, the active site presents a strong positive density peak that we interpret as a sulfate anion (Fig. 2). Sulfate, which is a common component of crystallization cocktails, has chemical coordination properties very similar to those of a phosphoryl group, and it often replaces it in protein crystals (35). Indeed, early structures of PPDK presented a sulfate ion at a position that was later found to be occupied by the phosphoryl group of the PEP analog (18). In S. carnosus EIC the sulfate ion is stabilized through interactions with Arg-297, Arg-333, and Asn-455, equivalent to Arg-561, Arg-617, and Asn-768 of PPDK, respectively, which stabilize the phosphoryl group of PEP. In addition, the conserved Arg-466 also contacts the sulfate. This residue may be functionally important in EI, in contrast to the situation seen in PPDK, where it adopts a conformation turned away from the PEP analogue. The side chains of Asp-456 and Glu-432 are oriented similar to the Mg2+-coordinating residues Asp-769 and Glu-745 of PPDK, although in our crystals we did not observe Mg2+. Finally Cys-503, which has been demonstrated to be required for acid/base catalysis in EI, occupies a position equivalent to that of Cys-831 in PPDK (36, 37). Taken together this suggests that the active site observed in our crystal structure reflects a catalytically competent configuration, as would be expected for the dimeric state of the enzyme (see below).


Figure 2
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 2.
The C-terminal domain (EIC) and the PEP binding site. Stereo view of the PEP binding site from S. carnosus EI and the PPDK superimposed. Residues involved in binding and catalysis are represented as sticks. Side-chain carbon atoms and the corresponding residue numbers are displayed in green for EI and in gray for PPDK. The sulfate ion present in the EI, and the substrate analogue phosphonopyruvate from the PPDK structure are displayed.

 
The EI Dimer: Implications for Catalysis—Autophosphorylation at His-190 is a prerequisite for the HPr-directed phosphotransferase activity of EI and requires the C-terminal domain, which contains the PEP binding site. This autophosphorylation in turn requires EI dimerization (5, 38-41), which is also mediated by the C-terminal domain (42, 43). In our crystals two EI monomers in adjacent asymmetric units show an extensive interface between the C-terminal domains (Fig. 3A). The amino acids contributing to this interface (Fig. S5, which is published as supplemental data) are highly conserved in the EI family despite being in loop regions. Similarly, the crystal structures of T. tengcongensis EIC and PPDK show dimers in the asymmetric unit, and these dimers are formed through interactions involving the same region of the C-terminal PEP-binding domain (14, 19) (see Fig. 3B). This suggests that our crystal structure indeed represents an EI dimer, with dimers formed by two symmetry-related molecules in the crystal lattice.


Figure 3
View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 3.
The EI dimer. A, ribbon diagram of the EI dimer with a color scheme as in Fig. 1. The sulfate ions at the PEP binding sites are depicted as CPK models. The spatial separation of the P-His and PEP-binding domains is clearly visible. B, ribbon diagram of the dimeric C-terminal region of EI from S. carnosus (left) and T. tengcongensis (right) showing the structural conservation of the dimer interaction region. Double arrow lines have been drawn along the interaction surfaces to help identify the two monomers.

 
The spatial arrangement of the domains in the EI dimer suggests that autophosphorylation occurs on one subunit rather than in trans between different subunits, consistent with biochemical data (37, 41). It also suggests that large conformational changes are required for phosphotransfer to occur between the PEP and the HPr binding sites, with the P-His domain playing a critical part in mediating the communication between the two reaction centers.

For PPDK, it has been suggested that a swiveling mechanism is required for the transfer of the phosphoryl group from PEP to its final acceptor (AMP in this case). In this scenario a rotational movement of the P-His domain would present the phosphorylation target or donor residue to two different active centers located in different portions of the molecule (18). Based on the biochemical and structural similarities of the two proteins the authors have proposed a similar mechanism for PEP synthase and EI (18). A recent structure analysis of PPDK from Zea mays has identified a previously unobserved conformation that appears to be primed for the autophosphorylation reaction, which seems to confirm the proposed swiveling mechanism. In this structure the P-His domain is closer to the PEP-binding domain, and only minor rearrangements would be required for the transfer of the phosphoryl group from PEP to the intermediate acceptor histidine (His-458 in Z. mays) (44). In Fig. 4A we have superimposed the C-terminal domains of Z. mays PPDK and EI and then superimposed the P-His domains, treating the EI derived P-His as an independent rigid body. The result is a plausible model for the conformational transition required for His-190 phosphorylation (Fig. 4A). To attain this conformation from the one observed in our crystal structure, only minor changes in the loop region between the long linker helix and the C-terminal domain would be required. In this scenario, the linker helix would then function like a lever arm to mediate the conformational transition, although (partial) melting of the helix cannot be excluded. In the proposed movement, the HPr-binding domain would clash with the upper ridge of the beta-barrel, which indicates that the HPr and the P-His domain must detach from each other during this part of the reaction; this could be mediated by changes in the linker peptides connecting the P-His- to the HPr-binding domain (Figs. 1 and 4A). Noteworthy, in a recent contribution Roseman and coworkers (11, 12) have analyzed the changes in the association constant and hydrodynamic properties of E. coli EI during PEP and magnesium binding. Their results show a shortening of the molecular diameters and an increase of the sedimentation coefficients upon PEP and magnesium binding for both the monomeric and dimeric forms of EI. In agreement with these results the conformational transition modeled in Fig. 4 results in shorter and more compact structures.

Based on the knowledge now available a tentative model for the conformational changes during the EI catalytic cycle is presented in Fig. 4B. As discussed by Herzberg and coworkers (18), the driving force for the proposed swiveling motions may emerge from changes in charge-charge interactions between domains. On the other hand, the apparent lack of interdomain interactions in the present structure would be compatible with a dynamic transition between alternate conformations of similar energy, where the P-His domain would be constantly swiveling and with conformations becoming progressively restricted upon PEP and magnesium binding, as the sedimentation data seem to indicate (11). The weak electron density of the P-His region in the present structure could again indicate the potential conformational flexibility of this domain.

An open question remains regarding the regulation of the EI activity. How could the dimeric conformation stimulate enzyme activity? As mentioned above, the large distance between the P-His domain and the PEP-binding in the EI dimer seems to preclude trans-phosphorylation of the neighboring monomer as the autophosphorylation mechanism. Trans-phosphorylation is an activation mechanism frequently observed in mammalian tyrosine protein kinases (31, 45). In an alternative scenario, dimerization may help assemble a catalytically active configuration at the PEP binding site of the C-terminal domain. Such a configuration appears to correspond to both the S. carnosus EI and the T. tengcongensis EIC crystal structures. Although a structure of the EI PEP-binding domain in its monomeric conformation would be required to test the latter possibility, a number of observations suggest that the assembly of the catalytic site on the C-terminal domain might actually be a possible mechanism of activation. In Salmonella typhimurium EI, the mutation Gly-346 -> Ser (Gly-357 in EI from S. carnosus) has been reported to inhibit dimerization (43). Glycine 357 is located in the loop between beta3C and {alpha}3aC precisely at the interface between two C-terminal domains. It is involved in a main-chain/main-chain contact with Asp-465 of the opposite subunit, and introduction of a side chain at this position would certainly not be compatible with the interface observed in the structure. Interestingly, the two mutations reduce the Vmax to 4% and 2% that of the wild-type enzyme, respectively. At the same time, the affinity for PEP is decreased by 30-fold in both cases. The exact nature of the link between dimerization and activation will certainly require further investigation. Our structure provides a framework to rationalize and address these issues.


Figure 4
View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 4.
Model of the conformational changes during catalysis. A, modeling the transition to the autophosphorylating conformation. Full-length EI as found in the crystal structure is represented with the different domains colored as in Fig. 1A. The relative position of the P-His-190-binding (cyan) and HPr-binding (red) domains after superposition with the structure of Z. mays PPDK (44) are presented. The arrow indicates the direction of movement that would be required to evolve from the former to the latter conformation. Steric hindrance between the HPr-binding domain and the C-terminal region indicates that the HPr-binding and P-His domains might slightly detach during this part of the reaction. His-190 is represented as CPK model. The sulfate ion at the PEP binding site is shown as CPK. B, a model for the conformational changes associated with the catalytic cycle of EI. The C-terminal domain is represented in green, the P-His domain in blue, the HPr-binding domain in red, and the linker regions in gray. The HPr protein is represented in light green. Yellow dots mark the location of the reaction centers: the PEP binding site, the His-190 of the P-His domain and the acceptor His-15 of HPr. The phosphoryl group that is transferred from PEP to HPr is represented by a red dot. Panel I represents the structure as observed in the crystal (only one monomer is shown). Once PEP is bound to EIC, P-His must approach EIC (IIa). This possible intermediate conformation is suggested by the structure of Z. mays PPDK (44) and would require only small conformational changes at the level of the linker helix that could act like a lever arm. Autophosphorylation at His-190 would require binding of PEP to the C-terminal region and a closer contact between the P-His and the C-terminal domains. This rearrangement would probably require a slight displacement of the HPr-binding domain (IIb). After completion of the autophosphorylation cycle the C-terminal and P-His domains dissociate, and the HPr-binding domain (with HPr bound) adopts a tight conformation with respect to the P-His domain (III). In the scheme, HPr is shown as entering the cycle at the last stage (III), but association during other stages cannot be excluded.

 

   FOOTNOTES
 
The atomic coordinates and structure factors (code 2HRO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S5. Back

1 Both authors contributed equally to this work. Back

2 Present address: Basilea Pharmaceutica AG, Grenzacher Strasse 487, 4005 Basel, Switzerland. Back

3 To whom correspondence should be addressed. Tel.: 49-6221-387-401; Fax: 49-6221-387-519; E-mail: scheffzek{at}embl.de.

4 The abbreviations used are: PTS, PEP-dependent sugar phosphotransferase system; PEP, phosphoenolpyruvate; EI and EII, Enzymes I and II; HPr, heatstable histidine phospho carrier protein; EIN, N-terminal protease-resistant domain; EIC, protease-sensitive C-terminal domain; P-His, phosphohistidine; PPDK, pyruvate phosphate dikinase; CPK, Corey-Pauling-Koltun; MAD, multiple-wavelength anomalous dispersion; DTPA-BMA, diethylene-triaminepentaacetic acid bismethylamide. Back

5 R. Rosenstein and F. Götz, (University of Tübingen, Tübingen, Germany) personal communication. Back

6 W. L. DeLano (2002) DeLano Scientific, San Carlos, CA, www.pymol.org. Back


   ACKNOWLEDGMENTS
 
We thank W. Burmeister (EMBL, Grenoble) for providing the gadolinium compound Gd·DTPA-BMA used for MAD phasing, S. Gemeinhardt for technical assistance, S. Welti for preparation of the schematic displayed in Fig. 4B, R. Rosenstein, Department of Microbial Genetics, University of Tübingen (Germany) for supplying a revised amino acid sequence of EI derived from the genome sequence of S. carnosus, I. D'Angelo and M. Hothorn for discussion, J. Wray and A. Parret for critical comments on the manuscript, and the beamline scientists and particularly H. Belrhali for technical support during MAD data collection at BM14CRG.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Postma, P. W., Lengeler, J. W., and Jacobson, G. R. (1993) Microbiol. Rev. 57, 543-594[Abstract/Free Full Text]
  2. Meadow, N. D., Fox, D. K., and Roseman, S. (1990) Annu. Rev. Biochem. 59, 497-542[CrossRef][Medline] [Order article via Infotrieve]
  3. Kundig, W., Ghosh, S., and Roseman, S. (1964) Proc. Natl. Acad. Sci. U. S. A. 52, 1067-1074[Free Full Text]
  4. Ginsburg, A., and Peterkofsky, A. (2002) Arch. Biochem. Biophys. 397, 273-278[CrossRef][Medline] [Order article via Infotrieve]
  5. Chauvin, F., Brand, L., and Roseman, S. (1996) Res. Microbiol. 147, 471-479[Medline] [Order article via Infotrieve]
  6. Saier, M. H., Jr., and Reizer, J. (1994) Mol. Microbiol. 13, 755-764[Medline] [Order article via Infotrieve]
  7. Warner, J. B., and Lolkema, J. S. (2003) Microbiol. Mol. Biol. Rev. 67, 475-490[Abstract/Free Full Text]
  8. Saier, M. H., Jr., Chauvaux, S., Deutscher, J., Reizer, J., and Ye, J. J. (1995) Trends Biochem. Sci. 20, 267-271[CrossRef][Medline] [Order article via Infotrieve]
  9. Reizer, J., Reizer, A., Saier, M. H., Jr., and Jacobson, G. R. (1992) Protein Sci. 1, 722-726[Medline] [Order article via Infotrieve]
  10. Weigel, N., Kukuruzinska, M. A., Nakazawa, A., Waygood, E. B., and Roseman, S. (1982) J. Biol. Chem. 257, 14477-14491[Abstract/Free Full Text]
  11. Patel, H. V., Vyas, K. A., Mattoo, R. L., Southworth, M., Perler, F. B., Comb, D., and Roseman, S. (2006) J. Biol. Chem. 281, 17579-17587[Abstract/Free Full Text]
  12. Patel, H. V., Vyas, K. A., Savtchenko, R., and Roseman, S. (2006) J. Biol. Chem. 281, 17570-17578[Abstract/Free Full Text]
  13. Reizer, J., Hoischen, C., Reizer, A., Pham, T. N., and Saier, M. H., Jr. (1993) Protein Sci. 2, 506-521[Medline] [Order article via Infotrieve]
  14. Oberholzer, A. E., Bumann, M., Schneider, P., Bachler, C., Siebold, C., Baumann, U., and Erni, B. (2005) J. Mol. Biol. 346, 521-532[CrossRef][Medline] [Order article via Infotrieve]
  15. Liao, D. I., Silverton, E., Seok, Y. J., Lee, B. R., Peterkofsky, A., and Davies, D. R. (1996) Structure 4, 861-872[Medline] [Order article via Infotrieve]
  16. Garrett, D. S., Seok, Y. J., Liao, D. I., Peterkofsky, A., Gronenborn, A. M., and Clore, G. M. (1997) Biochemistry 36, 2517-2530[CrossRef][Medline] [Order article via Infotrieve]
  17. Garrett, D. S., Seok, Y. J., Peterkofsky, A., Gronenborn, A. M., and Clore, G. M. (1999) Nat. Struct. Biol. 6, 166-173[CrossRef][Medline] [Order article via Infotrieve]
  18. Herzberg, O., Chen, C. C., Kapadia, G., McGuire, M., Carroll, L. J., Noh, S. J., and Dunaway-Mariano, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2652-2657[Abstract/Free Full Text]
  19. Herzberg, O., Chen, C. C., Liu, S., Tempczyk, A., Howard, A., Wei, M., Ye, D., and Dunaway-Mariano, D. (2002) Biochemistry 41, 780-787[CrossRef][Medline] [Order article via Infotrieve]
  20. Chauvin, F., Toptygin, D., Roseman, S., and Brand, L. (1992) Biophys. Chem. 44, 163-173[CrossRef][Medline] [Order article via Infotrieve]
  21. Dimitrova, M. N., Szczepanowski, R. H., Ruvinov, S. B., Peterkofsky, A., and Ginsburg, A. (2002) Biochemistry 41, 906-913[CrossRef][Medline] [Order article via Infotrieve]
  22. Chauvin, F., Brand, L., and Roseman, S. (1994) J. Biol. Chem. 269, 20263-20269[Abstract/Free Full Text]
  23. Kohlbrecher, D., Eisermann, R., and Hengstenberg, W. (1992) J. Bacteriol. 174, 2208-2214[Abstract/Free Full Text]
  24. Girard, E., Stelter, M., Anelli, P. L., Vicat, J., and Kahn, R. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 118-126[CrossRef][Medline] [Order article via Infotrieve]
  25. Kabsch, W. (1993) J. Appl. Crystallogr. 26, 795-800[CrossRef]
  26. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 849-861[CrossRef][Medline] [Order article via Infotrieve]
  27. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
  28. Jones, T. A., and Kjeldgaard, M. (1997) Methods Enzymol. 277, 173-208
  29. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]
  30. Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524[Medline] [Order article via Infotrieve]
  31. Sicheri, F., and Kuriyan, J. (1997) Curr. Opin. Struct. Biol. 7, 777-785[CrossRef][Medline] [Order article via Infotrieve]
  32. LiCalsi, C., Crocenzi, T. S., Freire, E., and Roseman, S. (1991) J. Biol. Chem. 266, 19519-19527[Abstract/Free Full Text]
  33. Lee, B. R., Lecchi, P., Pannell, L., Jaffe, H., and Peterkofsky, A. (1994) Arch. Biochem. Biophys. 312, 121-124[CrossRef][Medline] [Order article via Infotrieve]
  34. Seok, Y. J., Lee, B. R., Zhu, P. P., and Peterkofsky, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 347-351[Abstract/Free Full Text]
  35. Copley, R. R., and Barton, G. J. (1994) J. Mol. Biol. 242, 321-329[Medline] [Order article via Infotrieve]
  36. Garcia-Alles, L. F., Flukiger, K., Hewel, J., Gutknecht, R., Siebold, C., Schurch, S., and Erni, B. (2002) J. Biol. Chem. 277, 6934-6942[Abstract/Free Full Text]
  37. Garcia-Alles, L. F., Alfonso, I., and Erni, B. (2003) Biochemistry 42, 4744-4750[CrossRef][Medline] [Order article via Infotrieve]
  38. Saier, M. H., Jr., Schmidt, M. R., and Lin, P. (1980) J. Biol. Chem. 255, 8579-8584[Free Full Text]
  39. Kukuruzinska, M. A., Turner, B. W., Ackers, G. K., and Roseman, S. (1984) J. Biol. Chem. 259, 11679-11681[Abstract/Free Full Text]
  40. Misset, O., Brouwer, M., and Robillard, G. T. (1980) Biochemistry 19, 883-890[CrossRef][Medline] [Order article via Infotrieve]
  41. Seok, Y. J., Zhu, P. P., Koo, B. M., and Peterkofsky, A. (1998) Biochem. Biophys. Res. Commun. 250, 381-384[CrossRef][Medline] [Order article via Infotrieve]
  42. Zhu, P. P., Szczepanowski, R. H., Nosworthy, N. J., Ginsburg, A., and Peterkofsky, A. (1999) Biochemistry 38, 15470-15479[CrossRef][Medline] [Order article via Infotrieve]
  43. Brokx, S. J., Talbot, J., Georges, F., and Waygood, E. B. (2000) Biochemistry 39, 3624-3635[CrossRef][Medline] [Order article via Infotrieve]
  44. Nakanishi, T., Nakatsu, T., Matsuoka, M., Sakata, K., and Kato, H. (2005) Biochemistry 44, 1136-1144[CrossRef][Medline] [Order article via Infotrieve]
  45. Roskoski, R., Jr. (2005) Biochem. Biophys. Res. Commun. 331, 1-14[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Appl. Environ. Microbiol.Home page
R. Rosenstein, C. Nerz, L. Biswas, A. Resch, G. Raddatz, S. C. Schuster, and F. Gotz
Genome Analysis of the Meat Starter Culture Bacterium Staphylococcus carnosus TM300
Appl. Envir. Microbiol., February 1, 2009; 75(3): 811 - 822.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
J. Deutscher, C. Francke, and P. W. Postma
How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria
Microbiol. Mol. Biol. Rev., December 1, 2006; 70(4): 939 - 1031.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/43/32508    most recent
M513721200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Márquez, JoseA.
Right arrow Articles by Scheffzek, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Márquez, JoseA.
Right arrow Articles by Scheffzek, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement