Crystal Structure of Enzyme I of the Phosphoenolpyruvate Sugar Phosphotransferase System in the Dephosphorylated State*

The bacterial phosphoenolpyruvate (PEP) sugar phosphotransferase system mediates sugar uptake and controls the carbon metabolism in response to carbohydrate availability. Enzyme I (EI), the first component of the phosphotransferase system, consists of an N-terminal protein binding domain (EIN) and a C-terminal PEP binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here we report the 2.4 Å crystal structure of the homodimeric EI from Staphylococcus aureus. EIN consists of the helical hairpin HPr binding subdomain and the phosphorylatable βα phospho-histidine (P-His) domain. EIC folds into an (βα)8 barrel. The dimer interface of EIC buries 1833 Å2 of accessible surface per monomer and contains two Ca2+ binding sites per dimer. The structures of the S. aureus and Escherichia coli EI domains (Teplyakov, A., Lim, K., Zhu, P. P., Kapadia, G., Chen, C. C., Schwartz, J., Howard, A., Reddy, P. T., Peterkofsky, A., and Herzberg, O. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 16218–16223) are very similar. The orientation of the domains relative to each other, however, is different. In the present structure the P-His domain is docked to the HPr binding domain in an orientation appropriate for in-line transfer of the phosphate to the active site histidine of the acceptor HPr. In the E. coli structure the phospho-His of the P-His domain projects into the PEP binding site of EIC. In the S. aureus structure the crystallographic temperature factors are lower for the HPr binding domain in contact with the P-His domain and higher for EIC. In the E. coli structure it is the reverse.

to EIC to the active site His of HPr. The P-His domain has also been designated as a swiveling domain (10,16). EIC is a compact eight-stranded (␤/␣) 8 barrel. It contains the PEP binding site (17,18) and provides the dimerization interface of EI. The PEP binding domain EIC and the P-His subdomain of EIN exhibit 30% sequence identity with the respective domains of the pyruvate phosphate dikinase (PPDK, Swiss-Prot accession no. P22983 and PDB codes 1kbl and 2r82 (16, 19 -22), whereas the four-helix-bundle HPr binding domain of EIN is unrelated to the nucleotide binding domain of PPDK.
In vitro experiments indicated that a phosphoryl group can be transferred to EI not only from PEP but also from ATP and acetyl phosphate through the phosphorylated intermediate of acetate kinase (AcK) and that phosphoryl transfer between AcK and Enzyme I is reversible (23,24).
Equilibrium and rate constants of the EI monomer dimer transition vary strongly with protein and substrate concentrations, pH, temperature, and other in vitro parameters. The K a decreases with decreasing temperature (cold sensitivity) and increasing ionic strength (ionic interactions). K a increases in the presence of Mg 2ϩ and PEP (25,26). The phosphoryl group is transferred from PEP to the P-His domain of the same protein subunit, and intersubunit transfer does not occur (10,27). Nevertheless, only dimeric EI can be autophosphorylated, whereas phosphate is exchanged between HPr and the monomeric EIN domain (28). It has been suggested that EI activity controls overall PTS sugar transport (29) and that EI activity itself is controlled by the monomer-dimer equilibrium. It is not clear yet which physiological signal, for instance which allosteric effector, could shift the monomer-dimer equilibrium in vivo and, thus, regulate EI activity (26) and how the apolar dimerization interface of EIC is protected against denaturation/aggregation, for instance by heterodimerization with an as yet unknown protein. It is noteworthy that the compact C-terminal dimerization domain is thermally and proteolytically unstable in full-length EI of E. coli as well as when expressed separately, whereas the monomeric, highly flexible EIN domain is resistant (30).
Here we present the x-ray structure of EI from Staphylococcus aureus (SaEI, Swiss-Prot accession no. P51183) at 2.4 Å resolution. The structures of the SaEI and the E. coli EI (EcEI) (sub)domains are very similar. Their orientations relative to each other, however, are different. In the E. coli structure the P-His domain is oriented toward EIC thus that the phosphorus of the phosphohistidine and the oxygens of the PEP analog oxalate come to within 3-4 Å distance. In the present structure the P-His domain is oriented in the alternative conformation, namely docked to the HPr binding domain with its active site His perfectly oriented for an in-line transfer of the phosphate to the acceptor HPr.

EXPERIMENTAL PROCEDURES
Expression, Purification, and Crystallization-Plasmids encoding SaEI(H191A/C365S) were constructed as described previously (17). SaEI(H191A/C365S) was expressed in E. coli TH074(⌬ptsI), which carries a deletion of codons 131-258 of ptsI (31). Cells were grown in Luria-Bertani broth at 37°C to an A 550 of 0.8 and induced by 0.1 mM isopropyl-␤-thiogalactoside. After 2.5 h of induction cells were harvested, resuspended in buffer A (20 mM Tris-HCl, pH 8.5, 1 mM EDTA, 2 mM dithio-threitol), and disrupted with a French pressure cell. Insoluble material and membranes were removed by centrifugation (60 min, 150,000 ϫ g). The supernatant was applied on a High Q column (Bio-Rad) and eluted with a linear gradient of 0 -600 mM NaCl. The EI-containing fractions were pooled, dialyzed against buffer A, applied on a Resource Q (Amersham Biosciences) column, and eluted with a NaCl gradient. EI was further purified by gel filtration using a Superdex 200 (Amersham Biosciences) column equilibrated in buffer B (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM dithiothreitol, 20 mM CaCl 2 ). The peak fractions were pooled, dialyzed against buffer C (5 mM Tris-HCl pH 7.5, 2 mM dithiothreitol, 20 mM CaCl 2 ), and concentrated in a Centriprep-50 concentrator to 27 mg of protein/ml. 22.3 l of SaEI was mixed with 7.6 l of buffer C to a final protein concentration of 20 mg/ml, added to 10 l of crystallization buffer containing 0.2 M KCl, 20% (w/v) polyethylene glycol 3350, and preincubated at 32.5°C for 5 min. Crystals were grown at 20°C by vapor diffusion in hanging drops using 4 l of the preincubated solution. Crystals (ϳ200 ϫ 200 ϫ 200-m 3 size) were cryo-protected by the addition of 15% (v/v) 2-methyl-2,4-pentanediol and flash-cooled in a nitrogen stream at 110 K. Diffraction data to 2.4 Å resolution were collected on a Raxi-sIV image plate area detector mounted on a Rigaku RU300 rotating-anode generator operating at 100 mA and 50 kV. Data were integrated and scaled with XDS (32,33). Data collection statistics are reported in Table 1.
Structure Determination-SaEI crystallized in space group P3 2 21with one molecule in the asymmetric unit. The structure was solved by molecular replacement (MR) using the program PHASER (34) employing the EcEI P-His domain residues (2-21, 151-232) (PDB code 2hwg) (10), the EI HPr binding domain residues (36 -144) from S. carnosus (PDB code 2hro) (9), and the EIC from T. tengcongensis (PDB code 2bg5) (12) as search models. An initial model could be built manually into the resulting electron density. The model was completed by iterative cycles of restrained refinement and model building using COOT (35) and PHENIX (36). The final model includes residues 2-571 of 572. 94.9% of the residues are in the most favored, 3.2% are in the allowed, and 1.9% are in the disallowed region of the Ramachandran plot. Refinement statistics are given in Table 1. Figs. were prepared using PYMOL.

RESULTS AND DISCUSSION
Expression and Crystallization-Because the EIs of E. coli and T. tengcongensis did not afford diffraction quality crystals, EIs from seven other species were expressed in E. coli and purified for crystal screening. EI from Bacillus subtilis, Bacillus stearothermophilus, Borrelia burgdorferi, Enterococcus faecalis, Haemophilus influenzae, and S. aureus could be expressed in soluble form. They complemented glucose phosphotransferase activity of E. coli TH074 (ptsI Ϫ ) and displayed from 4 to 7% of E. coli EI phosphotransferase activity in the heterologous in vitro phosphotransferase assay with E. coli PTS protein subunits (37). The recombinant EI domain of the E. coli multidomain protein EI Ntr (ptsP, codons 171-748) formed inclusion bodies, and the EI domain of the Pseudomonas aeruginosa multidomain protein FruB (residues 301-956) was not expressed. After optimization, EI from S. aureus (SaEI) afforded the best crystals diffracting to 4.0 Å resolution (37). The same SaEI with an N-terminal histidine tag did not afford x-ray quality crystals.
To further improve the protein homogeneity of SaEI (judged by symmetry and sharpness of the gel filtration peak), two nonconserved cysteines predicted to be at the protein surface, Cys-365 and Cys-557 of the C-terminal domain, were mutated to Ser and Ala alone and in combination ( Fig. 1). Of eight mutants, the C365S mutation improved crystal quality to a diffraction of 3.5 Å. In addition to Cys-365, the phosphorylatable His-191 was mutated to alanine to avoid sample heterogeneity due to partial EI autophosphorylation. The double mutant EI(H191A/C365S) eventually afforded crystals that diffracted to 2.4 Å resolution.
Structure Determination-Crystals of SaEI(H191A/C365S) belonged to the trigonal space group P3 2 21 (a ϭ b ϭ 98.34, c ϭ 105.11) with one monomer in the asymmetric unit. Only a few crystals diffracting up to 2.4 Å could be obtained, whereas the majority of crystals grown in the same conditions diffracted poorly. The structure was determined by MR. A solution for the EIC domain could be easily determined by PHASER with EIC of T. tengcongensis (PDB code 2bg5) as the search model. The structure of the N-terminal domain, however, could be found neither with the model of the recombinant EIN (PDB code 1zym (11)) nor with EIN of the full-length EI (PDB code 2hwg (10)), probably because of the intrinsic mobility of the P-His subdomain relative to the HPr binding subdomain. An initial MR solution of poor Z-scores (Ͻ5) was obtained when the two subdomains (HPr binding domain of S. carnosus, PDB code 2hro; P-His domain of E. coli, PDB code 2hwg) were used as independent search models. Many runs of MR using differently trimmed search probes followed by evaluation of the resulting models by several rounds of refinement and inspection of annealed omit maps were necessary to identify correct solutions for the P-His and HPr binding subdomains. The correctness of the model was confirmed by calculation of an anomalous difference Fourier map, which showed peaks higher than 4 above the mean for 13 (10 in EIN, 3 in EIC) of 21 sulfur atoms.
Residues 2-571 could be built into the electron density ( Fig.  1). Residues Lys-208, Phe-301, Lys-416, and three short stretches corresponding to residues 151-155 (hinge I of EIN), 304 -308, and 341-356 (in EIC ␤/␣ loops 2 and 3) showed poor density in the 2F o Ϫ F c map. Only the main chain could be traced, whereas the side chains remained invisible. These residues were modeled as alanines for the structure refinement. Contrary to expectation, Cys-365, which was mutated to a serine to improve crystallization, is buried and inaccessible from the surface. Cys-365 is not conserved and is located in a loop far from the EIC-EIN interdomain interface and at a distance of 19 Å from the PEP binding site. In view of this it is unlikely that the C365S mutation has an effect on the domain conformation.
Overall Structure-The SaEI monomer model shows the three (sub)domains (Fig. 2); that is, the N-terminal HPr binding subdomain (green, residues 27-148), the P-His subdomain (yellow, residues 2-23, 150 -255), which transfers the phosphoryl groups from PEP to HPr (11), and the C-terminal PEP binding domain EIC (cyan/blue, residues 260 -571). The HPr binding domain consists of four helices arranged in two hairpins; the P-His domain exhibits an ␣/␤ fold, and the PEP binding domain exhibits a (␤␣) 8 barrel fold. HPr binding and P-His subdomains are linked by two crossovers of the polypeptide backbone (hinge I, residues 24 -37 and 145-156) and together form the EIN domain ( Figs. 1 and 2). The P-His domain is linked with the PEP binding domain through a short aperiodic segment (hinge II, residues 260 -271).
The N-terminal Domain-In the N-terminal domain of SaEI, the P-His and HPr binding subdomains assume the same relative orientation as in the crystal structure of the stand-alone EcEIN fragment (11) and in the NMR structure of the complex between EcEIN and EcHPr (14). NMR dipolar coupling measurements (38) indicated that this conformation remains unchanged irrespective of whether EIN, HPr, or both are phosphorylated or not. It appears that this is a minimal energy conformation of the ternary complex between HPr, HPr binding, and P-His domains. When HPr is docked in silico to the model of SaEI in the orientation given by the NMR solution structure (14,39) (Fig. 3C), the distance between N⑀2 of His-191 and N␦1 of His-15 of HPr can be reduced to 3.7 Å. This is less than the 4.1 Å O-P-O distance of the pentacoordinate phosphate in phosphoglucomutase near the transition state (40). To determine this distance, Ala-191 (mutation to prevent autophosphorylation) was replaced in silico with a histidine, and the rotamer conformation was adjusted such that a hydrogen bond between N␦1 of His-191 and O⑀1 of the invariant Glu-127 was formed (in analogy to similar motifs observed in other PTS proteins (41)(42)(43)). However, the excellent local fit notwithstanding, HPr could not bind to EI in exactly the conformation shown in the model (Fig. 3C) because of a clash with the flexible C-terminal extensions the EIC (␤␣) 8 barrel. This clash can be relieved by a small displacement of EIN and EIC around the hinge II between EIN and EIC (residues 260 -271). NOVEMBER 27, 2009 • VOLUME 284 • NUMBER 48

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As mentioned above, it was found that EcEI was in vitro phosphorylated not only by PEP but also by AcK with either ATP or acetyl phosphate as the phosphoryl donors (23,24). It was assumed that a phospho-AcK intermediate acts as phosphoryl donor toward EcEI. Such a mechanism appears unlikely today, first because AcK catalyzes the direct in-line transfer between ATP and acetate and not a double displacement with a covalent AcK-phosphate intermediate (44). Second, the P-His domain of EI and the substrate binding site of AcK lack any structural complementarity. One possibility to explain the observation is phosphorylation by Ac-P of the surface-accessible active site His-191 of the P-His domain.
Such nonenzymatic phosphorylation of active site histidines by Ac-P was observed with two-component response regulator proteins (45,46).
The C-terminal Domain-The PEP binding domain SaEIC features a (␤␣) 8 barrel with three extensions on the C-terminal side of the barrel. The atomic coordinates of the monomer superimpose well with those of the crystal structure of T. tengcongensis EIC (53% amino acid sequence identity) (12) and E. coli EIC (48% sequence identity, Fig. 1), resulting in a root mean square deviation of 0.95 Å for the paired C ␣ atoms. Two monomers from adjacent asymmetric units form the physiological dimer (Fig. 2, A and B). The contact interface between the Also indicated are the linker helix (ϳϳ), the mutated residues H191A and C365S, the active site Cys-502 of the PEP binding site, the three Ca 2ϩ binding residues of the PEP binding domain, and those residues (*) that are disordered in the x-ray structure.
two C-terminal domains buries 1833 Å 2 of solvent-accessible surface per monomer. Although the SaEIC and T. tengcongensis EIC protomers superimpose well, the protomer orientation in the dimer differs by a 7°rotation around the normal to the crystallographic dimer axis.
An anomalous difference Fourier map showed two peaks above 5 over mean within the dimer interface of SaEIC (Fig. 2C). We assigned the difference Fourier peaks to Ca 2ϩ ions because an anomalous signal at the CuK ␣ x-ray emission wavelength is expected of Ca 2ϩ only but not of any other positively charged ion of the crystallization buffer. 20 mM Ca 2ϩ was present because this concentration was beneficial for the homogeneity of EI during purification.
Ca 2ϩ is located in an electronrich cation hole at the edge of the dimer interface where it is coordinated by the side chain oxygens of Thr-397 and Asn-399 from one and Gln-477 of the second subunit (Fig.  2C). The three residues are conserved (S/T, N/Q/E, Q) but are not invariant in homologous EIs. It was shown that Mg 2ϩ alone and Mg 2ϩ plus PEP together shift the monomer-dimer equilibrium toward the dimeric form and stabilize EcEIC by 7 and 14°C, respectively, against thermal unfolding. The stoichiometry of Mg 2ϩ binding was estimated to be two Mg 2ϩ per monomer with K d values of 0.39 and 5.8 mM (25). The high affinity Mg 2ϩ occupies the PEP binding site of EcEIC, where it coordinates two oxygen atoms of the inhibitor oxalate and one oxygen of the His-189 phosphoryl group on the P-His domain (10). The low affinity Mg 2ϩ might complex to the interfacial site, which has been identified by the Ca 2ϩ . Ca 2ϩ improved purification and crystallization. Mg 2ϩ may affect other stability parameters by the same mechanism.   A and B, shown are crystallographic temperature factors. A, the P-His domain is bound to the HPr-binding domain, and as a consequence, the HPr-binding domain has small temperature factors (blue), and the P EP binding domain has large temperature factors (red). B, the P-His domain is bound to the PEP binding domain, and the temperature factors are small for the latter and large for the HP binding domain. C and D, semitransparent surface representation of EI to which HPr (magenta) is docked to the HPr binding domain (green) in the orientation, given by the NMR solution structure (PDB code 3eza) (14,39). C, His-191 (yellow) of the P-His domain projects toward His-15 (red) of HPr. D, His-191 projects toward the inhibitory oxalate (yellow) of the PEP binding domain (10). No correction was made for the steric clash between the EIC domain (cyan) and the in silico-docked HPr (magenta). NOVEMBER 27, 2009 • VOLUME 284 • NUMBER 48

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Conformation of the EI Dimer-The P-His domain is in contact with the HPr binding domain of EIN (Fig. 2, A and B). If HPr is in silico-docked to the HPr binding domain (Fig. 3C) in the orientation given by the NMR solution structure of the EIN-HPr complex (14,39), then the ternary complex between the P-His domain, the HPr binding domain, and HPr buries ϳ860 Å 2 of accessible surface area. The free energy of this interaction corresponds to an experimental complex dissociation constant of 5 M (38). For comparison, about 920 Å 2 of solvent-accessible surface are buried between the P-His and the EIC domains when the phosphorylated His-191 projects into the PEP binding site of EIC (Fig. 3, B and D) (10).
A comparison of the crystallographic temperature factors between SaEI and EcEI shows that the factors are low for the domains in contact with the P-His domain and increased in the uncomplexed domains (Fig. 3, A and B). For example, loops ␤␣2 and ␤␣3 of EIC are well ordered when in contact with the phosphorylated P-His domain as in EcEI (Fig. 3B) but display poor electron density without this contact as in SaEI (Fig. 3A). These two loops also participate in the EIC dimer interface. The free energy of the ternary contact (EIC⅐EIC⅐phospho-P-His domain) could account for the stronger protomer association of phospho-EI (26) in comparison with the binary contact (EIC⅐EIC) of dephosphorylated EI. The ␤␣2 and ␤␣3 loops of T. tengcongensis EIC are well defined even in the absence of a P-His domain. They may "freeze" at room temperature, which is 50°C below the physiological value of 75°C (47).
Conformation Changes-The three x-ray structures that have been solved so far show EI in three clearly different conformations. In SaEI the P-His domain and the HPr binding domain are orientated such that the phosphate can be transferred from His-191 to the active site His-15 of HPr (Fig. 4A, conformation I). In EcEI the P-His domain projects into the PEP binding site of EIC (Fig. 4B, conformation II) and is aligned to accept the phosphate from PEP (10). In S. carnosus EI (9) the three domains are by and large dissociated.
The E. coli and S. aureus models represent two alternative and functionally relevant conformations (I and II). Assuming that the shortest path between conformations I and II also is the energetically most favorable one, the transition can be decomposed into two small rigid body motions (Fig. 4, A and B). First, the P-His domain disengages from the PEP binding site. This step involves an anticlockwise rotation of 45°around hinge II (residues 260 -271) between the P-His and the EIC domain (Fig. 4B). In the second step the HPr binding domain, which was displaced away from EIC in conformation II, snaps back toward the P-His domain to afford conformation I (Fig. 4A). Snapping involves a 60°twist and a gentle bending of the hinge I (residues 24 -37 and 145-156) between the P-His and the HPr binding domains ( Figs. 1 and 4, A and B). It is noteworthy that the ␣␤ core of the P-His domain and the conspicuous C-terminal "linker helix" (residues 235-259, Figs. 1 and 4) move as a single rigid body. The short turn (residues 232-234) between the ␣␤ core and the linker helix does not function as a flexible hinge.
EI shares two homologous domains with the PPDK, namely the PEP binding (EIC) and the P-His domains (16). The HPr binding domain of EIN and the nucleotide binding domain of PPDK, however, have completely different folds and different locations relative to the PEP binding domain. PPDK is shown in Fig. 4, C and D, in an orientation such that the (␤␣) 8 cores of the PEP binding domains (cyan) of PPDK and EI are aligned. The HPr binding domain (green) with docked HPr (magenta) of EI is located at five o'clock above the picture plane (Fig. 4A), and the nucleotide binding domain of PPDK (green) is located at two o'clock behind the picture plane (Fig. 4C).
In EI the PEP binding domain and the HPr binding domain move relative to each other such that the distance between the phosphoryl donor PEP and the acceptor His-15 of HPr  (14,39). Conformation II, shown is EI with the P-His domain (yellow) projecting into the PEP binding site of EIC (cyan). X, hypothetical intermediate; the P-His and EIC domains have dissociated by a rotation around hinge II (red) that is located at the C-terminal end of the linker helix (schematic). In this intermediate HPr is not yet associated with the P-His domain. Conformation I, the HPr binding domain has snapped back to the P-His domain by a rotation and a bending motion of hinge I (red) located between the P-His domain and the HPr binding domain. PPDK, Conformation II, the P-His domain (yellow) projects into the PEP binding site (cyan). Conformation I, the P-His domain has swung to the nucleotide binding domain (NBD, green) by a rotation (swiveling motion) around the hinge (red) located at the N-terminal end of the linker helix (schematic). Only one protomer of EI and PPDK is shown.
varies between 17 Å for SaEI (conformation I, P-His domain projects toward His15 of HPr) and 35 Å for EcEI (conformation II, P-His domain projects into the PEP binding site) (Fig.  4, A and C). The P-His domain (yellow) first rotates around hinge II, and then the acceptor (complex of HPr binding domain and HPr) moves around hinge I toward the P-His domain (compare Fig. 4, B with A). The linker helix remains tightly associated with the ␤/␣ core of the P-His domain (yellow) of EI.
In PPDK, the PEP binding and nucleotide binding domains are coupled, and the 45 Å distance between the phosphoryl donor PEP and the acceptor AMP⅐PP does not change (Fig. 4, C  and D). Only the P-His domain moves from the donor to the acceptor. The conformational changes of PPDK are described in detail by Lim et al. (22). The P-His domain of PPDK first disengages from the PEP binding site and then rotates anticlockwise around the hinge (red) (Fig. 4, from D to C). The PPDK hinge is formed by two antiparallel peptide segments. The first connects the nucleotide binding domain with the P-His domain. The second connects the P-His domain with the N terminus of the linker helix (Fig. 4, C and D). The linker helix of PPDK remains tightly associated with the (␤/␣) 8 core of the PEP binding domain (cyan) and not with the P-His domain as in EI. Thus, only domain folds are conserved between EI and PPDK but not the structural elements that govern their dynamic interaction.
In summary, modest rigid body motions of the P-His domain relative to the EIC PEP binding domain and of the HPr binding relative to the P-His domain suffice to transfer phosphate from PEP to HPr. Compared with the compact structures of EcEI and SaEI, the three (sub)domains of S. carnosus EI (83% sequence identity with S. aureus) are stretched over a length of 110 Å (9). The P-His domain shows weak electron density and does not contact the PEP and HPr binding domains. The open structure may represent one of many short-lived conformers as postulated in Patel et al. (26), because there is no evidence that the shortest reaction pathway (outlined in Fig. 4) also is the energetically most favorable one. Alternatively this elongated conformation may be caused by crystal lattice contacts.