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Originally published In Press as doi:10.1074/jbc.M605886200 on August 12, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31544-31552, October 20, 2006
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Structures of R- and T-state Escherichia coli Aspartokinase III

MECHANISMS OF THE ALLOSTERIC TRANSITION AND INHIBITION BY LYSINE*

Masayo Kotaka{ddagger}1, Jingshan Ren{ddagger}1, Michael Lockyer§, Alastair R. Hawkins, and David K. Stammers{ddagger}2

From the {ddagger}Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, §Arrow Therapeutics Ltd., Trinity Street, Borough, London SE1 1DA, and Institute of Cell and Molecular Biosciences, Catherine Cookson Building, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom

Received for publication, June 20, 2006 , and in revised form, July 26, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Aspartokinase III (AKIII) from Escherichia coli catalyzes an initial commitment step of the aspartate pathway, giving biosynthesis of certain amino acids including lysine. We report crystal structures of AKIII in the inactive T-state with bound feedback allosteric inhibitor lysine and in the R-state with aspartate and ADP. The structures reveal an unusual configuration for the regulatory ACT domains, in which ACT2 is inserted into ACT1 rather than the expected tandem repeat. Comparison of R- and T-state AKIII indicates that binding of lysine to the regulatory ACT1 domain in R-state AKIII instigates a series of changes that release a "latch", the beta15-{alpha}K loop, from the catalytic domain, which in turn undergoes large rotational rearrangements, promoting tetramer formation and completion of the transition to the T-state. Lysine-induced allosteric transition in AKIII involves both destabilizing the R-state and stabilizing the T-state tetramer. Rearrangement of the catalytic domain blocks the ATP-binding site, which is therefore the structural basis for allosteric inhibition of AKIII by lysine.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The aspartate pathway catalyzes the synthesis of lysine, threonine, methionine, and isoleucine and is essential in plants and microorganisms. In addition, the pathway produces important metabolites including diaminopimelic acid, a key component for bacterial cell wall cross-linking, and dipicolinic acid, important for sporulation in Gram-positive bacteria (1). The absence of the aspartate pathway in animals renders its central enzymes attractive as potential targets for the development of selective antimicrobial drugs. The aspartate pathway has several isofunctional aspartokinases that catalyze the initial commitment step, and these are subject to differential regulation by feedback inhibition and repression at the genetic level via the end product amino acids. In Escherichia coli, isoforms I and II have aspartokinase-homoserine dehydrogenase (AK-HD)3 I and II activities (2). AK-HD I is both inhibited allosterically and repressed by threonine, whereas AK-HD II is repressed by methionine (3-5).

Aspartokinase III (AKIII, EC 2.7.2.4 [EC] ), in contrast, is mono-functional and is allosterically inhibited by lysine (6, 7). AKIII is homodimeric with subunits of 48.5 kDa, and the N-terminal catalytic domain shares low sequence identity with carbamate kinase (CK) and N-acetyl-L-glutamate kinase (NAGK) (8). Such enzymes belong to the amino acid kinase family (Pfam: PF00696) catalyzing phosphoryl transfer from ATP to a carboxylate or carbamate group. NAGK and CK have no regulatory domains, and crystal structures show dimers with an open {alpha}beta{alpha} sandwich fold (8-10). Dimerization of AKIII appears distinctive as it apparently involves the two ACT domains at the C terminus (11). ACT domains originally were postulated as regulatory domains in three proteins (including AKIII itself) from bioinformatics analyses (Pfam: PF01842) (12, 13). Although ACT domains share common properties of binding regulatory amino acids, their positioning within the polypeptide chain and mechanism of regulation varies. The structure of E. coli 3-phosphoglycerate dehydrogenase (3PGDH) (14) shows a single ACT domain with an archetypical beta{alpha}betabeta{alpha}beta topology. Pairs of ACT domains interact to form an eight-stranded beta-sheet with four helices on one side and two serine molecules, the allosteric regulator of 3PGDH buried in the subunit interface. Each serine makes extensive contacts with the ACT domain from both subunits (14, 15). The N-terminal ACT domains of the regulatory subunit of E. coli acetohydroxyacid synthase III form the subunit interface, and mutation studies have led to the proposal that valine binds between subunits in a manner similar to serine in 3PGDH (16). A double ACT-like domain is present in threonine deaminase as a tandem repeat (13, 17).

Studies on the allosteric regulation of AKIII by lysine show cooperativity, with a Hill coefficient near 2.0 (6, 18-21). A change in the oligomeric state of AKIII to a homotetramer on lysine binding has been suggested (18, 22, 23). Mutational analyses of the C-terminal AKIII region indicate this to be the site for lysine binding (24, 25). Until now, crystal structures of AKIII have not been available; a reported crystallization does not apparently yield a structure (26). The lack of AKIII crystal structures has led to attempts at homology modeling of the catalytic domain (11). During the preparation of this manuscript, the crystal structure of T-state Arabidopsis aspartate kinase (AK) co-crystallized with its effectors, lysine and S-adenosylmethionine, was published (27). We report crystal structures of E. coli AKIII in both active (R-state) and inactive (T-state) forms, allowing an understanding of the nature of the allosteric transition and mechanism of inhibition by the heterotrophic effector lysine.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Cloning, Expression, and Purification of AKIII—The gene for AKIII was amplified by PCR and ligated into the expression plasmid pET15b (Novagen) using NdeI/BamHI restriction sites. The resulting plasmid, coding for N-terminal His-tagged AKIII, was transformed into the B834 (DE3) met- strain for expression of SeMet-labeled AKIII using standard protocols (28). Briefly, an overnight culture of transformed cells was inoculated into LB medium supplemented with ampicillin and incubated at 37 °C. The cells were harvested when the A600 reached 0.2 and washed twice in SeMet base medium (Molecular Dimensions). The washed cells were then used to inoculate pre-warmed Athena minimal medium supplemented with nutrient mix (Molecular Dimensions), 40 µg/ml selenomethionine (Arcos), and ampicillin to a final A600 of 0.02. The culture was incubated at 37 °C until the A600 reached 0.5. At this point, the culture was induced with isopropyl 1-thio-beta-D-galactopyranoside, and the expression was carried out at 20 °C overnight. SeMet AKIII was purified by nickel affinity chromatography followed by gel filtration on Sephacryl S-200. At each step in the purification procedure, fractions were analyzed by SDS-PAGE and appropriate fractions pooled. The final yield of purified SeMet-labeled AKIII was approximately 75 mg/liter culture. The purity of SeMet-labeled AKIII determined by SDS-PAGE analysis was >99%, and the percentage of selenomethionine incorporation was determined by liquid chromatography-mass spectrometry as >99%.

For untagged AKIII, the gene was amplified by PCR and ligated into the expression plasmid pET3d (Novagen) using NcoI/BamHI sites. The resulting plasmid was transformed into E. coli BL21(DE3) pLysS for protein expression. Briefly, cells were grown at 37 °C in LB medium with ampicillin and chloramphenicol selection until the A600 reached 0.7. At this point, cells were induced with isopropyl 1-thio-beta-D-galactopyranoside for 5 h. Untagged AKIII was purified by DEAE-Sephacel chromatography (run at pH 7.2, elution with 0-1 M NaCl), ammonium sulfate fractionation (1 M), gel filtration on Sephacryl S-300, and finally fractionation on ceramic hydroxyapatite. At each step in the purification procedures, fractions were pooled after analysis with SDS-PAGE. The final yield of purified native AKIII was ~120 mg/liter culture. The purity of the native AKIII determined by SDS-PAGE analysis was >98%. Prior to crystallization, AKIII samples were buffer-exchanged into 150 mM KCl, 20 mM Tris HCl, pH 7.4, concentrated using Vivaspin centrifugal concentrators (Vivascience) to 25 mg/ml and stored at -80 °C.

Crystallization and Data Collection—Extensive screening of crystallization conditions for AKIII used the robotic system developed at the Oxford Protein Production Facility (29). Crystals in two different orthorhombic space groups, each of which could have two distinct sets of cell dimensions, were obtained (T-state forms 1A and 1B; R-state forms 2A and 2B) (see Tables 1 and 2). Crystals of form 1A with lysine, aspartic acid, and MgATP{gamma}S grew from Hampton PEG/Ion screen 36. Optimization using the sitting drop vapor-diffusion method yielded crystals in 0.2 M Na2 tartrate, 16-18% PEG 3350, 0.1 M Tris, pH 7.0, 2% MPD:ethylene glycol (1:1) at 4 °C. Crystals of form 1B with aspartic acid, MgATP{gamma}S, and lysine were from Hampton Natrix screen 10. Optimized conditions gave crystals in 0.05 M MgSO4, 0.1 M MES, pH 6.0, 5-7% PEG 3350, 5% MPD:ethylene glycol (1:1). Crystal form 2A with aspartic acid and MgADP was obtained from the published conditions by the sitting drop vapor-diffusion method (26) with the best crystals obtained from 0.2 M ammonium nitrate, 0.1 M Tris, pH 8.4, 12% PEG 3350. Form 2B resulted from freezing form 2A in 0.2 M ammonium nitrate, 0.1 M Tris, pH 8.4, 10% PEG 3350, and 20% glycerol.


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  		TABLE 1
Data collection, phasing, and refinement statistics for T-state AKIII

 


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  		TABLE 2
Data collection and refinement statistics for R-state AKIII

 
Data sets 1, 2, and 4 were collected at 100 K, whereas data set 3 was collected at 293 K. For data sets 1 and 2, the crystals were cryoprotected with 30% MPD:ethylene glycol (1:1) in reservoir buffer. Data set 1 for native T-state AKIII was collected at the European Synchrotron Radiation Facility beam-line ID14EH1 to 2.8 Å. Three-wavelength MAD data (data set 2) for T-state AKIII were collected at the European Synchrotron Radiation Facility beamline BM14 to 2.94 Å. Room temperature data from 19 crystals (data set 3) for R-state AKIII were collected in-house using copper K{alpha} radiation from a Rigaku MicroMax-007 generator equipped with a MAR345 image plate. High resolution data (data set 4) for R-state AKIII were collected at the European Synchrotron Radiation Facility beamline ID14EH2 to 2.5 Å. Data were processed with HKL2000 (data set 2) or DENZO (others) and merged using SCALEPACK (30); statistics are shown in Tables 1 and 2.

Structure Solution and Refinement—For the MAD data (data set 2) of T-state AKIII, SHELXD (31) and SOLVE (32) identified a total of 12 selenium sites. Automated model building was with RESOLVE (32), and the rest of the model was built manually with O (33). The structure was refined in CNS (34), using simulated annealing and B-factor refinement. Subsequent work on T-state AKIII structure used data set 1. Phasing and refinement statistics are given in Table 1.

The structure of R-state AKIII (data set 3) was solved by molecular replacement in CNS (34), using the T-state AKIII as the starting model. Cross-rotational and translational searches were performed with the N-terminal domain (residues 3-295) followed by the regulatory region (residues 296-445). The data set 3 partially refined structure was used to solve data set 4 by molecular replacement, which was refined in CNS as above to give the final statistics shown in Table 2. Ramachandran plot analysis using the program PROCHECK (35) showed that 87.7 and 11.5% of protein residues of T-state AKIII were in the most favored and additionally allowed regions, respectively. For R-state AKIII, it showed 88 and 11.3% of protein residues were in the most favored and additionally allowed regions, respectively. Glu-346 was in a disallowed region; nevertheless, the conformation was supported by excellent electron density.

Model Analysis and Identification of Conformational Changes—SHP (36) was used to overlap AKIII R- and T-state conformations. Analysis of domain movements was carried out using DynDom (37).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Structure of Lysine-bound AKIII—Lysine-bound AKIII (T-state) was solved to 3-Å resolution by SeMet MAD phasing and extended to 2.8-Å resolution for the native protein (Table 1). No electron density was observed for ATP{gamma}S, although this was included in the crystallization. The AKIII subunit is organized into an N-terminal catalytic domain (residues 3-291) and a C-terminal regulatory region (residues 300-449) (Fig. 1A). Two lysine molecules are present at the dimer interface, and one aspartate is in the catalytic domain of one subunit and a PO-4 ion in the other. The catalytic domain exhibits a typical amino acid kinase family fold with an eight-stranded, mainly parallel beta-sheet sandwiched by two layers of {alpha}-helices (8), which can be further divided into the N-terminal lobe (N-lobe) (residues 1-214) and the C-terminal lobe (C-lobe) (residues 215-291). One major difference observed between the catalytic domain of AKIII and NAGK is the replacement of the beta-hairpin, which forms a lid over the NAG site with two {alpha}-helices ({alpha}C and {alpha}D) from the insertion of residues 60-106 (Fig. 1A).

For the C-terminal regulatory region, earlier studies suggested that the regulatory region of AKIII consists of two ACT domains in a tandem repeat (11, 13, 38). The structure, however, confirms a more complex arrangement, in which the second ACT domain is inserted within the first via connections in two beta-strands as seen in the Arabidopsis AK (27). ACT1 (residues 308-384) exhibits the fold of a typical ACT domain with an extended 14-residue loop (residues 354-367) between beta15 and {alpha}K. ACT2 is made up of a C-terminal beta{alpha}betabeta{alpha}-fold (residues 386-339) and a beta-strand N-terminal to ACT1, beta12 (residues 300-306), completing the ACT domain architecture (Fig. 1, B and C). Like Arabidopsis AK, two lysine molecules were found at the dimer interface located between the ACT1 domains of the two subunits (Fig. 1C). The S-adenosylmethionine-binding site in Arabidopsis AK was not seen in our T-state AKIII structure. Superimposition of the T-state AKIII dimer with that of Arabidopsis AK gave an r.m.s.d. of 2.3 Å for 522 equivalenced C{alpha}s.

Consistent with reports of lysine-induced AKIII tetramer formation (22, 23), association of two dimers is apparent across a crystallographic 2-fold axis of the T-state AKIII (Fig. 1D). The AKIII tetramer is profoundly different from that previously proposed by analogy with other amino acid kinases (11), which involves a dimer interface between the N-terminal domains. Instead, the strategic positioning of {alpha}C and {alpha}D prevents AKIII tetramer formation by this route, and tetramer contacts are by H-bonding interactions at the loop connecting {alpha}D and {alpha}E across the dimers via OG of Ser-107 to the main chain oxygen of Leu-104' (Fig. 1E).

Structure of Substrate-bound AKIII—The structure of the substrate-bound (R-state) AKIII was initially solved with data from 19 unfrozen crystals to 3.25-Å resolution by molecular replacement using the T-state AKIII as search model. Exhaustive further screening of cryoprotection conditions led to a 2.5-Å data set with large unit cell dimension changes, which was solved using the previous partially refined model (see Table 2). The changes in unit cell dimensions reflected alterations in crystal packing rather than relative domain movements because the two structures overlapped well (r.m.s.d. for 417 equivalenced C{alpha}s of 1.3 Å). Clear electron density was observed for aspartate, ADP, Mg2+, and waters completing the coordination sphere of Mg2+ (Fig. 2A), as seen in other members of the amino acid kinase family (8-10).


Figure 1
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  		FIGURE 1.
A, AKIII dimers in the T- and R-state. One complete subunit of each dimer is gray. The N- and C-lobe of the N-terminal catalytic domain of the left subunit are blue and cyan, respectively, and the C-terminal regulatory domain is yellow. Helices {alpha}C and {alpha}D are green, and the beta15-{alpha}K loop of the regulatory domain is red. B, the topology of the C-terminal regulatory domain. C, dimerization of the C-terminal regulatory domain of T-state AKIII with bound lysine. The color scheme is as in B, with the regulatory domain of the second subunit shown partially transparent. D, tetramer formation of the T-state AKIII. The R- and T-state AKIII are superimposed with the subunits of the R-state dimers in yellow and orange and those of the T-state dimers in cyan and blue, respectively. The arrows indicate the rotation of the catalytic domains to form the tetramer in T-state. E, tetramer contacts of T-state AKIII.

 
The R-state AKIII dimer, located on a crystallographic 2-fold axis, formed interactions via the regulatory region (Fig. 1A). Substantial domain movement occurred on the allosteric transition from T- to R-state, and the catalytic domain moved relative to the regulatory region by a 36.3° rotation and a 0.6-Å translation hence disrupting the tetramer contacts (Fig. 1D). Furthermore, a large shift in the position of the beta15-{alpha}K loop of ACT1 was observed (Fig. 1A), resulting in an interaction with the N-lobe of the catalytic domain. The implications of such conformational changes for the allosteric inhibition mechanism of AKIII are discussed below.

Superimposition of the R-state AKIII with the homology model of the catalytic domain of AKIII (11) gave a 1.9-Å r.m.s.d. for 180 equivalenced C{alpha}s and showed significant discrepancies, e.g. {alpha}C and {alpha}D were not predicted, and key residues such as Arg-198 and Glu-250, where C{alpha}s were displaced by 8.7 and 12.1 Å, respectively.

The Active Site and Catalytic Mechanism—R-state AKIII (Fig. 2A) had aspartate bound in the catalytic domain N-lobe, with the {alpha}-carboxyl H-bonding to OG1 of Thr-45 and NE of Arg-198, whereas the NH+3 group interacted with Glu-119. The beta-carboxyl H-bonded to the main chain NH, the side chain OG of Ser-201, and a conserved water molecule, which also interacted with OG of Ser-39, NZ of Lys-8, and the beta-phosphoryl group of ADP. AKIII does not have the "lid" covering the substrate site as seen in other NAGK family members (8, 10). Aspartate has fewer contacts with AKIII than the substrates in other NAGK family members, explaining the broader AKIII substrate specificity (39).

ADP binds in the catalytic domain C-lobe, although unlike NAGK and CK-like carbamoyl-phosphate synthase structures, no adenine ring stacking interactions are observed (8, 10). Instead, adenine atom N-6 interacts with the oxygen atom of Tyr-227, providing selectivity against other nucleotides. In contrast to the ADP bound in NAGK and CK-like carbamoyl-phosphate synthase (8, 10), the 2' and 3' hydroxyl groups of the ribose ring are shielded from solvent by interactions with Arg-232 and Asp-222. The {alpha}-phosphoryl group forms direct interaction with the protein via the nonbridging oxygen atoms with Lys-257, a residue not conserved in the amino acid kinase family. The beta-phosphoryl group makes multiple connections with the protein via OG1 of Thr-221, the nitrogen atom of Gly-11, NZ of Lys-8, and indirectly to OG of Ser-39 via a water molecule, which also bridges the beta-phosphoryl group and aspartate. There is no equivalent interaction of Lys-217 of NAGK to the beta-phosphoryl group of ADP in AKIII (9). The two phosphoryl groups are complexed with Mg2+ by the nonbridging oxygen atoms. The Mg2+ ion only interacts indirectly with the protein to oxygen atoms of Gly-199, Lys-257, and Val-258 and OD2 of Asp-202 via water molecules.

At the center of the active site are Lys-8 and Asp-202, both proposed to be involved in phosphoryl transfer (8, 9). Lys-8 interacts directly with ADP via the beta-phosphoryl group and indirectly with the side chain of aspartate via a water molecule and is likely to abstract negative charge from the bipyramidal phosphorus transition state of ATP during catalysis (9). Asp-202 helps orientate the catalytic group Lys-8 via interactions of OD1 with Lys-8 and OD2 with two waters coordinating Mg2+, hence explaining the effects on Vmax and Km for aspartate observed for Lys-8 and Asp-202 mutants (11). Cysteine and histidine residues, predicted in the AKIII active site from chemical modification and pH profile studies, are however not present (39).

Allosteric Lysine-binding Site—In the AKIII dimer, two regulatory regions from adjacent subunits interacted to form two eight-stranded anti-parallel beta-sheets almost perpendicular to each other. In the T-state AKIII, two lysine molecules were found 17 Å apart at the dimer interface of ACT1 interacting with both subunits (Fig. 1C). The amino group of the lysine interacted with the oxygen atoms of Met-318 and Ser-321 in one subunit and the oxygen atom of Val-339' and OG of Ser-338' in the other subunit, whereas the carboxylate groups interacted with the nitrogen atoms of Phe-324 and Leu-325 and the nitrogen and oxygen atoms of Val-339' (Fig. 2B). Both carboxylate groups H-bonded with a water molecule, which capped off the lysine-binding site. NZ of the lysine formed a network of interactions with the oxygen atoms of Ser-345, Glu-346, and Asp-340' and the side chain of Asp-340'. Although the overall locations of serine in 3PGDH and lysine in AKIII are similar, surprisingly, the side chain and carboxylate positions of serine are reversed relative to lysine (14). Mutations of T344M, S345L, and T352I all gave partial resistance to lysine inhibition (24). From the structure it is clear that T344M and S345L would cause obstruction to the side chain pocket of the lysine-binding site. The presence of Tyr-420 in the ACT2 domain blocks the binding of lysine to this potential site.


Figure 2
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  		FIGURE 2.
A, final 2Fo - Fc map (contoured at 1{sigma}) of the bound aspartate, ADP, and Mg2+ in R-state AKIII at 2.5-Å resolution showing the completion of the coordination sphere of Mg2+ by waters. B, active site of R-state AKIII shown in stereo. Carbon atoms of ADP and aspartate are shown in yellow, and residues interacting with ADP and aspartate are gray. Potential H-bonds are indicated by dotted lines. C, allosteric inhibitor-binding site. Final 2Fo - Fc map of the bound lysine (contoured at 1{sigma}) in T-state AKIII. Carbon atoms of lysine are drawn in black, and residues interacting with lysine are in gray and cyan for different subunits. Potential H-bonds are indicated by dotted lines.

 
The Allosteric Transition and Mechanism of Lysine Inhibition of AKIII—To date, AKs are the only members of the amino acid kinase family known to contain an allosteric regulatory domain. In addition, E. coli AKIII is the first of the AKs for which structures of both R- and T-states have been determined. Such data allow us to understand details of the allosteric transition and to propose a mechanism of AKIII inhibition by lysine. The binding of lysine at ACTI induced a series of structural changes in AKIII allowing the transmission of a signal to the active site over a distance of ~24 Å (Fig. 3A). The key changes included the unlocking of a latch, thereby allowing the swiveling of the catalytic domains and in turn the formation of the R-state tetramer (Fig. 3A). Finally, an accompanying movement of a loop blocked the ATP-binding site thereby inhibiting AKIII activity. A more detailed description of the various conformational changes observed in the AKIII allosteric transition is given below.


Figure 3
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  		FIGURE 3.
Conformational changes observed in the R- to T-state transition of AKIII. A, schematic diagram depicting the R- to T-state transition of AKIII. ACT1 and ACT2 of the regulatory domain are represented by yellow rectangle and square, respectively, and the N- and C-lobe of the catalytic domain are represented by blue and cyan ovals. The beta15-{alpha}K loop is represented as a long red strip, and {alpha}H at the ATP-binding site is represented by a magenta strip. ATP and lysine are represented by an orange cross and a green hexagon, respectively. The arrows show the relative movement of the domains and loops upon transition to T-state. B, conformational changes in the ACT1 domain. ACT1 of the R- and T-state dimers are superimposed. The subunits of the R-state AKIII are gray and orange, respectively, whereas the subunits of the R-state AKIII are blue and green. For clarity, conformational changes observed are indicated on one subunit only, with the R-state in cyan and T-state in magenta. C, movement of the beta15-{alpha}K loop. The R- and T-state AKIII subunits are superimposed at the regulatory domain and are gray and blue, respectively. The beta15-{alpha}K loop is highlighted in cyan for the R-state and in magenta for the T-state. Potential H-bonds are indicated by dotted lines. D, conformational changes in the catalytic domain. The color scheme is as shown in C; only the C-lobe and central beta-sheet of the N-lobe of the catalytic domain are shown for clarity. The arrow indicates the direction of movement in the R- to T-state transition. E, conformational changes in the ACT2 domain. ACT2 of the R- and T-state dimers are superimposed. The color scheme is as shown in B. F, conformational changes in the active site shown in stereo. The active site region of the AKIII T-state structure is superimposed onto the R-state structure. The arrows indicate the direction of major movements of loops in the R- to T-state transition, which are colored as shown in C.

 
The binding of the lysine NZ at the dimer interface of ACT1 disrupts the interaction of the main chain nitrogen atom of Glu-346 of strand beta14 to the main chain oxygen atom of Asp-340' from beta14' causing relative movement of ACT1 domains from adjoining subunits. Conformational changes are seen in the beta15-{alpha}K and beta13-{alpha}J loops (residues 316-321) (Fig. 3B); the latter closes over the inhibitor and thereby forms the interactions with lysine described above.

Movement of the beta15-{alpha}K loop releases a latch that holds the N-lobe of the catalytic domain in R-state AKIII (Fig. 3C), an interaction that is lost in the T-state AKIII (Fig. 3C). Release of the beta15-{alpha}K loop allows rotation of the catalytic domain by ~36° and transition to the T-state (Fig. 3D). A number of conformational changes at the C-lobe of the catalytic domain accompany this domain rotation including residues 224-226 (beta8a-beta8b-hairpin loop), 242-247 (beta9), 252-270 ({alpha}H and {alpha}I), and 289-290 (beta11) (Fig. 3D).

Additional conformational changes are also seen in ACT2 upon R- to T-state transition. The movement of the ACT2 beta-sheet is also associated with movements of {alpha}L, {alpha}M, and the beta17-{alpha}L loop; the latter two changes result in the closure of the potential ligand-binding site of ACT2 (Fig. 3E). The T-state conformation of AKIII is then stabilized by the tetramer formation described above (Fig. 1D).

Upon transition to the T-state, conformational changes in the catalytic domain of AKIII are evident in the ATP-binding site. The movement of the beta8a-beta8b-hairpin loop causes a 12-Å shift in the position of Arg-232, abolishing its interaction with the ATP ribose hydroxyls. The shift in the position of {alpha}H results in a 6-Å shift in Lys-257, thereby disrupting its interaction with the ATP {alpha}-phosphoryl group. Most importantly, {alpha}H in the T-state obstructs the binding site for the adenine ring of ATP (Fig. 3F). Changes in the N-lobe of the catalytic domain are more minor. Release of the beta15-{alpha}K loop during the R- to T-state transition alters its interaction with the beta7-{alpha}G loop, which moves by 2 Å, hence disrupting H-bonding of Arg-198 with aspartate, although this does not prevent aspartate binding (Fig. 3F). However, loss of this interaction may affect the enzyme turnover rate despite the positions of the two key residues involved in the phosphoryl transfer, Lys-8 and Asp-202, being unaltered in the T-state (Fig. 3F). Therefore, in accordance with the previously observed results (20, 21), the binding of lysine and the subsequent R- to T-state transition of AKIII does have limited effects on aspartate binding. However, the major effects of conformational changes observed in the C-lobe of the catalytic domain appear to prevent the binding of ATP to the active site in the T-state.

Comparison with Other ACT Allosteric Enzymes—A common feature of allostery, signal transmission to a distal location following a triggering event such as ligand binding, can be carried out by many varied mechanisms. Not only is AKIII distinct from well characterized systems such as aspartate transcarbamoylase and chorismate mutase (40) but also from the more closely related ACT domain-containing enzymes, e.g. 3PGDH, phenylalanine hydroxylase, and acetohydroxyacid synthase III. Although binding of the regulatory amino acid at the subunit interface for AKIII and 3PGDH causes conformational changes in the catalytic domain, the structural consequences are different. For 3PGDH, effector binding prevents active site closure without affecting substrate binding (41), whereas for AKIII, binding of the ATP substrate is prevented. Also, changes in oligomeric state are seen in AKIII but not in 3PGDH. For phenylalanine hydroxylase, there is no evidence of effector binding to the ACT domain. Instead, this domain appears to be a module to facilitate allosteric regulation via transmission of finely tuned conformational changes (38, 42, 43). This function is thus similar to ACT2 in AKIII, which has both a structural role in maintaining the dimer interface as well as propagating the effects of lysine binding to the catalytic domain and yet is not involved in the binding of heterotrophic effectors. Acetohydroxyacid synthase III is unusual in that its ACT domain is on the N terminus of a separate regulatory subunit. The effects of valine binding to the ACT domain in this case are communicated to the catalytic subunit through the C-terminal domain of the regulatory subunit (16).

In addition to the information provided on the allosteric mechanism in AKIII, the structure has implications for drug design. The absence of the aspartate pathway in higher organisms makes inhibition of aspartokinases a target for novel antibacterial drugs. The AKIII structure serves as a template for modeling the AK part of the AK-HD I and AK II isoforms, where the conservation of the key residues in the active site of the three aspartokinases in E. coli, i.e. Lys-8, Thr-45, Glu-119, Arg-198, Asp-202, Asp-222, and Arg-232 of AKIII implies the possibility of designing a specific inhibitor of all three AK isoenzymes.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2J0W and 2J0X) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* Financial support was received from Arrow Therapeutics and the United Kingdom Medical Research Council. 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

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed. Tel.: 44-1865-287-565; Fax: 44-1865-287-547; E-mail: daves{at}strubi.ox.ac.uk.

3 The abbreviations used are: AK-HD, aspartokinase-homoserine dehydrogenase; 3PGDH, 3-phosphoglycerate dehydrogenase; AK, aspartate kinase; AKIII, aspartokinase III; CK, carbamate kinase; MES, 4-morpholineethanesulfonic acid; C-lobe, C-terminal lobe; PEG, polyethylene glycol; MPD, 2-methyl-2,4-pentanediol; N-lobe, N-terminal lobe; NAGK, N-acetyl-L-glutamate kinase; R-state, relaxed state; T-state, tense state; r.m.s.d., root mean square deviation; ATP{gamma}S, adenosine 5'-O-(thiotriphosphate); MAD, multiwavelength anomalous diffraction. Back


   ACKNOWLEDGMENTS
 
We thank the staff at beamlines BM14, ID14EH1, and ID14EH2 at the European Synchrotron Radiation Facility, Grenoble for help with data collection.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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