Crystal Structures of a New Class of Allosteric Effectors Complexed to Tryptophan Synthase*

Tryptophan synthase is a bifunctional α2β2 complex catalyzing the last two steps of l-tryptophan biosynthesis. The natural substrates of the α-subunit indole- 3-glycerolphosphate and glyceraldehyde-3-phosphate, and the substrate analogs indole-3-propanolphosphate anddl-α-glycerol-3-phosphate are allosteric effectors of the β-subunit activity. It has been shown recently, that the indole-3-acetyl amino acids indole-3-acetylglycine and indole-3-acetyl-l-aspartic acid are both α-subunit inhibitors and β-subunit allosteric effectors, whereas indole-3-acetyl-l-valine is only an α-subunit inhibitor (Marabotti, A., Cozzini, P., and Mozzarelli, A. (2000) Biochim. Biophys. Acta 1476, 287–299). The crystal structures of tryptophan synthase complexed with indole-3-acetylglycine and indole-3-acetyl-l-aspartic acid show that both ligands bind to the active site such that the carboxylate moiety is positioned similarly as the phosphate group of the natural substrates. As a consequence, the residues of the α-active site that interact with the ligands are the same as observed in the indole 3-glycerolphosphate-enzyme complex. Ligand binding leads to closure of loop αL6 of the α-subunit, a key structural element of intersubunit communication. This is in keeping with the allosteric role played by these compounds. The structure of the enzyme complex with indole-3-acetyl-l-valine is quite different. Due to the hydrophobic lateral chain, this molecule adopts a new orientation in the α-active site. In this case, closure of loop αL6 is no longer observed, in agreement with its functioning only as an inhibitor of the α-subunit reaction.

cavity of hemoglobin and decreases oxygen affinity by stabilizing the T state (1). In monomeric proteins noncompetitive inhibitors can be regarded as allosteric effectors and their action takes place via stabilization of tertiary conformations. The discovery of new allosteric effectors and the elucidation of the molecular basis of their action is relevant for the understanding of the plasticity of the protein matrix and the influence of cellular components for the control of metabolic pathways.
Tryptophan synthase (TRPS) 1 (EC 4.2.1.20) is a tetrameric enzyme, consisting of two ␣and two ␤-subunits arranged in a linear ␣␤␤␣ mode (2), that catalyzes the last two steps of the biosynthesis of L-tryptophan in a concerted way. It is known that ligand-induced intersubunit signals keep the catalytic activities of the ␣and ␤-active sites in phase (3)(4)(5)(6)(7)(8). For recent reviews of the TRPS allosteric regulation, see Refs. 9 -11. In particular, the ␣-subunit substrates indole 3-glycerolphosphate (IGP) and glyceraldehyde phosphate and the substrate analogs indole-3-propanole phosphate (IPP) and glycerol 3-phosphate (GP) are able to influence the activity of the ␤-subunit (3)(4)(5)(6). Detailed crystallographic studies of the wild-type enzyme and its mutants, in the absence and presence of ␣-subunit ligands were carried out (2,(12)(13)(14)(15)(16). The indole moiety is located in a hydrophobic cleft with the nitrogen atom N 1 of the indole ring interacting with ␣Asp 60 , a residue participating in ␣-subunit catalysis (17). No ionizable residues are present in the subsite occupied by the phosphate moiety of the ligands. However, the negative charge of the phosphate group is partially neutralized by the positive dipole moment created by helix ␣H8Ј. (The secondary structure elements are used as defined by Schneider et al. (14)). The phosphate oxygen atoms form hydrogen bonds with several amino acids. In particular, the nitrogen amide protons of ␣Gly 234 and ␣Ser 235 interact with the phosphate oxygen atoms. Serine, ␣Ser 235 , is localized at the end of ␣-helix ␣H8Ј and was proposed to be involved in intersubunit regulation (18). Another important amino acid involved in the interaction with the phosphate group is ␣Gly 184 , located in loop ␣L6. This structural element plays a crucial role for the propagation of the conformational events triggered by ␣-subunit ligands toward the ␤-active site (14). Loop ␣L6 is highly mobile, as 1 The abbreviations used are: TRPS, tryptophan synthase; IGP, indole 3-glycerolphosphate; IPP; indole 3-propanolphosphate; GP, DL-␣glycerol 3-phosphate; IAAA, indole-3-acetyl-L-amino acid; IAD, indole-3-acetyl-L-aspartate; IAG, indole-3-acetylglycine; IAV, indole-3-acetyl-L-valine; TRPS IPP , tryptophan synthase IPP complex; TRPS IGP ,  tryptophan synthase IGP complex; TRPS IAD , tryptophan synthase IAD  complex; TRPS IAG , tryptophan synthase IAG complex; TRPS IAV , tryptophan synthase IAV complex; ␣L6, loop 6 of the ␣-subunit of tryptophan synthase (14); ␣L2, loop 2 of the ␣-subunit of tryptophan synthase (14); ␤H6, helix 6 of the ␤-subunit of tryptophan synthase (14); COMM domain, domain (␤Gly 102 -␤Gly 189 ) for the communication between ␣and ␤-subunit (14); Bicine, N,N-bis(2-hydroxyethyl)glycine; r.m.s., root mean square. evidenced by the lack of electron density in native enzyme structures (2, 14 -16) and by limited proteolysis and protein engineering studies (19 -21), and switches between an open and closed conformation (14). In the TRPS complexes with IPP or GP ␣L6 is closed, and there is a hydrogen bond between ␣Thr 183 located in ␣L6 and the catalytic residue ␣Asp 60 . The closed state of the ␣-subunit is communicated to the ␤-subunit via several interactions with the COMM domain, a rigid but moveable domain (␤Gly 102 -␤Gly 189 ) of the ␤-subunit (14). A critical element of the intersubunit interface is the hydrogen bond between ␣Gly 181 and ␤Ser 178 , as functionally demonstrated (22) and structurally characterized in the accompanying paper (23).
In the course of an investigation aimed at the development of new inhibitors of the ␣-subunit of tryptophan synthase (24), eventually useful as herbicides (15,25), a new class of ␣-subunit ligands of tryptophan synthase was discovered, the indole-3-acetyl amino acids (IAAA). These molecules are also physiologically interesting, since several indole-3-acetyl amino acid conjugates are storage forms for the auxin indole-3-acetic acid (26), a very important plant hormone (27). In particular, it was found that some IAAA, as indole-3-acetylglycine (IAG) and indole-3-acetyl-L-aspartate (IAD), are potent allosteric effectors of tryptophan synthase, whereas indole-3-acetyl-L-valine (IAV) and indole-3-acetyl-L-alanine are inhibitors of the ␣-subunit activity (24). In the present work, the three-dimensional structures of the complexes between tryptophan synthase and IAG, IAD, and IAV, respectively, are presented. The investigation of the interaction of these derivatives with the ␣-subunit of tryptophan synthase is relevant both for the understanding of the residues that are involved in the intersubunit communication and the design of more potent allosteric effectors and herbicides.

MATERIALS AND METHODS
Crystallization and Substrate-Complex Preparation-IAG, IAD, and IAV were purchased from Sigma and used without further purification. TRPS was purified as described previously (28). The protein was stored at a concentration of 10 mg/ml in a solution containing 50 mM Na-Bicine, pH 7.8, 10 mM Na-EDTA, 1 mM dithioerythritol, and 20 M pyridoxal phosphate. Crystals were grown in the dark at room temperature within 1 week using the hanging drop method by mixing equal volumes (2-4 l) of protein and reservoir solutions. The latter contained 9 -12% polyethylene glycol 8000, 1.5 mM spermine, 1 mM EDTA, 50 mM Na-Bicine buffer, pH 7.8. The IAD, IAG, and IAV complexes were generated by soaking TRPS crystals for 10 -30 min in cryo-protectant solution (100 mM Na-Bicine buffer, pH 7.8, 15% (w/v) polyethylene glycol 8000, and 20% (v/v) glycerol) containing 10 -25 mM IAD, IAG, or IAV, respectively before flash-cooling in liquid nitrogen. To increase the IAV solubility 15% (v/v) Me 2 SO were added to the soak solution.
X-ray Data Collection and Refinement-Diffraction data of the TRP-S IAAA complexes were collected at beamlines BW7B at EMBL c/o DESY, Hamburg (IAD, IAG) and at ID14 -1 at the European Synchrotron Radiation Facility, Grenoble, France (IAV) using wavelengths of 0.842 or 0.934 Å, and a MAR 345 image plate or MAR CCD detector, respectively. The crystals were kept at 100 K during measurements. The diffraction data were processed with XDS and scaled with XSCALE (29). Data statistics are given in Table I. Refinement was started to 2.5-Å resolution with CNS 1.0 (30) by performing rigid-body and simulated-annealing steps. The coordinates of the wild-type TRPS IPP structure (Protein Database accession code 1Q0P (16)) were used as a starting model, omitting the coordinates of loops ␣L2 and ␣L6, IPP, the cofactor pyridoxal phosphate, and all water molecules. In each case the final model was obtained by cyclic rounds of manual model building with the program O (31) and Maximum-Likelihood refinement with the program REFMAC (32) using all reflections to maximum resolution. Water molecules were incorporated by "ARP" (33) using the automatic  Table II). The figure was prepared using "BOBSCRIPT" (40), "MOL-SCRIPT" (41), and "RASTER3D" (42,43). A, TRPS IAD structure. B, TRPS IAG structure. The second conformation of ␣Glu 49 is shown in orange. C, TRPS IAV structure.
FIG. 2. Stereo plot of the structure superposition of TRPS IPP , TRPS IAD , and TRPS IAV . The C ␣ -atom trace is shown for TRPS IPP ; the ␣-subunit is colored in gray, loops ␣L2 and ␣L6 in cyan, and the ␤-subunit in pink. The IPP, IAD, and IAV ligand C-atoms are colored in yellow, green, or orange, respectively. Nitrogen atoms are colored in blue, oxygen atoms are colored in red, and phosphate atoms are colored in magenta. The figure was prepared using MOLSCRIPT (41) and RASTER3D (42). not to remove this part from the final model. Also residues ␣Phe 54 , ␤Lys 167 , ␤Arg 175 , and ␤Tyr 279 are modeled as alanine. The higher flexibility of this region is reflected in significantly higher temperature factors. In case of the TRPS IAD structure a number of amino acids have two conformations. Data and refinement statistics are given in Table I. The coordinates and structure factor amplitudes have been deposited with the Protein Database (34) (accession codes 1K3U, 1K7E, 1K7F).
Structure Superposition-To investigate the influence of the different ␣-active site ligands on the allosteric communication, we superimposed the TRPS IAAA structures with each other and also with the wild-type TRPS IPP (16) structure. The superposition was done with the program O (31) using all common C ␣ -atom coordinates of both structures except the C ␣ atoms belonging to the COMM domain (␤Gly 102 -␤Gly 189 ). The resulting r.m.s. deviations were calculated with the program BRAGI (35).

RESULTS AND DISCUSSION
The IAAAs bind at the IGP substrate binding site (13,16) in the TRPS ␣-subunit (Fig. 1, A-C). No indication for a different binding site is found in the electron density of any of the three IAAA ligand complexes. Also, the overall TRPS topology is not effected by the binding of the indole-3-acetyl amino acids compounds. Fig. 1 shows the electron density at the ␣-active site for the IAD, IAG, and IAV complexes. All three ligands could be placed easily in the electron density. The distances of the hydrogen bonds between the IAAA ligands and the protein are shown in Table II. A superposition of the new TRPS IAAA structures with the TRPS IPP structure was calculated, excluding the C ␣ -atoms of loop ␣L2, ␣L6, and the COMM domain (Fig. 2), and, in the case of the TRPS IAV structure, also the C ␣ -atoms of the missing loop ␤L8 were not used in structure superposition calculation either. We chose the TRPS IPP structure for the comparison because the IPP ligand, which binds with high affinity to the TRPS ␣-subunit (36), served as a lead compound for the ␣-subunit effector/inhibitor development (24). The result of this superposition is shown in Figs. 2 and 3. There are no larger structural differences between the IAG and IAD TRPS complexes (Fig. 2). In particular, both TRPS IAAA structures adopt, apart from some surface loops, the same subunit conformations observed in the TRPS IPP structure, as shown by the superposition between the TRPS IAD and TRPS IPP structure (Fig. 3). This is also valid for the loop ␣L6, which is in the closed conformation in all three structures.
A common feature for all three IAAA complexes is the substitution of the IPP/IGP phosphate group on the N-terminal side of ␣-helix ␣H8Ј with a carboxylate group. In each case the two carboxylate oxygen atoms mimic two IPP phosphate oxygen atoms, but in different combinations. In the IAG complex the carboxylate oxygen atoms replace the OP1 and OP2 phosphate oxygen atoms, in the IAV complex the OP2 and OP3 oxygen atoms, and in the IAD complex the OP1 and OP3 oxygen atoms. In the case of the IAD ligand the additional side chain carboxylate group also replaces one phosphate oxygen and a water molecule that is found near the phosphate group in the TRPS IPP/IGP structures. The different IAAA ligand binding modes indicate less specific binding of the carboxylate compared with the IPP/IGP phosphate group. This is in line with the finding that the binding affinity of IAD and IAG is about 50-fold lower than of phosphate-containing ligands (24). Based on the common orientation of the negatively charged carboxylate group at the N-terminal side of ␣-helix ␣H8Ј, the indole ring of the IAG and IAD ligands adopts a similar orientation as in the TRPS IPP/IGP structures, although the acetyl side chain has a different orientation than the propyl side chain of IPP. As a consequence, both IAAA compounds have a shorter hydrogen bond between the indole nitrogen atom and the carboxylate group of aspartate, ␣Asp 60 , and the acetyl oxygen atom of the IAG and IAD ligands points in the same direction as the IGP OH3Ј hydroxyl group. Since IAG lacks the IAD aspartate side chain, the IAG ligand has less negative charges and is shifted by approximately 0.6 Å from the IPP/IGP phosphate binding loop toward the catalytically important glutamate ␣Glu 49 (8,37,38). Interestingly, in the TRPS IAG complex ␣Glu 49 has two conformations, an "inactive" one interacting with ␣Tyr 173 (2.6 Å), and an "active" one that forms a 2.7-Å hydrogen bond with the acetyl oxygen of IAG (see Table II and Fig. 1). This nomenclature reflects the finding that an active site conformation of ␣Glu 49 was also observed in case of the wild-type TRPS IGP (16) and the mutant ␣D60N IGP (39), ␤S178P GP (23), and ␤S178P IAG (23) complexes. In the case of the IGP and GP molecules the ␣-ligand serves as a proton donor, whereas the hydrogen bond between the IAG acetyl oxygen and the ␣Glu 49 carboxylate group implies that the ␣Glu 49 carboxylate group is protonated. This has a direct consequence for the mechanism of the ␣-reaction: the cleavage of the C3-C3Ј carbon-carbon bond in IGP (17,39) is activated by tautomerization of the indole ring to yield the indolenine tautomer. This tautomerization is probably facilitated by two basic groups B 1 H and B 2 , by "push-pull" general acid-base catalysis. B 1 H protonates the indole ring at C3, while B 2 abstracts the proton of the N1 nitrogen atom of the indole ring. The aldol cleavage is catalyzed by B 3 , which abstracts a proton from the 3Ј-hydroxyl to yield indole and glyceraldehyde 3-phosphate. Since the TRPS IAG structure implies that the ␣Glu 49 carboxylate group is protonated, we have for the first time experimental evidence that ␣Glu 49 may act as the putative residues B 1 -H and B 3 of the ␣-reaction.
The binding mode of the IAV compound is very different from that of the IAG and IAD ligands, since the hydrophobic valine side chain is bound in a hydrophobic pocket near the ␣-active site, formed by the ␣Phe 22 , ␣Ile 64 , and ␣Leu 100 side chains. The re-orientation of the IAV molecule leads to a rotation of the indole ring by nearly 90°around the C 7 -C 8 carbon bond. In this conformation the indole nitrogen forms only a weak hydrogen bond (3.1 Å) to aspartate, ␣Asp 60 , and a new hydrogen bond to the carbonyl oxygen of alanine, ␣Ala 59 (2.6 Å). As a consequence, the acetyl carbon atoms point toward the side chain of phenylalanine, ␣Phe 212 , which, therefore, is also shifted. As a direct consequence of this different binding mode and side chain movement, loop ␣L6 is not closed in the TRPS IAV struc-ture. Apart form the side chain shift of phenylalanine, ␣Phe 212 , the side chain of threonine, ␣Thr 183 , would clash with the re-oriented IAV indole ring in a closed loop ␣L6 conformation. This missing loop closure is the reason for the, compared with the TRPS IAG and TRPS IAD structures, different COMM domain conformation, as indicated by the large r.m.s. deviation values for several parts of the COMM domain (Fig. 3). This second COMM domain conformation is in good agreement (r.m.s. deviation plot not shown) with the observed open conformation in the wild-type TRPS IGP structure (16). Interestingly, in contrast to the TRPS IAG and TRPS IAD , but in agreement with the TRP-S IGP structures, there is no electron density for a sodium ion, although the soaking conditions (especially the pH) were the same for all three IAAA complexes. This observation might support our belief that the enzyme loses the sodium ion upon opening of the COMM domain (16). The reason for the reduced sodium affinity of the open COMM domain conformation might be the higher flexibility of the sodium binding loop (␤Asp 305 -␤Pro 307 ) as indicated by higher temperature factors in both sodium-free TRPS structures (data not shown), which is a consequence of the loss of side chain interactions of these loop region with COMM domain residues (16). CONCLUSIONS The analysis of the structural features of the TRPS IAAA complexes allows to better define the mechanism for intersubunit regulation mediated by ␣-subunit ligands, and, in particular, the residues that participate in the communication of allosteric signals. IAG and IAD share the IPP/IGP binding mode at the ␣-subunit, in which the terminal carboxylate moiety replaces the phosphate group, the acetyl oxygen atom points in the same direction as the IGP OH3Ј hydroxyl group, the indole ring interacts with ␣Asp 60 , and loop ␣L6 is in the closed state. The hydrogen bond between the acetyl oxygen atom of IAG and the carboxylate oxygen atom of ␣Glu 49 indicates that the latter is protonated. This has important implications for the mechanism of the ␣-reaction as it suggests a double role for ␣Glu 49 as a push-pull acid-base catalyst. The binding mode of IAV is very different, due to rearrangement of the lateral chain of the amino acid in a hydrophobic cavity. This results in loss of the hydrogen bond between the indole nitrogen and ␣Asp 60 . In this complex, the loop ␣L6 of TRPS remains in the open conformation and the intersubunit communication is lost.