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Dihydroorotase from Escherichia coli: Loop Movement and Cooperativity between Subunits
doi:10.1016/j.jmb.2005.01.067
J. Mol. Biol. (2005) 348, 523–533
C OMMUNICATION
Dihydroorotase from Escherichia coli: Loop Movement and Cooperativity between Subunits Mihwa Lee, Camilla W. Chan, J. Mitchell Guss Richard I. Christopherson* and Megan J. Maher* School of Molecular and Microbial Biosciences University of Sydney Sydney, New South Wales 2006 Australia
Escherichia coli dihydroorotase has been crystallized in the presence of the ˚ product, L-dihydroorotate (L-DHO), and the structure refined at 1.9 A resolution. The structure confirms that previously reported (PDB entry 1J79), crystallized in the presence of the substrate N-carbamyl-D,L-aspartate (D, L-CA-asp), which had a dimer in the asymmetric unit, with one subunit having the substrate, L-CA-asp bound at the active site and the other having L-DHO. Importantly, no explanation for the unusual structure was given. Our results now show that a loop comprised of residues 105–115 has different conformations in the two subunits. In the case of the L-CA-aspbound subunit, this loop reaches in toward the active site and makes hydrogen-bonding contact with the bound substrate molecule. For the LDHO-bound subunit, the loop faces in the opposite direction and forms part of the surface of the protein. Analysis of the kinetics for conversion of L-DHO to L-CA-asp at low concentrations of L-DHO shows positive cooperativity with a Hill coefficient nZ1.57(G0.13). Communication between subunits in the dimer may occur via cooperative conformational changes of the side-chains of a tripeptide from each subunit: Arg256His257-Arg258, near the subunit interface. q 2005 Elsevier Ltd. All rights reserved.
*Corresponding authors
Keywords: dihydroorotase; cooperativity; flexible loop; carbamyl aspartate; dihydroorotate
Dihydroorotase (DHOase; E.C. 3.5.2.3) is a zinc metalloenzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate (L-CA-asp) to L-dihydroorotate (L-DHO) in the third step of the pathway for de novo biosynthesis of pyrimidine nucleotides (Scheme 1). In prokaryotes, DHOase is a dimeric enzyme with identical subunits of approximately 40 kDa. 1 In higher eukaryotes, DHOase activity is associated with a trifunctional enzyme, carbamyl phosphate synthetase-dihydroorotase-aspartate transcarbamylase (CAD), which catalyzes the first three steps of the pathway.2 CAD contains carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroAbbreviations used: ATCase, aspartate transcarbamylase; CA-asp, N-carbamyl aspartate; CAD, carbamyl phosphate synthetase-dihydroorotaseaspartate transcarbamylase; CPSase, carbamyl phosphate synthetase; DHO, dihydroorotate; DHOase, dihydroorotase; DTT, dithiothreitol. E-mail addresses of the corresponding authors: [email protected]; [email protected]
orotase activities arranged NHC 3 -CPSase-DHOasebridge-ATCase-COOK.3,4 From extensive amino acid sequence alignments, Holm and Sander proposed that DHOase belongs to the amidohydrolase superfamily of enzymes that catalyzes a diverse set of hydrolytic reactions.5 The superfamily is divided into two subsets, predicted to have a common structural core consisting of eight alternating b-sheet/a-helices and a signature pattern of four conserved histidine residues and one aspartate residue. The first subset includes the enzymes urease and phosphotriesterase, that have binuclear metal centers, where the two metal ions are bridged by a solvent-derived hydroxide and a carboxylated lysine residue. The second subset includes adenosine deaminase that lacks the carboxylated lysine residue and has a single, fivecoordinate zinc ion at the active site, ligated by three conserved histidine residues, one aspartate residue and a water molecule.6 DHOase was originally proposed to belong to the second subset of amidohydrolases because it was believed to bind a single zinc atom.5
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
524 A recently reported phylogenetic analysis of the DHOases proposed two major classes that may have arisen from ancestral gene duplication.7 Type I DHOases, the oldest and largest, are found in all domains of life (Eukarya, Eubacteria, and Archaea) and include trifunctional (e.g. mammalian CAD) and monofunctional enzymes (e.g. Bacillus, Lactobacillus and Streptococcus). Type II DHOases (e.g. E. coli) are predominately eubacterial and exhibit significant differences in their primary amino acid sequences. They are predicted to have secondary structural characteristics similar to the type I DHOases. These enzymes are generally smaller, being 50 and ten residues shorter at the amino and carboxyl termini, respectively, with several internal insertions and deletions compared with the type I enzymes. DHOases from hamster and the eubacterium Aquifex aeolicus have been crystallized8,9 and the first structure of a DHOase, that from E. coli, was ˚ resolution (PDB entry reported recently to 1.7 A 1J7910). In agreement with the predictions of Holm and Sander,5 the overall architecture of the enzyme resembles that of urease. The protein folds into a TIM-barrel motif, with eight strands of parallel b-sheet flanked on the outer surface by a-helices. The enzyme was crystallized in the presence of the racemic substrate, D,L-CA-asp. Surprisingly, the structure showed CA-asp bound at the active site of one subunit (B) and DHO bound at the active site of the other (A). The asymmetric unit contained a single homodimer, with the coordinates submitted to the PDB including residues A4–A346, B4–B108
Loop Movement in E. coli dihydroorotase
and B113–B346 (PDB entry 1J79; residues B109–B112 were omitted). In the published structure of E. coli DHOase, the active sites of both subunits contain binuclear zinc centers. The zinc atoms are coordinated by histidine and aspartate residues. A carboxylated lysine residue (Lys102) bridges the active site metals. In the case of subunit B, one of the carboxylate groups of the bound CA-asp molecule provides an additional metal bridging interaction. A water molecule bridges the metal atoms in subunit A, with the DHO molecule lying adjacent, in the active site pocket. There are common interactions between the CA-asp and DHO molecules and the polypeptide, including hydrogen bonds between atoms O61 or O71 (for CA-asp and DHO, respectively; Scheme 1) and residues Arg20 and Ala266, atom N1 and residue Ala266, atoms O2, N3 and residue Leu222.10 Enzymic activity can be attributed in part to kinetic energy stored in the protein molecules. This energy is expressed as vibrational motions, movements of structural domains such as hinge bending or shearing motions and allosteric transitions, accompanied by relatively large motions of subunits within oligomeric proteins.11 One of the movements associated with catalysis is the rearrangement of loops that constitute active site lids. These movements may be rigid body reorientations around flexible hinges or local changes in the conformation of the polypeptide chain.12 Typically, these loops have different conformations depending on the catalytic state of the
Scheme 1. Structures of L-CA-asp and L-DHO.
525
Loop Movement in E. coli dihydroorotase
enzyme. Such loop movements may be part of the catalytic mechanism of the enzyme. For example, the movements of polypeptide loops in carboxypeptidase A13 and lactate dehydrogenase14 appear from crystallographic studies to bring new functional groups into position for catalysis. For phosphoglycerate kinase, domain closure may sequester substrates and exclude water so that the appropriate acceptor group is phosphorylated by ATP.15 Crystallographic studies on the triosephosphate isomerases from chicken muscle and yeast show that residues 168–177 leave the active site open to solvent, but close down on the bound substrate, dihydroxyacetone phosphate. This loop ˚ toward the active site as the moves by 10 A substrate binds and may prevent unwanted side reactions with water, yielding methylglyoxal.16 The review by Gerstein et al.12 proposes a mechanism by which domain (including loop) closure is achieved. Open and closed states of an enzyme are only slightly different in energy and at room temperature are in dynamic equilibrium. The relative stabilities of the open and closed states depend on bound substrate. If substrate binds to one domain, the second domain forms contacts that stabilize the closed conformation. Conversion to product lessens these interactions and makes the open form more stable, which facilitates product release. We report here the structure of E. coli DHOase crystallized in the presence of the product, L-DHO, ˚ resolution. Despite crystallizing the refined at 1.9 A enzyme in the presence of product (DHO) rather than substrate (CA-asp), we make an identical observation to that of Thoden et al.,10 detecting asymmetry between active sites in the dimer, with CA-asp bound to the active site of subunit B and DHO bound to the active site of subunit A. Importantly, we are able to resolve the conformations of residues 109–112 for both subunits, showing that this tetrapeptide comprises part of a loop (residues 105–115) that has different conformations for each subunit of the dimer. This loop in subunit B, reaches in toward the active site, with two residues at the tip of the loop (ThrB109 and ThrB110), making hydrogen-bonding contact with the bound substrate, CA-asp (“loop in”). The corresponding residues in subunit A do not interact with the bound product, DHO, and instead form part of the protein surface (“loop out”). In addition, the structure reported here shows that the contents of the respective subunit active sites may be communicated between subunits by shifts in the conformations of three pairs of residues (A/ B256–258), close to the dimer interface. Analysis of the kinetics of conversion of DHO to CA-asp at low DHO concentrations indicates positive cooperativity between subunits. E. coli DHOase crystallized with L-DHO Refinement of the DHOase structure using data collected from a single crystal grown in the
presence of L-DHO converged with residuals RZ0.168 and RfreeZ0.211. The asymmetric unit contains a single homodimer, both subunits being comprised of residues 4–346. Overall the model includes 686 amino acid residues, four zinc atoms, 775 water molecules and one molecule each of L -CA-asp and L -DHO. The model has good geometry with 89.2% of residues in most favored regions of the Ramachandran plot (Table 1).17 Subunits A and B are related by approximate 2-fold non-crystallographic symmetry and interact across an extensive, predominantly non-polar inter˚ 2) of the surface face. Approximately 16% (2285 A area of each subunit is buried as a result of dimer formation. The extent of this buried surface is large according to a recent survey of 18 homodimer structures,18 consistent with the fact that this dimer persists in solution. Comparison of the structures of subunits A and B The individual subunits within the DHOase dimer have different structures: (a) they contain different entities bound at their active sites (L-CAasp for subunit B and L-DHO for subunit A); (b) they differ in the conformation of a loop comprised of residues 105–115; (c) they differ in the orientations of residues 256–258. The active sites of both monomers contain two Zn atoms coordinated by His and Asp residues. In addition, a carboxylated lysine residue (Lys102) bridges the Zn atoms. For the CA-asp-bound subunit, one of the carboxylate groups of CA-asp provides an additional bridging interaction between the Zn atoms (atoms O4 and O5, Scheme 1). For the DHO-bound subunit, the Zn atoms are bridged by a water molecule (W694) with the DHO lying adjacent, in the active site cleft. Although subunits A and B have different ligands at their active sites, the coordination numbers of the corresponding zinc atoms are identical. For both subunits the Zn1 and Zn2 atoms are 5-coordinate and 4-coordinate, respectively. However, the metal– metal separations in the two subunits are different, ˚ for the CA-asp-bound subunit (B) being 3.85(14) A ˚ and 3.55(14) A for the DHO-bound subunit (A). This is consistent with the Thoden et al. structure,10 a longer Zn–Zn distance correlating with the presence of a bidentate, carboxylate bridge from the bound CA-asp molecule. Residues A/B105–115 form a loop, which has different conformations in each subunit (Figure 1). In the DHO-bound subunit (A), this loop forms part of the surface of the molecule, faces away from the active site and is solvent-exposed. In the CA-aspbound subunit (B), the loop of residues B105–115 curls in toward the active site. Contacts involving residues A/B105–115, both intra- and intermolecular are listed in Supplementary Data. The residues preceding A105 participate in b-sheet secondary structure by making hydrogen-bonding interactions between the O or N atoms of pairs of
526
Loop Movement in E. coli dihydroorotase
Table 1. Crystallographic data and refinement statistics for E. coli DHOase crystallized with L-DHO Crystallographic data X-ray wavelength Temperature (K) Space group Unit-cell parameters ˚) a (A ˚) b (A ˚) c (A ˚) Resolution range (A Mosaicity (deg.) Unique observations Total observations Redundancya Completeness (%)a I/s(I)a Rmergea,b Refinement statistics ˚) Resolution range (A Residues included in model Total no. protein residues No. of Zn atoms No. of water molecules No. of other entities Total no. of atoms Reflections Rcrysta,c Rfreea,d ˚) rms DKbond lengths (A rms DKbond angles (8) ˚ 2) Average protein B-value (A ˚ )e ESU (A Ramachandran plot, residues in:f Most favored regions (%) Additional allowed regions (%) Generously allowed regions (%)
1.5418 100 P212121 51.6 78.8 180.2 20.0–1.90 0.44 54,214 135,973 2.5 (1.6) 92.0 (83.8) 17.4 (2.8) 0.056 (0.256) 20.0–1.9 A/B4–346 686 4 775 2 (CA-asp; DHO) 6246 54,182 0.168 (0.222) 0.211 (0.238) 0.01 1.2 40.7 0.10 89.2 10.1 0.7
E. coli DHOase was overexpressed and purified from XL1 Blue/DHO pBSC, a gift from Professor F. M. Raushel (Texas A & M University, TX, USA). Purification procedures were adapted from the previous report.1 Crystals were grown at 277 K by vapor-diffusion from hanging drops containing equal volumes (2 ml) of protein (8.8 mg mlK1) and reservoir solution (15–20% (w/v) polyethylene glycol 3350, 0.1 M Mes (pH 6.0–6.5), 75 mM MgCl2, 150 mM KCl) and L-DHO (0.45 ml, 100 mM). Crystals were cryo-protected by immersion in well solution containing ethylene glycol (20%). Diffraction data were collected on a Mar345 image-plate detector with CuKa X-rays from a Rigaku RU-200 rotating anode generator focused using Osmic mirror optics. The diffraction data were processed and scaled with the HKL suite of programs, DENZO and SCALEPACK.28 Crystals of E. coli DHOase grown in the presence of L-DHO were isomorphous with the DHOase structure reported10 (PDB code 1J79) therefore this structure was used as the initial model. Refinement consisted of simulated annealing and positional refinement in CNS,29 followed by refinement with REFMAC5 with TLS,30,31 including positional and temperature factor refinement with manual model building using the program O.32 Positive electron density in difference Fourier maps within the active site cavities of each subunit was modeled with the respective ligands (DHO or CA-asp), the coordinates of which were created using the Dundee PRODRG server.33 Finally, residues B109–112, which were missing from the initial model coordinates (PDB code 1J79), were built into residual positive difference electron density adjacent to the active site of subunit B. The stereochemical quality of the structure was validated using PROCHECK34 and WHATCHECK.35 a Values in parentheses are for the highest resolution shell. b RmergeZSjIh-hIhij/ShIhi. c R valuesZSjFobsKFcalcj/SFobs. d 5% of the reflections were reserved for the calculation of Rfree. e Estimated standard uncertainty in atomic position, based on maximum likelihood.31 f Calculated using PROCHECK.34
residues: AlaA101-LeuA137, LeuA103-LeuA137 and LeuA103-HisA139. Within the loop, three hydrogen-bonding interactions, between residue pairs AlaA106-ThrA143, HisA114-TyrA79 and GlyA115-AspA82 anchor residues A105–115 to the remainder of the polypeptide chain. The A105–115 loop is further stabilized by intra-loop hydrogen bonds between AsnA111-GlyA115 and AsnA107ValA116. For the CA-asp-bound, loop in subunit, residues AlaB101 and LeuB103 also participate in hydrogen-bonding interactions characteristic of bsheet secondary structure. The HisA114–TyrA79 interaction is replaced by one between AsnB111 and
TyrB79. ThrB109 and ThrB110 at the face of the loop hydrogen bond to both carboxylate groups of CAasp, bound at the active site (CA-asp O5/ ˚ ; CA-asp O62/ThrB110O g1 ThrB109O g1 2.6 A ˚ 2.7 A). An extensive network of intra-loop hydrogen bonds between residues B104–108, B110–112 and B115–116 further stabilizes this loop conformation. Because of the different conformations of the 105–115 loops, subunits A and B have different solvent-exposed surface areas, being 14,218 and ˚ 2, respectively. This difference is accounted 14,017 A for by the solvent-exposed surface areas of the loop residues 105–115 for each subunit, which are 224
Loop Movement in E. coli dihydroorotase
527
Figure 1. Stereoviews showing the superposition of the residue 105–115 loops for DHOase subunits A and B. The zinc atoms are shown as gray spheres. Subunits A and B are colored blue and yellow, respectively. CA-asp, bound at the active site of subunit B is shown with its carbon atoms colored green. Thr109 and Thr110 are highlighted for both subunits. The hydrogen-bonding interactions between ThrB109, ThrB110 and the carboxylate groups of CA-asp are highlighted.
˚ 2 for subunits A and B, respectively. In and 18 A subunit B, loop B105–115 virtually fills a cavity of ˚ 3, present in subunit A (the volume of volume 227 A ˚ 3; Figure 2). the subunit B cavity is 22 A Superposition of subunits A and B, omitting residues A/B105–115 gives a root mean squared difference in the positions of 332 common Ca atoms ˚ . This value increases to 1.61 A ˚ if all of 0.31 A residues A/B4–346 are included. The former superposition shows the greatest distance between pairs
˚ , for residue Thr109. The of Ca atoms to be 13.9 A major changes in positions are concentrated between residues 105–115 (data not shown). The different conformations of the A/B105–115 loop in each subunit lead to different crystal packing interactions (Supplementary Data). In subunit A (loop out), SerA112 hydrogen-bonds with a neighboring symmetry-related molecule (subunit B* generated by symmetry operation Kx, yC1/2, KzC1/2). In particular, there are hydrogen
Figure 2. The active site cavities of the DHOase dimer. Ca traces of both subunits are shown with subunit A colored blue and subunit B colored yellow. Cavities were calculated using VOIDOO24,25 and are represented as red nets.
528 ˚) bonds between SerA112N/GluB*292O31 (2.8 A ˚ ). Residues and SerA112Og/GluB*292O32 (2.7 A B105–115 do not have such interactions as they face into the active site. However, by superposing subunit A onto subunit B, we can predict the crystal packing interactions that would result if the loop out conformation existed for both subunits. There would be steric clashes between ThrB109, AsnB111 and SerB112 and GlnA*289, GlnA*5 and ValA*6 of a symmetry-related molecule (by operation xC1/2, KyC1/2, Kz). It seems that the packing of molecules in the crystals used for determination of the present structure preferentially stabilizes a loop out conformation for subunit A. A loop out conformation for subunit B is precluded by unfavorable steric clashes with neighboring symmetryrelated molecules. However, a loop in conformation for both subunits simultaneously is not prevented, nor are loop conformations between in and out. The crystal packing effectively “freezes out” one of the many combinations of loop conformations for the dimer that are possible in solution. Transformation from the loop in to the loop out conformation (or the reverse) involves changes in torsion angles for virtually all residues in the 105–115 loop. In particular, residues 107, 109 and 111 undergo distinct changes in orientation, with their dihedral angles moving from one allowed region of the Ramachandran plot to another; from the b-sheet secondary structure to left-handed a-helix for residues 107 and 111 and to right-handed a-helix for residue 109 (data not shown). The combination of differences in dihedral angles and inter-residue contacts observed for the different loop conformations in subunits A and B indicates a hinge mechanism of movement in combination with changes in secondary structure. Residues 256–258 have different associated dihedral angles in the two subunits. Interestingly, the Ca positions of these residues in each subunit are identical within the limits of precision (data not shown). The relative conformations of the sidechains of residues 256–258 are different in subunits
Loop Movement in E. coli dihydroorotase
A and B (Figure 3). These side-chains face toward the active site in subunit A and away in subunit B, the reverse of the orientations of the 105–115 loop. In subunit A, the loop faces out, leaving a large cavity in the vicinity of the active site. The sidechains of residues A256–258 face toward this cavity and participate in a network of hydrogen bonds with ordered water molecules. In subunit B, with the loop in conformation, there is no such cavity. The side-chains of residues B256–258 face away from the active site, and do not interact with ordered water that is virtually excluded. Residues A/B256–258 do not interact directly at the interface between the subunits. However, there are three water molecules (W253, W412, W710) that may link these tripeptides via hydrogen bonds and provide communication between subunits. The orientations of side-chains A/B256–258 reflect the conformations of the 105–115 loops, in turn determined by bound CA-asp or DHO. Comparisons with the structure by Thoden et al.10 (PDB code 1J79) As detailed in the introduction, the coordinates of E. coli DHOase submitted to the PDB by Thoden et al.10 included residues A4–A346, B4–B108 and B113–B346. Our structure of E. coli DHOase differs from that published by Thoden et al. in its inclusion of residues B109–112. In addition, there are two differences in sequence between the 1J79 coordinates and our structure. In the header of the 1J79 coordinates, the authors note three differences between the sequence of the model protein in the crystals and the sequence of E. coli DHOase as entered under SWISS-PROT entry P05020. That is, the 1J79 model states that: “Gly69 is modeled as Pro, Ile119 is modeled as Val and Asn243 is modeled as Gln”. We therefore sequenced the DHOase pBSC plasmid to confirm the amino acid sequence of the crystallized protein. We find a single amino acid change only, of Ile119 to Val, versus the P05020 entry. Therefore we have modeled residues 69 and 243 as
Figure 3. Stereoview showing the superposition of residues 256–258 for DHOase subunits A and B. The bound CA-asp and DHO ligands are shown, with water molecules in the active site cavity (spheres) and the Zn atoms (gray spheres). Subunit A (blue bonds and water molecules) and subunit B (yellow bonds and water molecules).
Loop Movement in E. coli dihydroorotase
Gly and Asn, respectively, different from Thoden et al., but consistent with “omit” electron density maps where residues 69 and 243 did not contribute to the calculation of structure factors. Least squares superposition of our structure with that reported by Thoden et al.10 omitting residues A/B105–115 for both sets of coordinates, gives a root mean squared difference in 664 common Ca ˚ , increasing to 0.36 A ˚ when all positions of 0.21 A residues except B109–112 are included (682 atoms; residues B109–112 are missing in the 1J79 model). The conformations of residues A105–115 are identical in the two structures. The conformations of residues B105–108 and B113–115 (the portions of the B105–115 loop represented in both structures) are different. In particular, the 1J79 structure places the entire AlaB108 residue in the same region of space as the side-chain of AsnB107 in the present structure. This difference presumably results from difficulties encountered by Thoden et al. in interpreting electron density maps in this region of the structure (discussed further below). AlaB108 in the 1J79 structure faces away from the active site, with
529 its carboxylate end protruding into the solvent. The predicted locations of missing residues B109–112 from this model would be at the surface of the molecule, not near the active site. The 1J79 coordinates include eight water molecules (E1163, E1238, E1240, E1303, E1485, E1550, E1645, E1670) that occupy the same region of space as the B105–115 loop of the present structure. To investigate this further, we obtained the structure factors deposited with PDB entry 1J79. Our coordinates, omitting residues B109–112, were refined against these structure factors and electron density maps calculated. The resulting 2FoKFc and FoKFc electron density maps showed disconnected density in this region, consistent with the interpretation by Thoden et al.10 of ordered water molecules (Figure 4). The reason why this region of the structure could be resolved from crystals grown in our laboratory and not by Thoden et al.10 may be linked to the stereochemical purity of the ligands (CA-asp or DHO) used in the crystallization. Thoden et al. crystallized E. coli DHOase in the presence of racemic D,L-CA-asp,10 whereas the
Figure 4. Stereoviews of the region of space occupied by residues B105–115 for (a) the present structure and (b) the structure reported by Thoden et al.10 In both representations, the Zn atoms are depicted as gray spheres, with the carbon atoms of the protein in yellow and carbon atoms of the bound CA-asp ligand in green. The 2FoKFc electron density maps are represented as fawn nets and are contoured at 1.0 s. The hydrogen bonding interactions between ThrB109, ThrB110 and the bound CA-asp molecule are indicated for (a). In (b) electron density maps were calculated using structure factors deposited with PDB coordinates 1J79.
530 crystals in the present work were grown with enantiomerically pure L-DHO. Kinetics The dependence of DHOase activity upon DHO concentration was measured over a wide range (0.1–200 mM) to determine whether the asymmetry observed in the present structure was reflected in some form of cooperativity. The data obtained from several experiments were plotted in double reciprocal form, and showed upward curvature, consistent with positive cooperativity (Figure 5). These data were fitted to the Hill equation and the following parameter values were obtained: VZ1.90(G0.06) pmol CA-asp/minute, K s Z 20.8(G1.3) mM and nZ1.57(G0.13). These parameters can be compared with two previous experiments using a spectrophotometric assay, that determined KmZ75.6 and 50.0 mM.1,19 Our values were substituted into the Hill equation and used to draw the theoretical curve through the experimental data (Figure 5). The value for the Hill coefficient of 1.57 is consistent with a two-site enzyme showing strong positive cooperativity. This cooperativity is consistent with the structural differences reported here between the subunits in the DHOase dimer, when crystallized in the presence of L-DHO.
Loop Movement in E. coli dihydroorotase
What is the mechanism for movement of the 105-115 loop? The interactions of CA-asp and DHO with the DHOase polypeptide chain are detailed in Supplementary Data. The two carboxylate groups of CA-asp, bridge the active site Zn atoms (with atoms O4 and O5) and hydrogen bond to ArgB20, ThrB109, ThrB110 and AlaB266 (with atoms O61 and O62; Scheme 1, Supplementary Data). The DHO molecule has a carboxylate group in common with CA-asp (atoms C7, O71 and O72). A leastsquares superposition of subunit A onto subunit B superposes the respective active sites and the DHO and CA-asp ligands. We can then predict interactions between DHO and loop residues B105–115 if DHO were bound at the active site of a subunit with a loop in conformation. This superposition shows that the carboxylate group common to CA-asp and DHO lies in an equivalent position and the respective projected distances of DHO atoms O71 and O72 to residues ArgB20, ThrB110 and AlaB266 are the same as for the equivalent atoms of CA-asp. In short, if DHO occupied the active site of a loop in subunit, atoms O71 and O72 would be in the correct position to hydrogen bond to ThrB110. However, this superposition predicts a potential close contact between DHO and ThrB109 (DHO O4/ ˚ ). This distance is too short to ThrB109Og1Z2.3 A be considered a hydrogen-bond20 and significantly shorter than the observed interaction between CAasp and ThrB109 (CA-asp O5/ThrB109O g1Z ˚ ). A possible mechanism for the conversion 2.6 A from loop in to loop out is as follows: the cyclization of CA-asp to form DHO converts the C4/O4/O5 carboxylate group to a carbonyl group in DHO, that can no longer bridge the Zn atoms at the active site. This causes the DHO molecule to move up and away from the active site, putting steric pressure on residue Thr109 and releasing the loop to the out conformation. What does loop movement mean to the mechanism of E. coli DHOase?
Figure 5. Lineweaver-Burk plot showing the dependence of DHOase activity upon the concentration of DHO. Positive cooperativity is indicated by upward curvature of the theoretical line generated from the Hill equation using the values VZ1.90(G0.06) pmol CAasp/minute, nZ1.57(G0.13), and KsZ20.76(G1.28) mM. Assays contained 5.7 ng DHOase in 25 ml. E. coli DHOase was assayed in the degradative direction (DHO/ CA-asp) by established procedures.26,27 To detect cooperativity, 14C-labelled DHO concentrations covered a wide range, from 0.1–200 mM. Reaction velocities were calculated from three time points and data were fitted by non-linear regression using PRISM (version 3.2, Graphpad Software, San Diego, CA) to the Hill equation: nZ VSn =ðKs C Sn Þ, where v is the reaction velocity, V is the maximal velocity, S is the concentration of DHO, Ks is the apparent dissociation constant and n is the Hill coefficient.
The introduction summarizes functions proposed for loop movements in other enzymes. It is possible that movement of the 105–115 loop into the active site of E. coli DHOase upon CA-asp binding fulfills multiple roles. Thoden et al. reported a mechanism for the DHO biosynthetic reaction in which direct molecular contacts between CA-asp and the binuclear Zn site were proposed to make the C5 atom of CA-asp more electrophilic and therefore promote bond formation, in addition to electrostatically stabilizing the proposed tetrahedral intermediate.10 It is possible that the hydrogen-bonding interaction between CA-asp and ThrB109 in the present structure provides further stabilization. Studies on porcine pepsin A, an aspartyl protease, have examined the role of Thr77, one of a tripeptide of so-called “flap residues” (Tyr75, Gly76, Thr77) shown to contribute directly to
531
Loop Movement in E. coli dihydroorotase
specificity by hydrogen-bonding to substrate residues. Kinetic analysis of the enzyme, following sitedirected mutagenesis of Thr77 to Ser, Val and Gly showed that the hydroxyl group provides an essential hydrogen-bonding interaction that contributes to a delicate network that aligns the substrate and is indirectly responsible for the proper geometry of the transition state. In addition, this interaction appears to lock the flap loop closed when a substrate is bound and in this way encloses the catalytic environment.21 In the same way, hydrogen-bonding interactions involving Thr109, Thr110 and both carboxylate groups of CA-asp in the present structure may serve to poise CA-asp in an optimum conformation for conversion to DHO. Closure of the 105–115 loop on CA-asp binding does enclose the substratebinding pocket and this may further aid catalysis by excluding water from the active site. The cavity present at the active site in the DHO-bound subunit (A) is filled with water molecules, some of which are excluded in the CA-asp-bound subunit (B). This exclusion of water from the active site during catalysis may prevent unwanted side reactions. Relevance of loop movements and cooperativity to the mechanisms of DHOases DHOases may have limited sequence homology but in general are found in native form as dimers.7 For example, E. coli DHOase is a dimer, as is the hamster DHOase domain isolated after proteolysis of the trifunctional CAD protein, or expressed in E. coli.22 The exceptions are the monomeric DHOases from parasitic organisms Crithidia fasciculata and Plasmodium berghei. 23 DHOase sequences are aligned in Figure 6. Residues corresponding to 105–115 of the E. coli enzyme are conserved in DHOases from Plasmodium falciparum, Arabidopsis thaliana, Helicobacter pylori and Saccharoymyces cerevisiae but the human and hamster DHOase domains have different sequences. The residues at each end of the loop, Pro105 and Gly115 in the E. coli protein, are also conserved in some species and may provide flexibility for the transition between loop in and loop out conformations. Interestingly, Thr110 has the same identity in all sequences aligned here and
Thr109 is conserved an all but the mammalian sequences. It is possible that hydrogen-bonding interactions between loop residues and bound CAasp are important for catalysis in many DHOases. Conclusions We have refined the structure of E. coli DHOase, ˚ crystallized in the presence of L-DHO, at 1.9 A resolution. The structure confirms many features reported by Thoden et al.10 In particular, we observe asymmetry between active sites in the dimer, with the substrate, CA-asp, bound to one active site and DHO bound to the other. Three new aspects of the structure and mechanism of E. coli DHOase have been described here: (1) residues 105–115 form loops with different conformations for each subunit within the dimer. This loop in subunit B, reaches in toward the active site, with ThrB109 and ThrB110 making hydrogenbonding contacts with the bound substrate, CAasp. The corresponding residues in subunit A do not interact with the bound product, DHO, and instead form part of the protein surface. (2) The kinetics for conversion of DHO/CA-asp at low DHO concentrations show positive cooperativity with a Hill coefficient of 1.57. (3) The asymmetry in binding CA-asp and DHO at the two active sites may be communicated between subunits by shifts in the conformations of three pairs of residues (A/B256–258), close to the dimer interface. The asymmetry in the structures of the active sites of the two subunits, indicates that there is conformational communication between them. Such a conformational link could also mediate cooperativity. Our kinetic experiments show positive cooperativity with respect to DHO. The minimal explanation is that the cooperativity is mediated by the same mechanism that provided the asymmetric dimer. In summary, binding of CA-asp accompanies movement of the 105–115 loop to the loop in conformation, held in place by hydrogenbonding interactions with ThrB109 and ThrB110. This closes the active site cavity, excludes water and correlates with conformational changes in the side-chains of residues ArgB256, HisB257 and ArgB258. These residues interact via water molecules across the dimer interface and may
Figure 6. Alignment of DHOase sequences. Abbreviations are as follows: ECOLI, Escherichia coli, accession code P05020; PFALC, Plasmodium falciparum, Q8IKA9; ARATH, Arabidopsis thaliana, O04904; HELPY, Helicobacter pylori, P56465; YEAST, Saccharoymyces cerevisiae, P20051; HUMAN, Homo sapiens, P27708; MESAU, Mesocricetus auratus, P08955. The positions of Lys102 and Thr110 (numbering for the E. coli protein) are indicated as *. Regions of high sequence similarity are shaded blue. Regions with lower similarity are shaded green and yellow. Residues corresponding to 105–115 for the E. coli protein are boxed.
532 mediate communication between subunits, which is borne out in cooperativity in the kinetics. The structure described here may represent one of the many combinations of loop conformations and occupancies of active sites possible for the DHOase dimer in solution.
Loop Movement in E. coli dihydroorotase
9.
Protein Data Bank accession numbers The atomic coordinates, structure amplitudes and phases have been deposited at the RCSB Protein Data Bank with accession code 1XGE.
10.
11.
Acknowledgements Support from the Australian Research Council (DP0209273) is gratefully acknowledged. M.J.M. is an ARC Australian Postdoctoral Fellow. The authors thank Professor F. Raushel for the XL1 Blue/DHO pBSC plasmid and Dr D.T.C. Huang for initial work on protein expression and purification.
12. 13.
14.
Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jmb.2005.01.067
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Edited by I. Wilson (Received 6 December 2004; received in revised form 20 January 2005; accepted 26 January 2005)
J. Mol. Biol. (2005) 348, 523–533
C OMMUNICATION
Dihydroorotase from Escherichia coli: Loop Movement and Cooperativity between Subunits Mihwa Lee, Camilla W. Chan, J. Mitchell Guss Richard I. Christopherson* and Megan J. Maher* School of Molecular and Microbial Biosciences University of Sydney Sydney, New South Wales 2006 Australia
Escherichia coli dihydroorotase has been crystallized in the presence of the ˚ product, L-dihydroorotate (L-DHO), and the structure refined at 1.9 A resolution. The structure confirms that previously reported (PDB entry 1J79), crystallized in the presence of the substrate N-carbamyl-D,L-aspartate (D, L-CA-asp), which had a dimer in the asymmetric unit, with one subunit having the substrate, L-CA-asp bound at the active site and the other having L-DHO. Importantly, no explanation for the unusual structure was given. Our results now show that a loop comprised of residues 105–115 has different conformations in the two subunits. In the case of the L-CA-aspbound subunit, this loop reaches in toward the active site and makes hydrogen-bonding contact with the bound substrate molecule. For the LDHO-bound subunit, the loop faces in the opposite direction and forms part of the surface of the protein. Analysis of the kinetics for conversion of L-DHO to L-CA-asp at low concentrations of L-DHO shows positive cooperativity with a Hill coefficient nZ1.57(G0.13). Communication between subunits in the dimer may occur via cooperative conformational changes of the side-chains of a tripeptide from each subunit: Arg256His257-Arg258, near the subunit interface. q 2005 Elsevier Ltd. All rights reserved.
*Corresponding authors
Keywords: dihydroorotase; cooperativity; flexible loop; carbamyl aspartate; dihydroorotate
Dihydroorotase (DHOase; E.C. 3.5.2.3) is a zinc metalloenzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate (L-CA-asp) to L-dihydroorotate (L-DHO) in the third step of the pathway for de novo biosynthesis of pyrimidine nucleotides (Scheme 1). In prokaryotes, DHOase is a dimeric enzyme with identical subunits of approximately 40 kDa. 1 In higher eukaryotes, DHOase activity is associated with a trifunctional enzyme, carbamyl phosphate synthetase-dihydroorotase-aspartate transcarbamylase (CAD), which catalyzes the first three steps of the pathway.2 CAD contains carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroAbbreviations used: ATCase, aspartate transcarbamylase; CA-asp, N-carbamyl aspartate; CAD, carbamyl phosphate synthetase-dihydroorotaseaspartate transcarbamylase; CPSase, carbamyl phosphate synthetase; DHO, dihydroorotate; DHOase, dihydroorotase; DTT, dithiothreitol. E-mail addresses of the corresponding authors: [email protected]; [email protected]
orotase activities arranged NHC 3 -CPSase-DHOasebridge-ATCase-COOK.3,4 From extensive amino acid sequence alignments, Holm and Sander proposed that DHOase belongs to the amidohydrolase superfamily of enzymes that catalyzes a diverse set of hydrolytic reactions.5 The superfamily is divided into two subsets, predicted to have a common structural core consisting of eight alternating b-sheet/a-helices and a signature pattern of four conserved histidine residues and one aspartate residue. The first subset includes the enzymes urease and phosphotriesterase, that have binuclear metal centers, where the two metal ions are bridged by a solvent-derived hydroxide and a carboxylated lysine residue. The second subset includes adenosine deaminase that lacks the carboxylated lysine residue and has a single, fivecoordinate zinc ion at the active site, ligated by three conserved histidine residues, one aspartate residue and a water molecule.6 DHOase was originally proposed to belong to the second subset of amidohydrolases because it was believed to bind a single zinc atom.5
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
524 A recently reported phylogenetic analysis of the DHOases proposed two major classes that may have arisen from ancestral gene duplication.7 Type I DHOases, the oldest and largest, are found in all domains of life (Eukarya, Eubacteria, and Archaea) and include trifunctional (e.g. mammalian CAD) and monofunctional enzymes (e.g. Bacillus, Lactobacillus and Streptococcus). Type II DHOases (e.g. E. coli) are predominately eubacterial and exhibit significant differences in their primary amino acid sequences. They are predicted to have secondary structural characteristics similar to the type I DHOases. These enzymes are generally smaller, being 50 and ten residues shorter at the amino and carboxyl termini, respectively, with several internal insertions and deletions compared with the type I enzymes. DHOases from hamster and the eubacterium Aquifex aeolicus have been crystallized8,9 and the first structure of a DHOase, that from E. coli, was ˚ resolution (PDB entry reported recently to 1.7 A 1J7910). In agreement with the predictions of Holm and Sander,5 the overall architecture of the enzyme resembles that of urease. The protein folds into a TIM-barrel motif, with eight strands of parallel b-sheet flanked on the outer surface by a-helices. The enzyme was crystallized in the presence of the racemic substrate, D,L-CA-asp. Surprisingly, the structure showed CA-asp bound at the active site of one subunit (B) and DHO bound at the active site of the other (A). The asymmetric unit contained a single homodimer, with the coordinates submitted to the PDB including residues A4–A346, B4–B108
Loop Movement in E. coli dihydroorotase
and B113–B346 (PDB entry 1J79; residues B109–B112 were omitted). In the published structure of E. coli DHOase, the active sites of both subunits contain binuclear zinc centers. The zinc atoms are coordinated by histidine and aspartate residues. A carboxylated lysine residue (Lys102) bridges the active site metals. In the case of subunit B, one of the carboxylate groups of the bound CA-asp molecule provides an additional metal bridging interaction. A water molecule bridges the metal atoms in subunit A, with the DHO molecule lying adjacent, in the active site pocket. There are common interactions between the CA-asp and DHO molecules and the polypeptide, including hydrogen bonds between atoms O61 or O71 (for CA-asp and DHO, respectively; Scheme 1) and residues Arg20 and Ala266, atom N1 and residue Ala266, atoms O2, N3 and residue Leu222.10 Enzymic activity can be attributed in part to kinetic energy stored in the protein molecules. This energy is expressed as vibrational motions, movements of structural domains such as hinge bending or shearing motions and allosteric transitions, accompanied by relatively large motions of subunits within oligomeric proteins.11 One of the movements associated with catalysis is the rearrangement of loops that constitute active site lids. These movements may be rigid body reorientations around flexible hinges or local changes in the conformation of the polypeptide chain.12 Typically, these loops have different conformations depending on the catalytic state of the
Scheme 1. Structures of L-CA-asp and L-DHO.
525
Loop Movement in E. coli dihydroorotase
enzyme. Such loop movements may be part of the catalytic mechanism of the enzyme. For example, the movements of polypeptide loops in carboxypeptidase A13 and lactate dehydrogenase14 appear from crystallographic studies to bring new functional groups into position for catalysis. For phosphoglycerate kinase, domain closure may sequester substrates and exclude water so that the appropriate acceptor group is phosphorylated by ATP.15 Crystallographic studies on the triosephosphate isomerases from chicken muscle and yeast show that residues 168–177 leave the active site open to solvent, but close down on the bound substrate, dihydroxyacetone phosphate. This loop ˚ toward the active site as the moves by 10 A substrate binds and may prevent unwanted side reactions with water, yielding methylglyoxal.16 The review by Gerstein et al.12 proposes a mechanism by which domain (including loop) closure is achieved. Open and closed states of an enzyme are only slightly different in energy and at room temperature are in dynamic equilibrium. The relative stabilities of the open and closed states depend on bound substrate. If substrate binds to one domain, the second domain forms contacts that stabilize the closed conformation. Conversion to product lessens these interactions and makes the open form more stable, which facilitates product release. We report here the structure of E. coli DHOase crystallized in the presence of the product, L-DHO, ˚ resolution. Despite crystallizing the refined at 1.9 A enzyme in the presence of product (DHO) rather than substrate (CA-asp), we make an identical observation to that of Thoden et al.,10 detecting asymmetry between active sites in the dimer, with CA-asp bound to the active site of subunit B and DHO bound to the active site of subunit A. Importantly, we are able to resolve the conformations of residues 109–112 for both subunits, showing that this tetrapeptide comprises part of a loop (residues 105–115) that has different conformations for each subunit of the dimer. This loop in subunit B, reaches in toward the active site, with two residues at the tip of the loop (ThrB109 and ThrB110), making hydrogen-bonding contact with the bound substrate, CA-asp (“loop in”). The corresponding residues in subunit A do not interact with the bound product, DHO, and instead form part of the protein surface (“loop out”). In addition, the structure reported here shows that the contents of the respective subunit active sites may be communicated between subunits by shifts in the conformations of three pairs of residues (A/ B256–258), close to the dimer interface. Analysis of the kinetics of conversion of DHO to CA-asp at low DHO concentrations indicates positive cooperativity between subunits. E. coli DHOase crystallized with L-DHO Refinement of the DHOase structure using data collected from a single crystal grown in the
presence of L-DHO converged with residuals RZ0.168 and RfreeZ0.211. The asymmetric unit contains a single homodimer, both subunits being comprised of residues 4–346. Overall the model includes 686 amino acid residues, four zinc atoms, 775 water molecules and one molecule each of L -CA-asp and L -DHO. The model has good geometry with 89.2% of residues in most favored regions of the Ramachandran plot (Table 1).17 Subunits A and B are related by approximate 2-fold non-crystallographic symmetry and interact across an extensive, predominantly non-polar inter˚ 2) of the surface face. Approximately 16% (2285 A area of each subunit is buried as a result of dimer formation. The extent of this buried surface is large according to a recent survey of 18 homodimer structures,18 consistent with the fact that this dimer persists in solution. Comparison of the structures of subunits A and B The individual subunits within the DHOase dimer have different structures: (a) they contain different entities bound at their active sites (L-CAasp for subunit B and L-DHO for subunit A); (b) they differ in the conformation of a loop comprised of residues 105–115; (c) they differ in the orientations of residues 256–258. The active sites of both monomers contain two Zn atoms coordinated by His and Asp residues. In addition, a carboxylated lysine residue (Lys102) bridges the Zn atoms. For the CA-asp-bound subunit, one of the carboxylate groups of CA-asp provides an additional bridging interaction between the Zn atoms (atoms O4 and O5, Scheme 1). For the DHO-bound subunit, the Zn atoms are bridged by a water molecule (W694) with the DHO lying adjacent, in the active site cleft. Although subunits A and B have different ligands at their active sites, the coordination numbers of the corresponding zinc atoms are identical. For both subunits the Zn1 and Zn2 atoms are 5-coordinate and 4-coordinate, respectively. However, the metal– metal separations in the two subunits are different, ˚ for the CA-asp-bound subunit (B) being 3.85(14) A ˚ and 3.55(14) A for the DHO-bound subunit (A). This is consistent with the Thoden et al. structure,10 a longer Zn–Zn distance correlating with the presence of a bidentate, carboxylate bridge from the bound CA-asp molecule. Residues A/B105–115 form a loop, which has different conformations in each subunit (Figure 1). In the DHO-bound subunit (A), this loop forms part of the surface of the molecule, faces away from the active site and is solvent-exposed. In the CA-aspbound subunit (B), the loop of residues B105–115 curls in toward the active site. Contacts involving residues A/B105–115, both intra- and intermolecular are listed in Supplementary Data. The residues preceding A105 participate in b-sheet secondary structure by making hydrogen-bonding interactions between the O or N atoms of pairs of
526
Loop Movement in E. coli dihydroorotase
Table 1. Crystallographic data and refinement statistics for E. coli DHOase crystallized with L-DHO Crystallographic data X-ray wavelength Temperature (K) Space group Unit-cell parameters ˚) a (A ˚) b (A ˚) c (A ˚) Resolution range (A Mosaicity (deg.) Unique observations Total observations Redundancya Completeness (%)a I/s(I)a Rmergea,b Refinement statistics ˚) Resolution range (A Residues included in model Total no. protein residues No. of Zn atoms No. of water molecules No. of other entities Total no. of atoms Reflections Rcrysta,c Rfreea,d ˚) rms DKbond lengths (A rms DKbond angles (8) ˚ 2) Average protein B-value (A ˚ )e ESU (A Ramachandran plot, residues in:f Most favored regions (%) Additional allowed regions (%) Generously allowed regions (%)
1.5418 100 P212121 51.6 78.8 180.2 20.0–1.90 0.44 54,214 135,973 2.5 (1.6) 92.0 (83.8) 17.4 (2.8) 0.056 (0.256) 20.0–1.9 A/B4–346 686 4 775 2 (CA-asp; DHO) 6246 54,182 0.168 (0.222) 0.211 (0.238) 0.01 1.2 40.7 0.10 89.2 10.1 0.7
E. coli DHOase was overexpressed and purified from XL1 Blue/DHO pBSC, a gift from Professor F. M. Raushel (Texas A & M University, TX, USA). Purification procedures were adapted from the previous report.1 Crystals were grown at 277 K by vapor-diffusion from hanging drops containing equal volumes (2 ml) of protein (8.8 mg mlK1) and reservoir solution (15–20% (w/v) polyethylene glycol 3350, 0.1 M Mes (pH 6.0–6.5), 75 mM MgCl2, 150 mM KCl) and L-DHO (0.45 ml, 100 mM). Crystals were cryo-protected by immersion in well solution containing ethylene glycol (20%). Diffraction data were collected on a Mar345 image-plate detector with CuKa X-rays from a Rigaku RU-200 rotating anode generator focused using Osmic mirror optics. The diffraction data were processed and scaled with the HKL suite of programs, DENZO and SCALEPACK.28 Crystals of E. coli DHOase grown in the presence of L-DHO were isomorphous with the DHOase structure reported10 (PDB code 1J79) therefore this structure was used as the initial model. Refinement consisted of simulated annealing and positional refinement in CNS,29 followed by refinement with REFMAC5 with TLS,30,31 including positional and temperature factor refinement with manual model building using the program O.32 Positive electron density in difference Fourier maps within the active site cavities of each subunit was modeled with the respective ligands (DHO or CA-asp), the coordinates of which were created using the Dundee PRODRG server.33 Finally, residues B109–112, which were missing from the initial model coordinates (PDB code 1J79), were built into residual positive difference electron density adjacent to the active site of subunit B. The stereochemical quality of the structure was validated using PROCHECK34 and WHATCHECK.35 a Values in parentheses are for the highest resolution shell. b RmergeZSjIh-hIhij/ShIhi. c R valuesZSjFobsKFcalcj/SFobs. d 5% of the reflections were reserved for the calculation of Rfree. e Estimated standard uncertainty in atomic position, based on maximum likelihood.31 f Calculated using PROCHECK.34
residues: AlaA101-LeuA137, LeuA103-LeuA137 and LeuA103-HisA139. Within the loop, three hydrogen-bonding interactions, between residue pairs AlaA106-ThrA143, HisA114-TyrA79 and GlyA115-AspA82 anchor residues A105–115 to the remainder of the polypeptide chain. The A105–115 loop is further stabilized by intra-loop hydrogen bonds between AsnA111-GlyA115 and AsnA107ValA116. For the CA-asp-bound, loop in subunit, residues AlaB101 and LeuB103 also participate in hydrogen-bonding interactions characteristic of bsheet secondary structure. The HisA114–TyrA79 interaction is replaced by one between AsnB111 and
TyrB79. ThrB109 and ThrB110 at the face of the loop hydrogen bond to both carboxylate groups of CAasp, bound at the active site (CA-asp O5/ ˚ ; CA-asp O62/ThrB110O g1 ThrB109O g1 2.6 A ˚ 2.7 A). An extensive network of intra-loop hydrogen bonds between residues B104–108, B110–112 and B115–116 further stabilizes this loop conformation. Because of the different conformations of the 105–115 loops, subunits A and B have different solvent-exposed surface areas, being 14,218 and ˚ 2, respectively. This difference is accounted 14,017 A for by the solvent-exposed surface areas of the loop residues 105–115 for each subunit, which are 224
Loop Movement in E. coli dihydroorotase
527
Figure 1. Stereoviews showing the superposition of the residue 105–115 loops for DHOase subunits A and B. The zinc atoms are shown as gray spheres. Subunits A and B are colored blue and yellow, respectively. CA-asp, bound at the active site of subunit B is shown with its carbon atoms colored green. Thr109 and Thr110 are highlighted for both subunits. The hydrogen-bonding interactions between ThrB109, ThrB110 and the carboxylate groups of CA-asp are highlighted.
˚ 2 for subunits A and B, respectively. In and 18 A subunit B, loop B105–115 virtually fills a cavity of ˚ 3, present in subunit A (the volume of volume 227 A ˚ 3; Figure 2). the subunit B cavity is 22 A Superposition of subunits A and B, omitting residues A/B105–115 gives a root mean squared difference in the positions of 332 common Ca atoms ˚ . This value increases to 1.61 A ˚ if all of 0.31 A residues A/B4–346 are included. The former superposition shows the greatest distance between pairs
˚ , for residue Thr109. The of Ca atoms to be 13.9 A major changes in positions are concentrated between residues 105–115 (data not shown). The different conformations of the A/B105–115 loop in each subunit lead to different crystal packing interactions (Supplementary Data). In subunit A (loop out), SerA112 hydrogen-bonds with a neighboring symmetry-related molecule (subunit B* generated by symmetry operation Kx, yC1/2, KzC1/2). In particular, there are hydrogen
Figure 2. The active site cavities of the DHOase dimer. Ca traces of both subunits are shown with subunit A colored blue and subunit B colored yellow. Cavities were calculated using VOIDOO24,25 and are represented as red nets.
528 ˚) bonds between SerA112N/GluB*292O31 (2.8 A ˚ ). Residues and SerA112Og/GluB*292O32 (2.7 A B105–115 do not have such interactions as they face into the active site. However, by superposing subunit A onto subunit B, we can predict the crystal packing interactions that would result if the loop out conformation existed for both subunits. There would be steric clashes between ThrB109, AsnB111 and SerB112 and GlnA*289, GlnA*5 and ValA*6 of a symmetry-related molecule (by operation xC1/2, KyC1/2, Kz). It seems that the packing of molecules in the crystals used for determination of the present structure preferentially stabilizes a loop out conformation for subunit A. A loop out conformation for subunit B is precluded by unfavorable steric clashes with neighboring symmetryrelated molecules. However, a loop in conformation for both subunits simultaneously is not prevented, nor are loop conformations between in and out. The crystal packing effectively “freezes out” one of the many combinations of loop conformations for the dimer that are possible in solution. Transformation from the loop in to the loop out conformation (or the reverse) involves changes in torsion angles for virtually all residues in the 105–115 loop. In particular, residues 107, 109 and 111 undergo distinct changes in orientation, with their dihedral angles moving from one allowed region of the Ramachandran plot to another; from the b-sheet secondary structure to left-handed a-helix for residues 107 and 111 and to right-handed a-helix for residue 109 (data not shown). The combination of differences in dihedral angles and inter-residue contacts observed for the different loop conformations in subunits A and B indicates a hinge mechanism of movement in combination with changes in secondary structure. Residues 256–258 have different associated dihedral angles in the two subunits. Interestingly, the Ca positions of these residues in each subunit are identical within the limits of precision (data not shown). The relative conformations of the sidechains of residues 256–258 are different in subunits
Loop Movement in E. coli dihydroorotase
A and B (Figure 3). These side-chains face toward the active site in subunit A and away in subunit B, the reverse of the orientations of the 105–115 loop. In subunit A, the loop faces out, leaving a large cavity in the vicinity of the active site. The sidechains of residues A256–258 face toward this cavity and participate in a network of hydrogen bonds with ordered water molecules. In subunit B, with the loop in conformation, there is no such cavity. The side-chains of residues B256–258 face away from the active site, and do not interact with ordered water that is virtually excluded. Residues A/B256–258 do not interact directly at the interface between the subunits. However, there are three water molecules (W253, W412, W710) that may link these tripeptides via hydrogen bonds and provide communication between subunits. The orientations of side-chains A/B256–258 reflect the conformations of the 105–115 loops, in turn determined by bound CA-asp or DHO. Comparisons with the structure by Thoden et al.10 (PDB code 1J79) As detailed in the introduction, the coordinates of E. coli DHOase submitted to the PDB by Thoden et al.10 included residues A4–A346, B4–B108 and B113–B346. Our structure of E. coli DHOase differs from that published by Thoden et al. in its inclusion of residues B109–112. In addition, there are two differences in sequence between the 1J79 coordinates and our structure. In the header of the 1J79 coordinates, the authors note three differences between the sequence of the model protein in the crystals and the sequence of E. coli DHOase as entered under SWISS-PROT entry P05020. That is, the 1J79 model states that: “Gly69 is modeled as Pro, Ile119 is modeled as Val and Asn243 is modeled as Gln”. We therefore sequenced the DHOase pBSC plasmid to confirm the amino acid sequence of the crystallized protein. We find a single amino acid change only, of Ile119 to Val, versus the P05020 entry. Therefore we have modeled residues 69 and 243 as
Figure 3. Stereoview showing the superposition of residues 256–258 for DHOase subunits A and B. The bound CA-asp and DHO ligands are shown, with water molecules in the active site cavity (spheres) and the Zn atoms (gray spheres). Subunit A (blue bonds and water molecules) and subunit B (yellow bonds and water molecules).
Loop Movement in E. coli dihydroorotase
Gly and Asn, respectively, different from Thoden et al., but consistent with “omit” electron density maps where residues 69 and 243 did not contribute to the calculation of structure factors. Least squares superposition of our structure with that reported by Thoden et al.10 omitting residues A/B105–115 for both sets of coordinates, gives a root mean squared difference in 664 common Ca ˚ , increasing to 0.36 A ˚ when all positions of 0.21 A residues except B109–112 are included (682 atoms; residues B109–112 are missing in the 1J79 model). The conformations of residues A105–115 are identical in the two structures. The conformations of residues B105–108 and B113–115 (the portions of the B105–115 loop represented in both structures) are different. In particular, the 1J79 structure places the entire AlaB108 residue in the same region of space as the side-chain of AsnB107 in the present structure. This difference presumably results from difficulties encountered by Thoden et al. in interpreting electron density maps in this region of the structure (discussed further below). AlaB108 in the 1J79 structure faces away from the active site, with
529 its carboxylate end protruding into the solvent. The predicted locations of missing residues B109–112 from this model would be at the surface of the molecule, not near the active site. The 1J79 coordinates include eight water molecules (E1163, E1238, E1240, E1303, E1485, E1550, E1645, E1670) that occupy the same region of space as the B105–115 loop of the present structure. To investigate this further, we obtained the structure factors deposited with PDB entry 1J79. Our coordinates, omitting residues B109–112, were refined against these structure factors and electron density maps calculated. The resulting 2FoKFc and FoKFc electron density maps showed disconnected density in this region, consistent with the interpretation by Thoden et al.10 of ordered water molecules (Figure 4). The reason why this region of the structure could be resolved from crystals grown in our laboratory and not by Thoden et al.10 may be linked to the stereochemical purity of the ligands (CA-asp or DHO) used in the crystallization. Thoden et al. crystallized E. coli DHOase in the presence of racemic D,L-CA-asp,10 whereas the
Figure 4. Stereoviews of the region of space occupied by residues B105–115 for (a) the present structure and (b) the structure reported by Thoden et al.10 In both representations, the Zn atoms are depicted as gray spheres, with the carbon atoms of the protein in yellow and carbon atoms of the bound CA-asp ligand in green. The 2FoKFc electron density maps are represented as fawn nets and are contoured at 1.0 s. The hydrogen bonding interactions between ThrB109, ThrB110 and the bound CA-asp molecule are indicated for (a). In (b) electron density maps were calculated using structure factors deposited with PDB coordinates 1J79.
530 crystals in the present work were grown with enantiomerically pure L-DHO. Kinetics The dependence of DHOase activity upon DHO concentration was measured over a wide range (0.1–200 mM) to determine whether the asymmetry observed in the present structure was reflected in some form of cooperativity. The data obtained from several experiments were plotted in double reciprocal form, and showed upward curvature, consistent with positive cooperativity (Figure 5). These data were fitted to the Hill equation and the following parameter values were obtained: VZ1.90(G0.06) pmol CA-asp/minute, K s Z 20.8(G1.3) mM and nZ1.57(G0.13). These parameters can be compared with two previous experiments using a spectrophotometric assay, that determined KmZ75.6 and 50.0 mM.1,19 Our values were substituted into the Hill equation and used to draw the theoretical curve through the experimental data (Figure 5). The value for the Hill coefficient of 1.57 is consistent with a two-site enzyme showing strong positive cooperativity. This cooperativity is consistent with the structural differences reported here between the subunits in the DHOase dimer, when crystallized in the presence of L-DHO.
Loop Movement in E. coli dihydroorotase
What is the mechanism for movement of the 105-115 loop? The interactions of CA-asp and DHO with the DHOase polypeptide chain are detailed in Supplementary Data. The two carboxylate groups of CA-asp, bridge the active site Zn atoms (with atoms O4 and O5) and hydrogen bond to ArgB20, ThrB109, ThrB110 and AlaB266 (with atoms O61 and O62; Scheme 1, Supplementary Data). The DHO molecule has a carboxylate group in common with CA-asp (atoms C7, O71 and O72). A leastsquares superposition of subunit A onto subunit B superposes the respective active sites and the DHO and CA-asp ligands. We can then predict interactions between DHO and loop residues B105–115 if DHO were bound at the active site of a subunit with a loop in conformation. This superposition shows that the carboxylate group common to CA-asp and DHO lies in an equivalent position and the respective projected distances of DHO atoms O71 and O72 to residues ArgB20, ThrB110 and AlaB266 are the same as for the equivalent atoms of CA-asp. In short, if DHO occupied the active site of a loop in subunit, atoms O71 and O72 would be in the correct position to hydrogen bond to ThrB110. However, this superposition predicts a potential close contact between DHO and ThrB109 (DHO O4/ ˚ ). This distance is too short to ThrB109Og1Z2.3 A be considered a hydrogen-bond20 and significantly shorter than the observed interaction between CAasp and ThrB109 (CA-asp O5/ThrB109O g1Z ˚ ). A possible mechanism for the conversion 2.6 A from loop in to loop out is as follows: the cyclization of CA-asp to form DHO converts the C4/O4/O5 carboxylate group to a carbonyl group in DHO, that can no longer bridge the Zn atoms at the active site. This causes the DHO molecule to move up and away from the active site, putting steric pressure on residue Thr109 and releasing the loop to the out conformation. What does loop movement mean to the mechanism of E. coli DHOase?
Figure 5. Lineweaver-Burk plot showing the dependence of DHOase activity upon the concentration of DHO. Positive cooperativity is indicated by upward curvature of the theoretical line generated from the Hill equation using the values VZ1.90(G0.06) pmol CAasp/minute, nZ1.57(G0.13), and KsZ20.76(G1.28) mM. Assays contained 5.7 ng DHOase in 25 ml. E. coli DHOase was assayed in the degradative direction (DHO/ CA-asp) by established procedures.26,27 To detect cooperativity, 14C-labelled DHO concentrations covered a wide range, from 0.1–200 mM. Reaction velocities were calculated from three time points and data were fitted by non-linear regression using PRISM (version 3.2, Graphpad Software, San Diego, CA) to the Hill equation: nZ VSn =ðKs C Sn Þ, where v is the reaction velocity, V is the maximal velocity, S is the concentration of DHO, Ks is the apparent dissociation constant and n is the Hill coefficient.
The introduction summarizes functions proposed for loop movements in other enzymes. It is possible that movement of the 105–115 loop into the active site of E. coli DHOase upon CA-asp binding fulfills multiple roles. Thoden et al. reported a mechanism for the DHO biosynthetic reaction in which direct molecular contacts between CA-asp and the binuclear Zn site were proposed to make the C5 atom of CA-asp more electrophilic and therefore promote bond formation, in addition to electrostatically stabilizing the proposed tetrahedral intermediate.10 It is possible that the hydrogen-bonding interaction between CA-asp and ThrB109 in the present structure provides further stabilization. Studies on porcine pepsin A, an aspartyl protease, have examined the role of Thr77, one of a tripeptide of so-called “flap residues” (Tyr75, Gly76, Thr77) shown to contribute directly to
531
Loop Movement in E. coli dihydroorotase
specificity by hydrogen-bonding to substrate residues. Kinetic analysis of the enzyme, following sitedirected mutagenesis of Thr77 to Ser, Val and Gly showed that the hydroxyl group provides an essential hydrogen-bonding interaction that contributes to a delicate network that aligns the substrate and is indirectly responsible for the proper geometry of the transition state. In addition, this interaction appears to lock the flap loop closed when a substrate is bound and in this way encloses the catalytic environment.21 In the same way, hydrogen-bonding interactions involving Thr109, Thr110 and both carboxylate groups of CA-asp in the present structure may serve to poise CA-asp in an optimum conformation for conversion to DHO. Closure of the 105–115 loop on CA-asp binding does enclose the substratebinding pocket and this may further aid catalysis by excluding water from the active site. The cavity present at the active site in the DHO-bound subunit (A) is filled with water molecules, some of which are excluded in the CA-asp-bound subunit (B). This exclusion of water from the active site during catalysis may prevent unwanted side reactions. Relevance of loop movements and cooperativity to the mechanisms of DHOases DHOases may have limited sequence homology but in general are found in native form as dimers.7 For example, E. coli DHOase is a dimer, as is the hamster DHOase domain isolated after proteolysis of the trifunctional CAD protein, or expressed in E. coli.22 The exceptions are the monomeric DHOases from parasitic organisms Crithidia fasciculata and Plasmodium berghei. 23 DHOase sequences are aligned in Figure 6. Residues corresponding to 105–115 of the E. coli enzyme are conserved in DHOases from Plasmodium falciparum, Arabidopsis thaliana, Helicobacter pylori and Saccharoymyces cerevisiae but the human and hamster DHOase domains have different sequences. The residues at each end of the loop, Pro105 and Gly115 in the E. coli protein, are also conserved in some species and may provide flexibility for the transition between loop in and loop out conformations. Interestingly, Thr110 has the same identity in all sequences aligned here and
Thr109 is conserved an all but the mammalian sequences. It is possible that hydrogen-bonding interactions between loop residues and bound CAasp are important for catalysis in many DHOases. Conclusions We have refined the structure of E. coli DHOase, ˚ crystallized in the presence of L-DHO, at 1.9 A resolution. The structure confirms many features reported by Thoden et al.10 In particular, we observe asymmetry between active sites in the dimer, with the substrate, CA-asp, bound to one active site and DHO bound to the other. Three new aspects of the structure and mechanism of E. coli DHOase have been described here: (1) residues 105–115 form loops with different conformations for each subunit within the dimer. This loop in subunit B, reaches in toward the active site, with ThrB109 and ThrB110 making hydrogenbonding contacts with the bound substrate, CAasp. The corresponding residues in subunit A do not interact with the bound product, DHO, and instead form part of the protein surface. (2) The kinetics for conversion of DHO/CA-asp at low DHO concentrations show positive cooperativity with a Hill coefficient of 1.57. (3) The asymmetry in binding CA-asp and DHO at the two active sites may be communicated between subunits by shifts in the conformations of three pairs of residues (A/B256–258), close to the dimer interface. The asymmetry in the structures of the active sites of the two subunits, indicates that there is conformational communication between them. Such a conformational link could also mediate cooperativity. Our kinetic experiments show positive cooperativity with respect to DHO. The minimal explanation is that the cooperativity is mediated by the same mechanism that provided the asymmetric dimer. In summary, binding of CA-asp accompanies movement of the 105–115 loop to the loop in conformation, held in place by hydrogenbonding interactions with ThrB109 and ThrB110. This closes the active site cavity, excludes water and correlates with conformational changes in the side-chains of residues ArgB256, HisB257 and ArgB258. These residues interact via water molecules across the dimer interface and may
Figure 6. Alignment of DHOase sequences. Abbreviations are as follows: ECOLI, Escherichia coli, accession code P05020; PFALC, Plasmodium falciparum, Q8IKA9; ARATH, Arabidopsis thaliana, O04904; HELPY, Helicobacter pylori, P56465; YEAST, Saccharoymyces cerevisiae, P20051; HUMAN, Homo sapiens, P27708; MESAU, Mesocricetus auratus, P08955. The positions of Lys102 and Thr110 (numbering for the E. coli protein) are indicated as *. Regions of high sequence similarity are shaded blue. Regions with lower similarity are shaded green and yellow. Residues corresponding to 105–115 for the E. coli protein are boxed.
532 mediate communication between subunits, which is borne out in cooperativity in the kinetics. The structure described here may represent one of the many combinations of loop conformations and occupancies of active sites possible for the DHOase dimer in solution.
Loop Movement in E. coli dihydroorotase
9.
Protein Data Bank accession numbers The atomic coordinates, structure amplitudes and phases have been deposited at the RCSB Protein Data Bank with accession code 1XGE.
10.
11.
Acknowledgements Support from the Australian Research Council (DP0209273) is gratefully acknowledged. M.J.M. is an ARC Australian Postdoctoral Fellow. The authors thank Professor F. Raushel for the XL1 Blue/DHO pBSC plasmid and Dr D.T.C. Huang for initial work on protein expression and purification.
12. 13.
14.
Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jmb.2005.01.067
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Edited by I. Wilson (Received 6 December 2004; received in revised form 20 January 2005; accepted 26 January 2005)