Structure, Vol. 9, 439–450, May, 2001, 2001 Elsevier Science Ltd. All rights reserved.
PII S0969-2126(01)00604-9
The Crystal Structure of E. coli Pantothenate Synthetase Confirms It as a Member of the Cytidylyltransferase Superfamily Frank von Delft,1,6 Ann Lewendon,2 Venugopal Dhanaraj,1,7 Tom L. Blundell,1 Chris Abell,3 and Alison G. Smith4,5 1 Department of Biochemistry 80 Tennis Court Road Cambridge, CB2 1GA United Kingdom 2 Pantherix West of Scotland Science Park Glasgow, G20 0SP United Kingdom 3 University Chemical Laboratory Lensfield Road Cambridge, CB2 1EW United Kingdom 4 Department of Plant Sciences Downing Street Cambridge, CB2 3EA United Kingdom
Summary Background: Pantothenate synthetase (EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitamin B5) synthesis. It catalyzes the condensation of pantoate with -alanine in an ATP-dependent reaction. Results: We describe the overexpression, purification, and crystal structure of recombinant pantothenate synthetase from E. coli. The structure was solved by a selenomethionine multiwavelength anomalous dispersion experiment and refined against native data to a final Rcryst of 22.6% (Rfree ⫽ 24.9%) at 1.7 A˚ resolution. The enzyme is dimeric, with two well-defined domains per protomer: the N-terminal domain, a Rossmann fold, contains the active site cavity, with the C-terminal domain forming a hinged lid. Conclusions: The N-terminal domain is structurally very similar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamily. This relationship has been used to suggest the location of the ATP and pantoate binding sites and the nature of hinge bending that leads to the ternary enzyme-pantoate-ATP complex. Introduction Pantothenic acid (vitamin B5) is the precursor of the phosphopantetheine moiety of coenzyme A (CoA) and the acyl carrier protein (ACP), cofactors required in many essential reactions, particularly of lipid metabolism. 5
Correspondence:
[email protected] Present address: Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California, 92037, USA. 7 Dedicated to the memory of Dr. V. Dhanaraj, who tragically died early but who “so much loved his protein crystals.” 6
Pantothenic acid is synthesized in microorganisms, plants, and fungi, but not in animals, and so the enzymes of the pathway are potential targets for agents against these organisms. The pathway is best understood in E. coli, where it comprises four enzymatic reactions (Figure 1) [1]. Pantothenate is produced by the condensation of pantoate, which is derived from ␣-ketoisovalerate, the oxo-acid of valine, and -alanine, produced by the decarboxylation of L-aspartate. The reaction is catalyzed by pantothenate synthetase (D-pantoate:-alanine ligase [AMP-forming]; EC 6.3.2.1), which requires ATP and proceeds via a pantoyl-adenylate intermediate that is attacked by -alanine [2, 3]. Pantothenate synthetase (PS) from E. coli was purified to homogeneity and characterized by Miyatake et al. [4]. It had a high pH optimum of 10, with a KM of 150 M for -alanine, 63 M for pantoate, and 100 M for ATP, lower than had previously been reported for partially purified enzyme preparations. Based on ultracentrifugation, gel filtration, and SDS disc gel electrophoresis, the authors proposed PS to be a tetramer of 18–20 kDa protomers. Subsequently, mutants in the PS-encoding gene, panC, were isolated, and the gene was mapped close to panB and panD [5, 6] (encoding, respectively, ketopantoate hydroxymethyltransferase and L-aspartate ␣-decarboxylase from the same pathway). The panBCD gene cluster was sequenced, allowing identification of the three pan open reading frames by insertional mutagenesis and characterization of the mutant phenotypes [7]. Miyatake et al. [4] were probably dealing with a degradation product, since panC from E. coli encodes a 31.5 kDa protein similar in size to the many panC orthologs that, at ⵑ50% pairwise sequence identity, are readily identified in various sequenced bacterial genomes and have also been isolated from the eukaryote Saccharomyces cerevisiae [8] and the higher plants Oryza sativa and Lotus japonicus [8]. PS from the latter, cloned from cDNA and expressed in E. coli, was shown by gel filtration to be dimeric. An indication of the three-dimensional fold of PS was reported by Bork et al. [9] in a modeling study on the TagD gene product from B. subtilis, encoding CTP:glycerol-3-phosphate cytidylyltransferase (CGT). There are two motifs that had been shown to be characteristic of class I aminoacyl-tRNA synthetases (tRS) [11], one of which is the so-called HIGH sequence motif [10]. Based on the conservation of this motif, Bork et al. [9] proposed a cytidylyltransferase superfamily, which includes CGT, the tRS nucleotide binding domain, and PS. All of these enzymes catalyze the formation of an acyl-adenylate (or acyl-cytidylate) species via P␣ of the nucleotide triphosphate, with release of pyrophosphate. The tRS domain was already known [12] to adopt a Rossmann fold (e.g., [13]), and Bork et al. [9] constructed a detailed model of CGT. A fold could therefore also be proposed for PS, Key words: adenylate intermediate; hinge bending; Rossmann fold; selenomethionine MAD experiment; vitamin B5
Structure 440
Figure 1. The Pantothenate Pathway in E. coli
although only the N-terminal, HIGH-containing fragment (residues 21–60) was aligned to the rest of the superfamily, due to extremely low sequence identity. In this paper, we describe the overexpression, purification, and crystal structure of recombinant PS from E. coli. Both the native form and a selenomethionine (SeMet) derivative of the protein have been purified and crystallized, and the crystal structure was solved at 1.7 A˚. Its relationship to the rest of the cytidylyltransferase superfamily is discussed, and a model is proposed for a conformational change during substrate binding. Results and Discussion Overexpression and Purification of Recombinant PS from E. coli The E. coli panC gene was isolated by functional complementation of the panC lesion of E. coli AT1371 and cloned into vector pUC19 to generate plasmid pEC. This construct allowed overexpression of recombinant enzyme protein, which could be purified in high yield. Both native and selenomethionine-substituted (SeMet) enzyme were purified using the procedure described in Experimental Procedures. Both forms had a subunit MW of 30 kDa on SDS-PAGE, close to that predicted from the gene sequence, with a solution MW of 65 kDa, estimated by size exclusion chromatography (data not shown), indicating that PS from E. coli is a dimer, like that from L. japonicus [8]. Electrospray mass spectroscopy was used to determine the extent of incorporation of SeMet into the protein. The observed mass for the native protein was 31,601.2 ⫾ 3.4 (predicted mass of 31,597), whereas that for the SeMet enzyme was 32,073.4 ⫾ 5.0 (predicted mass of 32,067). The difference in mass between the two forms was 472 ⫾ 8.2, which corresponds to the expected difference if all ten methionines in the polypeptide were replaced with SeMet. Crystals of PS were tie-shaped, and native crystals typically had dimensions of 500 ⫻ 200 ⫻ 40 m; SeMet crystals were approximately half this size. Structure Solution Data collected from crystals of the native protein indicated that they belonged to space group P21 with unit cell dimensions a ⫽ 66.03, b ⫽ 78.08, c ⫽ 77.13 A˚, and  ⫽ 103.7⬚. The structure of PS was determined by a SeMet multiwavelength anomalous dispersion (MAD) ex-
periment and was refined at 1.7 A˚ resolution (1.9 A˚ effective resolution) to a final model data residual Rcryst of 22.6% (Rfree ⫽ 24.9%) (Table 1). The absence of a well-defined “white line” in the X-ray fluorescence spectrum recorded during data collection indicated that the selenium had been partially oxidized during crystallization [14]. The Bijvoet signal of the peak wavelength (where f⬘⬘ was largest, Table 1) was strong enough to allow the complete selenium substructure to be solved with SnB [15] for 20% of all starting trials; all 20 sites were confirmed by heavy atom refinement with SHARP [16]. Phases calculated using all three MAD datasets, as well as an isomorphous native, resulted in easily interpretable electron density maps (Figure 2a), even before solvent flattening with SOLOMON [17] and in spite of significant radiation-induced crystal decay for the final MAD dataset (“Edge”, in Table 1). The final model contains all 566 expected residues of the dimer in the asymmetric unit, although density was poor for the C-terminal residue of both protomers A and B. Residues B187–193 appeared only after two cycles of density reconstruction using BUSTER [18], whereas residues B63–68 and A-B251–259 have temperature factors approaching 60 A˚2. Over 80% of the side chains assume conformations that correspond to previously observed rotamers [19], 4 have double conformations, and 12 were omitted altogether. The majority (92.2%) of the residues have favorable Ramachandran angles, and none is an outlier. Furthermore, no serious or otherwise unjustifiable deviations from expected geometry were reported by OOPS [20], PROCHECK [21], and WHATCHECK [22]. Figure 2b shows the stereo C␣ trace of one PS subunit. General Architecture PS is dimeric (Figure 2c), agreeing with the results of size exclusion chromatography. Each protomer consists of two well-defined domains, residues 1–176 and 177– 283 (Figure 2d) connected by a hinge region. The N-terminal domain has an ␣ architecture with six parallel  strands with 1⬘-3-2-1-4-5 topology, which alternate with ␣ helices to form a central  sheet sandwiched between two layers of ␣ helices. The helices (␣1⬘, 1, 2, 3, and 4) pack against the  sheet in a right-handed way, as is generally seen for ␣ supersecondary structures [23], forming the typical nucleotide binding, or Rossmann, fold [13]. The secondary structural elements have been numbered accordingly (Figure 2d), with elements
Structure of E. coli Pantothenate Synthetase 441
Table 1. Crystallographic Data Quality, Phasing, Refinement, and Model Quality Space group Cell parameters (A˚) (Rmsd) (A˚)
P21 a ⫽ 66.03 b ⫽ 78.08 c ⫽ 77.13  ⫽ 103.7⬚ (0.02) (0.02) (0.02) (0.15⬚)
Data Quality Data set Wavelength (A˚) Limiting resolution (A˚) Rmeasa ⬍I/I⬎ (high resolution) Completeness (high resolution) Number of unique reflections (multiplicity) Wilson B factor (A˚2) Experimental f⬘/f″ (electrons)b
Remote 0.88500 2.0 0.078 17.0 (2.5) 0.96 (0.79) 49,287 (7.0) 31.1 ⫺1.3/3.2
Peak 0.97918 2.4 0.082 20.6 (4.6) 0.99 (0.98) 29,782 (7.2) 47.3 ⫺7.8/5.4
Edge 0.97939 3.0 0.067 26.9 (6.8) 1.0 (0.99) 15,381 (7.0) 80.1 ⫺9.7/2.7
Native 1.1 1.7 0.103 15.9 (2.1) 0.98 (0.87) 81,752 (6.8) 28.0 —
⫺/1.50 — ⫺/0.83 0.35/0.91
46.8/2.68 0.87 0.85/0.73
1.18/2.08 0.82 0.86/0.86
1.09/⫺ 0.85 0.85/⫺
Phasing Phasing powerc: Acentrics (iso/ano) Centrics Rcullisd for acentrics (iso/ano) Figure of Merite (cent/acent) Refinement (40–1.7 A˚) Rcrystf Rfreeg Number of reflections: working/testg Number of restraints Number of parameters
0.226 (highest resolution: 0.26) 0.249 (highest resolution: 0.29) 77,294/4,062 15,730 20,236
Model Contents and Quality Number of: Residues Hetero groups Non hydrogen atoms Solvent molecules Average B factor (A˚2) Subunit A Subunit B Solvent Ramachandran plot: % residues favorable % disallowed Rmsd (% outliersh): Bond lengths (A˚) Bond angles (⬚) Planarity (A˚)
566 1 Tris, 2 ethanol 5,059 622 33.9 36.4 47.8 92.7 None 0.018 (2.6) 1.41 (3.1) 0.007 (1.0)
Rmeas ⫽ [⌺hw⌺i|⬍Ih⬎ ⫺ Ih,i|]/⌺h⌺iIh,i, where w ⫽ [nh/(nh ⫺ 1)]1/2 and ⬍Ih⬎ ⫽ [⌺ni Ih,i]/nh. This is the multiplicity-weighted Rsymm [54]. Estimates from fluorescence scans using CHOOCH (Gwyndaf Evans, Laboratory of Molecular Biology, Cambridge, UK). c Phasing power ⫽ ⬍|FH|/⑀⬎; ⑀ ⫽ |FPH ⫺ |FP ⫹ FH||, the residual lack-of-closure (LOC); FH is the calculated heavy atom structure factor amplitude; FPH, FP are the trial structure factors with and without heavy atoms. Numbers as output by SHARP [16]. d Rcullis ⫽ ⬍LOC⬎/⬍|FPH ⫺ FP|⬎. e FOM ⫽ ⬍cos⌬␣j⬎; ⌬␣j is the phase angle error for phase angle j. f Rcryst ⫽ ⌺||Fobs| ⫺ |Fcalc||/⌺|Fobs|; Fobs and Fcalc are observed and calculated structure factor amplitudes respectively. g Rfree: cross validation Rcryst, i.e., calculated using randomly selected test data not used in refinement. h Percentage parameters flagged as outliers by PROCHECK [21]. a
b
that are insertions or additions to the “standard” fold denoted by primes. Strand 5 leads directly into the short  hairpin (6 and 7) and 310 helix motif (3107), which lies at the head of the C-terminal domain and is likely to be involved in phosphate binding. The rest of the domain has a simple two-layer organization: a helix-turn-helix (␣8 and 9), layered above a flat sheet of three antiparallel  strands (10–12). This sheet faces a prominent cleft in the N-terminal domain, the predicted catalytic region, so that the C-terminal domain acts as a hinged lid over
the N-terminal domain, a common arrangement in twodomain enzymes. There are four clusters of buried, hydrophobic side chains. Two occur in domain N, where there are clusters on both sides of the central  sheet, as a result of packing with the flanking ␣ helices. A third occurs in the C-terminal domain, where the core is the extended packing of helices ␣8 and ␣9 against the  sheet through hydrophobic interactions. A fourth hydrophobic cluster forms at the domain interface, involving residues of helices 310D1/2 and the loop between strands 10 and 11.
Structure 442
Figure 2. General Structure (a) Representative experimental electron density (solvent-flattened MAD phases) and final, refined, atomic model of the phosphate binding KMSKS motif residues in subunit A. (b) Stereo C␣-trace of one PS subunit, colored by position in the sequence, with every tenth residue numbered. (c) Stereo cartoon representation of the PS dimer, with subunits colored differently, and secondary structure numbered as in (d). The view is approximately along the two-fold axis (shown as a dot) relating the N-terminal domains. The figure was prepared with MOLSCRIPT [53]. (d) Schematic diagram of the secondary structural elements of one PS subunit;  strands are shown in blue, ␣ helices are shown in yellow, and 310 helices are shown in orange. For the Rossmann domain, ␣ pairs have the same number, and structural elements of the dimerization insertion are denoted by D. P176 denotes the approximate domain boundary.
Dimerization PS crystallizes as a dimer in the asymmetric unit, but the two protomers are not identical. Whereas the N-terminal domains are related by proper two-fold symmetry, the
two C-terminal domains differ in orientation relative to their respective N-terminal domains, requiring a further 14⬚ rotation to superpose them when the N-terminal domains are aligned. Therefore, the symmetry axes that
Structure of E. coli Pantothenate Synthetase 443
relate the two N- and C-terminal domains in the two protomers do not coincide, and they have rotations of 179.3⬚ and 177.6⬚, respectively. The N-terminal domains are more similar to each other than are the C-terminal domains (rmsd 0.37 A˚ versus 0.69 A˚). The side chains of the two faces of the 1340 A˚2 dimerization interface do not have the perfect symmetry that could be expected (1.22 A˚ rmsd for all atoms of residues 15–23), since the interface is asymmetrically disrupted along one edge by a crystal contact with the catalytic region of a symmetry-related molecule. Interface interactions (data not shown) are both polar and hydrophobic: an H-bonded polar cluster (Ser135, Asn139, and three waters) inside a nonpolar cage (Val109, Met166, and Phe168) form the core. This is surrounded by an intersubunit two-stranded antiparallel  sheet (D from each protomer; Figure 2c), a second polar cluster (Tyr108, Asp110, and Arg128), salt bridges (His106– Asp165 and Arg11–Asp169), and numerous water-mediated H bonding interactions. The residues in the interface are poorly conserved among the known PS sequences from both plants and microorganisms, which share about 50% pairwise similarity to one another over the entire protein [8]. In all the known plant sequences, the dimerization domain is preceded by an insertion of about 20 amino acids (between residues 99 and 100 relative to the E. coli sequence). Crystal Packing Interactions and Subunit Differences Protomers A and B contain some significant differences, the result of differing crystal packing environments. The average B factor of A is 4 A˚2 lower than that of B, since it is more tightly constrained by the crystal lattice; A has twice as much surface area as B involved in crystal contacts (1836 versus 953 A˚2, excluding the dimer interface). Similarly, B contains poorly ordered regions that are well ordered in A, where they are involved in crystal packing (residues 63–68 and 187–193). The second stretch of residues contains the putative phosphate binding residues (see section on “ATP Binding”), and their disorder in B is likely to be more characteristic of the apo-enzyme in vivo. The crystal contact that orders them in A is also responsible for the asymmetry of the dimer interface and the different relative orientations of the C-terminal domains, which are more closed in A than in B. Structural Homologs and Superfamily Definition The highest ranking structural matches to the C␣ coordinates of the N-terminal domain that were found by the DALI server [24] were the ATP binding domains of some of the class I aminoacyl-tRNA synthetases (tRS): those of the glutamyl-, glutaminyl-, and tyrosyl-tRNA synthetases (EtRS from Thermus thermophilus [25], QtRS from E. coli [26], and YtRS from Bacillus stearothermophilus [12]; Protein Data Bank (PDB) codes 1gln, 1gtr, and 2ts1, respectively). Since these are members of the cytidylyltransferase superfamily, this confirms the prediction of Bork et al. [9] that PS, too, is a member of this family. Recently, both phosphopantetheine adenylyltransferase (PPAT) from E. coli (PDB code 1b6t) and CTP:glycerol-3-phosphate cytidylyltransferase (CGT) from B.
subtilis (PDB code: 1coz) were likewise confirmed experimentally to be members of the superfamily [27, 28]. The superposition of the N-terminal domain of PS on PPAT, CGT, and the Rossmann fold domains of QtRS and EtRS is shown in Figures 3a and 3b. This particular subclass of Rossmann fold domains is distinguished by having five  strands in the central sheet (as opposed to the more usual six) and a cleft between  strands 1 and 4, which are usually more closely H bonded, at the adenosine binding site. Whereas the PPAT and CGT structures consist only of the minimum elements required, both PS and tRS have insertions between the two “halves” of the fold, between strand 3 and helix 4. In tRS, the insertion binds the 3⬘ terminus of the tRNA molecule, while, in PS, it contains the elements necessary for dimerization. A structure-based alignment of the amino acid sequences of PS, QtRS, EtRS, YtRS, CGT, and PPAT (Figure 3c) shows that few residues are conserved and sequence identities are low, the highest being 18% identity to CGT (Table 2); this is also the case between the various tRS sequences themselves. However, the two sequence motifs that have previously been noted in tRS, HIGH [10] and KMSKS [29], are boxed in Figure 3c; the HIGH motif binds the adenine portion of ATP (cytidine in CGT), whereas the KSMKS motif stabilizes the - and ␥-phosphate groups [30]. In contrast to the HIGH motif, the KMSKS motif is only evident in a structural alignment, and Bork et al. [9] were unable to identify it in PS with their sequence-based approach. What does appear conserved in the motif, apart from serine at position three, is a requirement for either basic or hydroxyl functionality in the last two amino acids, with the order varying across the superfamily, as the PS structure now shows. As reported by the DALI server [24], the organization of the C-terminal domain of PS is similar to fragments of many known folds (e.g., methionine aminopeptidase [1xgs], inositol monophosphatase [1imb], and aminopeptidase P [1az9]). However, in none of these matches does the fragment on its own form a whole domain, as it does in PS. Background to Substrate Binding The members of the superfamily catalyze chemically closely related reactions (Figure 4), namely, the nucleophilic attack of a substrate on the ␣-phosphate group of ATP- (or CTP-) releasing pyrophosphate, forming either an activated adenylate intermediate (PS and tRS) or an adenylated (cytidylylated) product (PPAT and CGT). Given this chemical similarity, and since, to date, neither substrate-soaking nor cocrystallization experiments have been successful, we have used structural observations of ligands bound to tRSs, PPAT, and CGT to make some deductions about substrate binding in PS. ATP Binding Of all the structures that have ATP bound , that of QtRS [22] is the closest to PS. The location of the ATP corresponds closely to the positions of the bound nucleotides in YtRS [12], PPAT [27], and CGT [28]. It is found in the cleft between strands 1 and 4 of the Rossmann fold
Structure 444
Figure 3. Structural Alignment and Superposition (a and b) Superposition of the PS domain N (red) with the matching secondary structure elements from (a), CGT (blue) and PPAT (gold), and from (b), QtRS (light blue) and EtRS (green). Also shown is ATP found in QtRS. The figure was prepared with MOLSCRIPT [53]. (c) Structure-guided alignment of the sequences of the homologous Rossmann domains, in JOY format [52]; this is a more complete and accurate alignment than the purely sequence-based alignment of Bork et al. [9]. The HIGH and KSMKS motifs are indicated by large boxes, and residues in QtRS, CGT, and PPAT that interact with ATP (or CTP) are indicated by small boxes. PS residues proposed to contact ATP are in shaded boxes and marked for complete (solid circle) or partial (asterisk) conservation among orthologous PS sequences. The three residues shaded in blue are in different one-dimensional, but equivalent three-dimensional positions. (See http://www.cryst.bioc.cam.ac.uk/ⵑjoy for key to symbols.)
Table 2. Similarity of PS with Homologous Structures
Sequence identity to PS (%) Rmsd of C␣ atoms (A˚) Number of C␣ aligned for rmsd
QtRS
EtRS
YtRS
CGT
PPAT
10.8 1.8 65
14.5 1.5 62
11.2 2.2 68
27.3 1.8 63
14.5 1.8 76
Sequence identity is calculated only for those residues used for rmsd calculation.
Structure of E. coli Pantothenate Synthetase 445
Figure 4. The Reactions of the Superfamily Comparisons of the reactions catalyzed by PS [2,3], QtRS [23], CGT [28], and PPAT [27]. The step shown is fundamentally the same for all reactions: attack of some nucleophilic, acidic oxygen atom on the ␣-P atom of a nucleotide triphosphate to release pyrophosphate. For PS and QtRS (and the other tRS), this is the first of two reaction steps; for PPAT and CGT, there is only one step.
and against the top of helix ␣1, the location of the HIGH motif. When this domain of QtRS is aligned with the N-terminal domain of PS (Figure 3b), the HxGH residues line up (Figure 3c), and the QtRS-bound ATP fits into the same cleft in PS (Figure 5a), moving by only 1.3 A˚ (rmsd between all ATP atoms before and after) when the
fit is optimized by energy minimization (in the program SYBYL, using the implemented TRIPOS force field [31]). Despite this ease in positioning the ATP into PS, there is a significant difference in the positions of the helices 3107 (in PS) and ␣I (in QtRS) relative to the Rossmann domain (Figure 5a). This is the location of the KMSKS
Figure 5. Domain Movement (a) The orientation of the PS C-terminal domain (yellow) relative to the N-terminal domain (bright red) in the crystal structure. (b) The orientation of the PS C-terminal domain (yellow) relative to the N-terminal domain (bright red) after the modeled rotation (arrow in [a]) that aligns helix 3107 (dark red ribbon) with helix ␣I of QtRS (dark green ribbon). For both PS and QtRS, KMSKS C␣ atoms are shown as spheres (gray and light green, respectively), and residues of strand 5 up to the helices are shown as C␣-trace (dark red and dark green, respectively). PS residues that were rotated are blue in (b). ATP (orange) is shown in (a), in the way it is bound to QtRS that has been aligned to PS, and in (b), in its position after minimization; it binds against the HIGH residues (pink spheres on helix ␣1 in [a]). The position of the positive charge that binds P␣ in QtRS (K270, dark green in [a]) is retained in this model of PS (S188 and Mg2⫹, gray and light blue, respectively, in [b]), and R189 (shown but not labeled) may also bind P␣. The figure was prepared with MOLSCRIPT [53].
Structure 446
Figure 6. Substrate Binding Model (a) Proposed ATP binding interactions. (b) Proposed binding of ATP and pantoate in the active site. The molecular surface (green) was calculated after energy minimization of the modeled PS-ATP complex, and the pocket appears perfect for the bulky pantoate. The apparent “clash” with the surface is caused by the H bonding of the pantoate carboxylate to both Q61 and Q155, since the surface was calculated with hydrogen atoms added. The putative line of attack by pantoate is shown (red dotted line). The figure was prepared with SYBYL [31]. (c) Schematic diagram of pantoate-PS interactions. Note the cluster of conserved, hydrophobic residues to accommodate the two methyl groups. Both hydroxy groups can be accommodated, and the carboxylate group is at a suitable distance for attack.
motif that binds the - and ␥-phosphate groups of ATP in both QtRS and CGT. However, by changing conservatively the φ/-angles of residues V175, P176, I177, and M178, which form the PS interdomain linker main chain, the C-terminal domain can be rotated (30.3⬚) to align
these residues with their QtRS counterparts and thus involve them in phosphate binding (Figure 5b). The domain rotation also brings OGSer188 close to the three atoms O1ATP, O␥3ATP, and OHTyr71, thus creating a suitable Mg2⫹ binding site that corresponds to the proposed Mg2⫹
Structure of E. coli Pantothenate Synthetase 447
binding region in PPAT [27]. This also appears to satisfy a functional requirement, since inspection of the residues that are structurally equivalent to PS Ser188 (Figure 3c) reveals that all tRS structures have lysine at this position (Lys270 in QtRS), whereas PS, CGT, and PPAT have serine/threonine. Placing the Mg2⫹ in PS as proposed means that the positive charge in this position is conserved between PS and the tRS structures (Figure 5b). Minimization, performed as before, of the modeled PS-ATP-Mg2⫹ complex, but with the C-terminal domain rotated and additional constraints for the Mg-O bonding distances of 2.05 A˚ (estimated from the Cambridge Structural Database [32]), gives rise to the ATP binding interactions shown in Figure 6a. No waters were added, so the proposed H bonding network is incomplete. The adenine ring is sandwiched between Gly36 and Gly149, with Val175, Ile177, and Met178 (from strand 5) forming the hydrophobic back of the pocket, and NE2His34 and OLeu186 forming hydrogen bonds with N7ATP and N6ATP, respectively. The ribose O2* is H bonded to both NHGly149 and CO2⫺Asp152 . In addition to the positive Mg2⫹ ion, Ser187 and two basic side chains, Lys151 and Arg189, bind the phosphate groups. This involvement of Arg189, which follows naturally from the modeled domain movement, implies that PS employs the KMSKS residues in a novel way, since in neither QtRS nor CGT does the last KMSKS residue bind ␥-phosphate. The Movement of the C-Terminal Domain For most enzymes that bind ATP and the substrate, the apo-enzyme has an open structure that is closed by “hinge bending” when substrates bind, so that water is excluded from the active site during catalysis (e.g., [33]). PS contains a similar domain flexibility, since in the crystal structure, the orientation of the C-terminal domain relative to the N-terminal domain differs between subunit A and B of PS, due to rotations about residues 176 and 177. Furthermore, crystals of PS are fragile and dissolve when ATP is included in the stabilizing solution. In the model for ATP binding by PS proposed above, the changes in torsion angle required to achieve the domain reorientation are small, lead to no unfavorable steric interactions (only Met178 requires repositioning), and do not violate backbone torsion angle restrictions. The conserved residues 187–189 are structurally equivalent to the phosphate binding motif in QtRS, EtRS, and CGT structures (Figure 3c), directly following strand 5 and forming the end of a short  hairpin and the N terminus of a helix (310 in PS); the NH groups at the positive pole of the helix dipole would enhance phosphate binding when aligned as in QtRS. In QtRS, CGT, and PPAT these helices and strands are in similar relative positions, with the bound triphosphates of QtRS and CGT in very similar horseshoe conformations. Alignment of the KMSKS residues in PS with those from QtRS and CGT brings them into closer proximity with the substrate phosphates. The resulting hydrophobic cavity accommodates adenine better, and there is increased positive charge available to stabilize the phosphate moiety. Although the positions of the KMSKS residues differ from one another in the available tRS structures, only
QtRS, with tRNA bound, is considered to represent the conformation that stabilizes the transition state of the adenylation reaction [30, 34]. In contrast, the observed position of the motif in PS corresponds to that in EtRS, which has neither nucleotide nor tRNA bound and is therefore unlikely to be in the active conformation. The proposed domain movement shifts the KMSKS residues in PS from the EtRS-like to the QtRS-like position; liganded and unliganded tryptophan tRS has also been shown to have such differing domain orientations [35]. L-Pantoate Binding There is no known structural binding motif for pantoate; consequently, its binding site cannot be defined as confidently as that of ATP. However, a Connolly surface (Figure 6b) reveals an appropriately sized pocket beside the ATP ribose group, with walls formed by fully conserved and predominantly hydrophobic residues. The most favorable conformer of pantoate can be readily modeled into this pocket (Figure 6c); Pro28, Met30, Ile133, Val134, and Leu137 provide hydrophobic interactions for the two methyl groups, and H bond interactions are available for the hydroxyl and carboxylate groups (Figure 6c). Minimization, as described previously, of the entire complex (PS, ATP, Mg2⫹, and pantoate) positions one of the pantoate carboxylate oxygens within 3.8 A˚ of the ␣-phosphate of ATP, in a geometry appropriate for the reaction to proceed. This tight binding of pantoate is consistent with the observation that pantoate analogs are not processed by PS if there is more than one additional methyl(ene) group attached to the existing methyl groups [3]. Additionally, the respective nucleophilic aminoacyl groups bind in equivalent sites on QtRS [36] (PDB code 1qtq) and YtRS [12]. Biological Implications In this paper, we have presented the X-ray crystal structure of PS, the final enzyme of the biosynthetic pathway for pantothenate (vitamin B5). Pantothenate is the precursor to coenzyme A and ACP, and so its biosynthetic pathway is an essential cellular process. The structure confirms biochemical data suggesting that the enzyme is a dimer and provides the means to study the mechanism of the enzyme. Pantothenate is thought to have existed in the prebiotic world [37], and, as such, enzymes for its synthesis may have evolved early on. It is thus of considerable interest that PS is a member of the superfamily that includes tRSs, another group of enzymes of ancient origin, and PPAT, an enzyme involved in the synthesis of coenzyme A, which is itself derived from pantothenate. Despite the very low levels of primary sequence identity between members of this superfamily, the conservation of structural domains has allowed detailed insights into substrate binding. This superfamily strikingly illustrates the extent to which proteins can diverge while retaining not only structure, but also function. Although the role of the KMSKS motif, namely, to bind phosphate, has apparently not changed, both its composition and exact mode of binding certainly have. It is ironic that it was once hoped that PS,
Structure 448
catalyzing an ATP-dependent amide bond formation, would aid our understanding of protein synthesis [2], whereas now it has been the tRS structures that have illuminated aspects of PS. Further insight will require structural information about liganded enzymes as well as mutagenesis coupled to kinetics, which will in turn provide the template for rational inhibitor design. Experimental Procedures Cloning and Expression of the E. coli panC Gene All recombinant DNA techniques were carried out according to Sambrook et al. [38]. The E. coli panC gene was isolated by screening a library of fragments from a partial HpaII digest of genomic DNA from E. coli K12 SK3430 for plasmids capable of complementing the panC lesion of E. coli AT1371 [6]. One clone was obtained that contained a 2.5 kb insert and also complemented the panB mutant YA139 [6]. A 1.3 kb PvuII-EcoRI fragment of this insert, containing the entire panC gene, was subcloned into SmaI-EcoRI-digested pUC19, such that the panC gene was under the control of the lacZ promoter. This plasmid was named pEC. For overexpression of pantothenate synthetase, E. coli AT1371 cells harboring pEC were grown overnight at 37⬚C on LB medium containing ampicillin (100 g/ml). The lacZ gene was induced by the addition of IPTG (70 g/ ml) at the start of the culture. Expression of Selenomethionine-Labeled Protein Selenium-labeled protein was produced as described by van Duyne [39], by methionine pathway feedback inhibition in the presence of 50 mg/l selenomethionine (SeMet). E. coli AT1371 cells harboring pEC were grown in minimal medium containing ampicillin (100 g/ ml) and the following additions to supplement the growth requirement of AT1371: L-arginine (127 mg/l), L-histidine (16 mg/l), L-proline (230 mg/l), and adenine (68 mg/l). Starter cultures lacked selenomethionine and inhibitory amino acids. For each purification, a 1 ml starter culture was used to inoculate 250 ml of culture medium. As before, overexpression was continuously induced with IPTG in an overnight culture at 37⬚C, and the cells were harvested by centrifugation. Purification of Recombinant Pantothenate Synthetase For the purification of both native and SeMet-substituted pantothenate synthetase, essentially identical procedures were followed. Harvested cells were sonicated in TD buffer (Tris-HCl [pH 7.5] [50 mM] and DTT [0.1 mM, 5 mM for SeMet protein]), and cell debris was removed by centrifugation. Ammonium sulfate was added to 29.1% (w/v), and precipitated protein was recovered by centrifugation. The pellet was resuspended in TD buffer, followed by overnight dialysis, and then subjected to anion exchange chromatography (Pharmacia Q-Sepharose, 16/10; 4⬚C; protein eluted in a 0–500 mM NaCl gradient in TD buffer over 75 min at 5 ml/min). Fractions containing pantothenate synthetase protein were loaded onto a size exclusion column (Pharmacia S200HR, 26/60; 500 mM NaCl), followed by dialysis and then affinity chromatography (Pharmacia Blue Sepharose HiLoad, 16/10; protein eluted with 5 column volumes ATP [10 mM] solution). ATP was completely removed by buffer exchange during concentration in an Amicon ultrafiltration cell, (5K MW cutoff membrane) to 20 mg/ml. Throughout the purification procedure, fractions were monitored by SDS-PAGE. Electrospray Mass Spectrometry To assess the incorporation of selenomethionine, 10 l of either native or SeMet-substituted protein (0.1 mg/ml) in aqueous formic acid (2.5%) and acetonitrile (50%) were injected (at 4 l/min) into an electro-spray quadrupole mass spectrometer (Micromass Quattro LC) calibrated with horse-heart myoglobin. Fifty averaged quadrupole scans were baseline-corrected, smoothed, and at least 20 peaks were transformed to calculate the main component mass. Crystallization and Cryoprotection PS was crystallized by hanging drop vapor diffusion in 24-well Linbro plates (Hampton Research). Hanging drops were composed of equal
volumes of protein and reservoir solutions (50 mM Tris-HCl [pH 8]; 4%–6% PEG 4000). Crystals grew to a size of 500 ⫻ 200 ⫻ 40 m in ⵑ2–4 days at 19⬚C and were soaked for 5 min successively in 4, 8, 12, etc., to 28% glycerol-enriched mother liquors (and 2 mM DTT for SeMet PS) before cryogenic mounting in cryoloops [40]. MAD Data Collection and Processing A three-wavelength MAD dataset was collected on beamline X-25 of the NSLS (Brookhaven National Laboratories) from a SeMet pantothenate synthetase crystal selected after extensive screening for resolution limits, low mosaicity, and low background scatter. Experimental fluorescence spectra (automatically remeasured every 180 frames) were used to select X-ray wavelengths that optimized the anomalous signals within and between datasets (Table 1). Each dataset comprised a complete 360⬚ oscillation for high observational multiplicity, with opposing 45⬚ segments of reciprocal space consecutively covered; exposure times were reduced to eliminate detector saturation and prolong crystal lifetime. A closely isomorphous native crystal (cell parameters agreeing within 0.4%) yielded a fourth dataset, collected in two passes aimed at measuring high- and lowangle reflections, respectively. Images were indexed, reflections were integrated with MOSFLM [41] and scaled and merged (each dataset separately) with SCALA [42], and datasets were scaled to one another with FHSCAL [43]. Location of Selenium Sites and Phasing Bijvoet differences of the peak dataset (where f⬘⬘ was largest) were normalized using DREAR [44] and used for solution of the selenium substructure with SnB [15]; default parameters were used for both programs. After SHARP [16] refinement of the 11 strongest SnB sites against the peak dataset, log-likelihood residual maps indicated the remaining 9 selenium sites. The SHARP selenium-sulfur pseudoatom was used in the final heavy atom model for refinement and phasing against all SeMet and native datasets simultaneously, followed by solvent flattening with SOLOMON [17], using a solvent content of 45%. Model Building and Refinement An initial model was built into experimental electron density maps using O [45], followed by three cycles of restrained isotropic, phased refinement (REFMAC [46]), and manual rebuilding in O into Aweighted [47] 2Fo-Fc maps. Half the ordered solvent could be automatically modeled by cycles of ARP [48] and REFMAC; but three ultimate cycles of rebuilding/refinement using BUSTER/TNT [18, 49] first revealed 300 additional water molecules (by ARP’s automatic criteria), and upon their inclusion, interpretable electron density for all remaining breaks in the model. The final solvent model has a smooth B factor distribution without outliers (assessed using ACT [43]). Although the data resolution limit was effectively 1.9 A˚, significant reflections were present even up to 1.7 A˚ and were included in all refinements. Structural Superpositions Structural homologs identified by the DALI server [24] were submitted pairwise with PS to the COMPARER server [50] for automatic three-dimensional alignments, which were refined with LSQMAN [51], using secondary structural elements 1, ␣1, 2, ␣2, 3, ␣3, 4, ␣4, and 5. Sequence alignments were manually adjusted accordingly and formatted with JOY [52]. Acknowledgments We thank Jennifer Ashurst for help with protein expression, Pietro Roversi for help with the BUSTER refinement, the Buster Development Group for making the program available, Hal Lewis and Lonnie Berman for beamline support, and Joe Patel for help with the figures. Research was carried out at the National Synchrotron Light Source, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Division of Materials Sciences and Division of Chemical Sciences under contract number DE-AC02-98CH10886. The project was supported by the Biotechnology and Biological Sciences Research Council of the UK.
Structure of E. coli Pantothenate Synthetase 449
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Accession Numbers The coordinates of PS have been deposited in the Protein Data Bank and assigned accession number 1iho.
