X-ray crystallographic and NMR studies of pantothenate synthetase provide insights into the mechanism of homotropic inhibition by pantoate Kalyan Sundar Chakrabarti*, Krishan Gopal Thakur, B Gopal and Siddhartha P Sarma Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India Keywords competitive inhibition; NMR; pantothenate biosynthesis; substrate binding; X-ray crystallography Correspondence S P Sarma, 207, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India Fax: +91 80 23600535 Tel: +91 80 22932839 E-mail: sidd@mbu.iisc.ernet.in *Present address Department of Biochemistry and Howard Hughes Medical Institute, MS009 Brandeis University, Waltham, MA, USA Database The 1HN, 15N, 13Ca, 13Cb and 13C¢ chemical shift values of the dimeric N-terminal domain of Escherichia coli pantothenate synthetase have been deposited in BioMagResBank (http://www.bmrb wisc.edu) under the accession number 6940 The structure factor file and the atomic coordinates of the dimeric N-terminal domain of Escherichia coli pantothenate synthetase bound to three molecules of pantoate have been deposited in the Protein Data Bank under the accession number 3GUZ (Received 26 July 2009, revised October 2009, accepted 24 November 2009) The structural basis for the homotropic inhibition of pantothenate synthetase by the substrate pantoate was investigated by X-ray crystallography and high-resolution NMR spectroscopic methods The tertiary structure of the dimeric N-terminal domain of Escherichia coli pantothenate synthetase, ˚ determined by X-ray crystallography to a resolution of 1.7 A, showed a second molecule of pantoate bound in the ATP-binding pocket Pantoate binding to the ATP-binding site induced large changes in structure, mainly for backbone and side chain atoms of residues in the ATP binding HXGH(34–37) motif Sequence-specific NMR resonance assignments and solution secondary structure of the dimeric N-terminal domain, obtained using samples enriched in 2H, 13C, and 15N, indicated that the secondary structural elements were conserved in solution Nitrogen-15 edited twodimensional solution NMR chemical shift mapping experiments revealed that pantoate, at 10 mm, bound at these two independent sites The solution NMR studies unambiguously demonstrated that ATP stoichiometrically displaced pantoate from the ATP-binding site All NMR and X-ray studies were conducted at substrate concentrations used for enzymatic characterization of pantothenate synthetase from different sources [Jonczyk R & Genschel U (2006) J Biol Chem 281, 37435–37446] As pantoate binding to its canonical site is structurally conserved, these results demonstrate that the observed homotropic effects of pantoate on pantothenate biosynthesis are caused by competitive binding of this substrate to the ATP-binding site The results presented here have implications for the design and development of potential antibacterial and herbicidal agents Structured digital abstract l MINT-7301221: PS (uniprotkb:P31663) and PS (uniprotkb:P31663) bind (MI:0407) by x-ray crystallography (MI:0114) l MINT-7301241: PS (uniprotkb:P31663) and PS (uniprotkb:P31663) bind (MI:0407) by nuclear magnetic resonance (MI:0077) doi:10.1111/j.1742-4658.2009.07515.x Abbreviations cPS, C-terminal domain of pantothenate synthetase; HSQC, heteronuclear single quantum coherence; nPS, dimeric N-terminal domain of Escherichia coli pantothenate synthetase; PS, pantothenate synthetase; TEV, tobacco etch virus; TROSY, transverse relaxation optimized spectroscopy FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS 697 Structure and binding studies of nPS K.S Chakrabarti et al Introduction Inhibition by substrates is an important feature of metabolic enzymes that remains poorly characterized in terms of structure An intriguing aspect of substrate-induced reversible inhibition is that it may vary substantially even among closely related homologues Pantothenate synthetases (PSs) from all species catalyze the condensation of a molecule of b-alanine with a molecule of pantoate in an ATP-dependent manner to form pantothenate [1,2] Pantothenate itself is an important cofactor that is essential for CoA biosynthesis, and functions in fatty acid synthesis and energy metabolism [2] The structure and function of PSs from several prokaryotic and eukaryotic organisms have been reviewed recently [2] Of special interest are the PSs from the bacteria Escherichia coli [3–6] and Mycobacterium tuberculosis [7], and from the plants Oryza sativa [8], Arabidopsis thaliana [9,10] and Lotus japonicus [8] Analysis of the primary sequences of this enzyme from E coli, M tuberculosis and A thaliana have shown that there is a high degree of conservation ( 40% sequence identity) among the enzymes from various sources, with the E coli and A thaliana enzymes sharing 42% [9] sequence identity, and the M tuberculosis and A thaliana enzymes sharing 36% sequence identity [11] Recently, it has been shown that, unlike their bacterial counterparts, the plant PSs are subject to allosteric control, and that this allosteric control arises from the homotropic effects of one of the substrates, i.e pantoate [8,9] One of the interesting features of the plant PSs is the presence of a conserved 24 residue insertion in the sequence [9] This sequence of amino acids, which is missing in the sequence of bacterial enzymes, is thought to play a significant role in the proposed allostery in plant PSs [9], through long-range interactions All PSs are known to be dimeric, with distinct N-terminal and C-terminal domains in each protomer Structural studies have shown that the catalytic site residues, as well as the dimerization interface, lie in the N-terminal domain of the bacterial enzyme Enzymatic and other biochemical studies have shown that for PS from M tuberculosis, pantoate, ATP and b-alanine have Km values of 0.13 mm, 2.6 mm and 0.8 mm, respectively Enzymatic studies of plant PSs have indicated that the affinity of these substrates echoes that observed for the bacterial enzyme However, pantoate at concentrations above mm has been shown to inhibit the plant enzyme [8,9] This inhibition by pantoate is thought to occur by binding of pantoate at a noncanonical site, although with much lower affinity ( 100-fold weaker) [8,9] Knowledge of the canonical 698 binding site comes from the well-documented structures of the M tuberculosis PS in the substrate-bound forms [11,12] ATP at high concentration ( mm) has been shown to offset the inhibitory effect of pantoate on this enzyme Furthermore, the plant PS exhibited negative cooperativity for b-alanine when pantoate was present at a high concentration To date, no information is available on the structural basis for this inhibition of this enzyme It is also hypothesized that this negative cooperativity has no regulatory function but serves to ensure robust synthesis of pantothenate from low amounts of pantoate [9] Here, we report the results of the structural studies of the pantoate-bound form of the N-terminal domain of E coli PS (nPS) The structure and substrate-binding interactions of E coli nPS have been studied using solution NMR as well as X-ray crystallography It is important to note that the structure of the E coli protein in the substrate-bound form has not been reported [13] Details of the structural basis for the biochemically observed inhibition of plant PS by pantoate, as well as of the alleviation of this inhibition by another substrate of the enzyme, ATP, are presented [8,9] Results Solution properties of the N-terminal domain of PS, the C-terminal domain of PS, and full-length PS Full-length PS is a dimer of 63 kDa The protein was found to aggregate at the concentrations used for the NMR studies, and did not provide spectra of adequate quality under a number of different sample conditions (phosphate buffer, pH 6.8; acetate buffer, pH 5.5; and Tris buffer, pH 7.5), in the presence of substrates (10 mm pantoate, ATP, and b-alanine), in the presence of protein stabilizers such as arginine, glutamate and proline [14–17], or even in the presence of detergents (Chaps [18] or dodecylphosphocholine) [17] Like PS (283 amino acids), nPS (residues 1–176) exists as a dimer in solution Evidence for this comes from gel filtration data and from the experimentally determined rotational correlation time (sc) of 17.25 ns for nPS, calculated from the measured average 15N T1 and T2 relaxation time constants [19] The C-terminal domain of PS (cPS; residues 177– 283) showed spectra of poor quality, similar in nature to that of the full-length PS under similar conditions Coconcentration of cPS with nPS did not prevent aggregation of cPS It is possible that the C-terminal FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS K.S Chakrabarti et al domain is responsible for the aggregation of the fulllength PS proteins at the concentration required for NMR experiments The studies leading to structural and dynamic characterization of the PS molecule were limited to the nPS protein The nPS protein has a V52A mutation, which was confirmed by MS and DNA sequencing Sequence-specific resonance assignments and secondary structure of nPS As mentioned above, nPS is a dimeric molecule ( 40 kDa) Under the circumstances, the backbone and side chain resonance assignments could only be obtained on samples that were enriched in 2H, 13C and 15 N Figure shows the assigned 1H–15N correlation spectrum of nPS Sequence-specific resonance assignment for the backbone 1HN, 13Ca, 13Cb, 13C¢ and 15N nuclei of the homodimeric nPS was obtained from transverse relaxation optimized spectroscopy (TROSY) [20] versions of HNCA, HN(CO)CA, HNCACB, HN(CO)CACB and HNCO [21–23] experiments The resonance assignments leading to the establishment of sequential connectivity for Glu5–Glu20 are shown in Fig S1 The backbone assignment for the amide H–15N pairs is 93% complete, whereas the backbone and side chain 13Ca and 13C¢ and 13Cb carbons are assigned to the extent of 97%, 93% and 93%, respectively (BMRB accession number 6940) [24] The H–15N correlation in the TROSY experiment is missing for Asp35, Asn101, Thr103, Glu104, Tyr108, Phe127, Thr132, Val134, Leu137, Leu140, and Phe153 These resonances were not visible even after denaturation and refolding to facilitate back-exchange of amide protons from bulk solvent (H2O) These resonances, especially the ones concentrated near the dimer Structure and binding studies of nPS interface, are missing, presumably owing to the fact that the residues undergo intermediate exchange on the NMR timescale The secondary structure of nPS was found to be, with some differences, nearly identical to the N-terminal domain in the crystal structure reported here (see below) and of the full-length structure reported earlier [13] The secondary carbon chemical shift [25,26] and short-range 1HN–1HN NOE pattern [27] that define the secondary structure are shown in Fig S2 The 310 helices formed by Pro59–Gln61 and Glu119–Ser122 that are present in the crystal structures of E coli and M tuberculosis PS, are missing in the solution structure Helix IX, which is a continuous helix (16 residues) in the PS crystal structure, is broken at Thr132 and resumes only at Phe138 in the solution structure In the absence of substrates, the crystal structure of the E coli PS has shown that the protein has an extensive dimer interface constructed from strands formed by Tyr108–Val111 from each subunit in an antiparallel b-sheet arrangement Under the conditions of these solution studies, the resonances for Tyr108 could not be identified However, we have been able to identify resonances for Val109, Asp110, and Val111 Prediction of backbone u and w dihedral angles from calculated 13C secondary chemical shifts, using the program talos [28], indicates that this region adopts a b-strand conformation under solution conditions The occurrence of a b-strand is corroborated by the absence of backbone HN–HN NOEs Mapping chemical shift changes upon substrate binding Binding of substrates to nPS was studied by both NMR and X-ray crystallography NMR studies that involved monitoring changes in chemical shift Fig (A) Sequence specifically assigned two-dimensional 1H–15N TROSY spectrum of triple-labeled nPS in 10 mM phosphate buffer, 10 mM NaCl, mM dithiothreitol, and 0.01% NaN3 (pH 6.8) For clarity, assignments of only resolved resonances have been indicated FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS 699 Structure and binding studies of nPS K.S Chakrabarti et al positions of backbone atom pairs in 1H–15N correlation spectra were conducted by addition of substrates in the concentration range used for biochemical characterization of PS [7–9] Changes in chemical shifts were observed at pantoate concentrations > mm, indicating weak binding coupled to an exchange rate that is intermediate on the NMR timescale Addition of d-pantoate (10 mm) to nPS caused deviations in chemical shifts of more than 0.03 p.p.m for 24 residues Addition of ATP brought about further changes in the heteronuclear single quantum coherence (HSQC) spectrum of nPS The changes in chemical shifts in the HSQC spectra for select residues that lie in the pantoate-binding and ATP-binding pocket of nPS are shown in Fig The residues that showed deviations in chemical shifts of more than 0.03 p.p.m upon binding pantoate and ATP fell into three categories Residues in class I were almost uniquely shifted in the presence of ATP (Fig 2, row 1) Residues in class II showed a net perturbation in chemical shifts in the presence of both pantoate and ATP (Fig 2, row 2) Finally, residues in class III showed deviations in the presence of pantoate but reverted back to the free protein resonance upon addition of ATP (Fig 2, row 3) The backbone atoms of Val111 and Gly113, which are involved in dimerization (see above), were also affected by substrate binding However, the backbone of His106, whose side chain is involved in dimerization, and a residue Fig The overlay of selected regions of H–15N correlation spectra, highlighting cumulative changes in chemical shifts of specific residues of nPS as a function of substrate(s) The resonances of free nPS are shown in red, those of nPS bound to D-pantoate are shown in blue, and those of nPS bound to D-pantoate and ATP are shown in green The arrows indicate the sign of change in chemical shift of the pantoate-bound (P; in blue) and pantoate + ATP-bound (PA; in green) protein as compared with the unbound protein Numbers in parentheses indicate the magnitude of change in p.p.m 700 FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS K.S Chakrabarti et al immediately before the dimer interface, Gly102, were unaffected by substrate binding (Fig 2, row 4) The absolute values of the deviations in chemical shift when compared to the unbound protein for both pantoate and ATP are shown in Fig 3A,B It can be clearly seen that there were two regions of the protein that were significantly perturbed upon the addition of pantoate These two regions correspond to the pantoate-binding and ATP-binding sites of PS [11] ATP caused additional changes in the spectrum, particularly for those residues that lie in the ATP-binding pocket Interestingly, in the presence of ATP, the His34 backbone HN chemical shift reverted back to the resonance position observed for the free form of the protein The observed changes in resonance positions in the HSQC spectrum upon addition of pantoate followed by ATP Structure and binding studies of nPS can be reconciled if one takes into consideration that a molecule of pantoate is bound in the ATP-binding pocket in addition to its canonical binding site, and that ATP displaces this molecule of pantoate b-Alanine and pantothenate did not bind nPS under the experimental conditions Attempts to study the binding of ATP alone to the protein were unsuccessful, as the protein precipitated upon addition of ATP in the absence of pantoate An important feature of the NMR study is the fact that a single set of resonances were observed for nPS in the absence or presence of substrates This strongly suggests that binding of pantoate and ATP preserves the dimer symmetry in solution In an effort to unambiguously identify and understand the nature of binding of pantoate at the ATP-binding site, we also structurally characterized the pantoate-bound form of nPS using X-ray crystallography, as described below A Solution and refinement of the structure using X-ray crystallography B The crystal structure of the pantoate–nPS complex was solved by molecular replacement using phaser [29] in ccp4 [30] The first solution with a log likelihood gain of 1995 was obtained using the edited coordinates of E coli PS (1IHO.pdb) The molecular replacement solution could be readily refined, and an initial examination of the (mf0 – Dfc) maps in coot clearly showed the position of pantoate within the active site cleft of nPS The data-processing parameters are listed in Table Maximum likelihood refinement, using refmac [31], was used to improve the quality of the electron density maps and facilitate further rebuilding and improvement of the molecular model, until no unexplained electron density remained, and the Rcryst and Rfree values converged at 18.9% and 24.4%, respectively The electron density was missing or weak for residues 113–124 in all cases, and this stretch of residues was therefore not rebuilt X-ray crystallographic structure of nPS bound to pantoate Fig The absolute value of deviation in chemical shift (p.p.m.) of residues of nPS upon binding to (A) D-pantoate as a function of sequence, and (B) D-pantoate and ATP as a function of sequence Residues that showed a deviation of more than 0.03 p.p.m were considered to have a direct interaction with the ligand(s) (see text for details) Cocrystallization trials of nPS with pantoate were performed over a range of pantoate concentrations Crystals of the nPS–pantoate complex obtained at ˚ 50 mm pantoate diffracted to a resolution of 1.7 A The crystal structure of the nPS–pantoate complex indicated that it is a dimer in the asymmetric unit The backbone structure of the dimeric N-terminal domain of the E coli PS determined here is identical to that determined earlier for the full-length protein [13] The FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS 701 Structure and binding studies of nPS K.S Chakrabarti et al Table Atomic refinement of models for nPS bound to two molecules of pantoate per monomer Data collection ˚ Wavelength (A) ˚ Resolution (A) Space group ˚ Unit cell parameters, a, b, c (A) Total reflections Unique reflections Completeness (%) I ⁄ r(I) Rmerge (%) Redundancy Refinement Rcryst (%) Rfree (%) rmsd from ideal bond ˚ length (A) ⁄ angles (°) No of protein residues No of water molecules No of ligand molecules ˚ Wilson B-factor (A2) Ramachandran plot (%) Most favored regions (%) Allowed regions (%) a 1.5418 34.78–1.67 (1.76–1.67)a P21212 61.89, 77.50, 78.85 109 170 (13 893) 43 231 (5676) 96.2 (87.5) 12.8 (2.0) 5.1 (46.4) 2.5 (2.4) 18.7 24.4 0.024 ⁄ 2.03 332 279 20.3 99.1 0.9 The high-resolution shell is shown in parentheses dimer interface in PS is extensive The dimer interface is stabilized by 19 hydrogen bonds and two salt ˚ bridges, with a total buried surface area of 1240 A2 (Table S3) The structure of the complex showed two molecules of pantoate bound to one monomer (chain A) and a single pantoate bound to the second monomer (chain B) Thus, pantoate binding occurs in the canonical pantoate-binding site (site I) in both monomers with full occupancy, and at the ATP-binding site (site II) in one monomer with full occupancy Figure 4A shows the dimeric nPS molecule bound to three molecules of pantoate The electron density for the two molecules of pantoate bound at the active site of nPS can be unambiguously distinguished in Fig 4B The crystal structures of the protein were of high stereochemical quality, in that all backbone u and w dihedral angles occupy allowed regions of the Ramachandran map [32,33] Comparison of the monomer of the substrate-free form of E coli PS [13] with the monomers of nPS in the pantoate-bound form at full occupancy indicates that the backbone Ca atoms show ˚ an overall deviation of 0.51 A Pantoate bound at site I The physicochemical nature of binding of pantoate to its canonical binding site on nPS is identical to that 702 observed in the case of the M tuberculosis protein [11] In fact, the residues of nPS involved in binding pantoate are highly conserved in the two proteins Figure 5A shows that the important substrate–protein interactions that are observed for the M tuberculosis PS–pantoate complex are conserved in the case of the E coli protein too Figure shows that the side chain atoms of Gln61, i.e Oe1 and Ne2, are hydrogen-bonded to the O3 and O4 atoms of pantoate, respectively, whereas the similar atoms of Gln155 are hydrogenbonded to the O2 and O3 atoms of pantoate, respectively These two residues are conserved in the E coli and M tuberculosis proteins, and are involved in identical conserved interactions with pantoate Furthermore, the pantoate molecule is hydrogen-bonded to other protein residues via networks of water-mediated hydrogen bonds The backbone nitrogen and carbonyl oxygen of Thr29 and the side chain oxygen of Ser54 are hydrogen-bonded to the O4 atom of pantoate via a network that consists of two water molecules The backbone carbonyl oxygen of Phe56 is also hydrogenbonded to the O4 atom of pantoate via one of the water molecules in the same network The phenolic oxygen of Tyr71 is hydrogen-bonded to the O1 atom of pantoate via a water molecule included in a network of five water molecules Also hydrogen-bonded to the O1 atom of pantoate via this cluster of water molecules is the Ne nitrogen of the imidazole ring of His37 In addition to the hydrogen-bonding interactions, the pantoate molecule is also involved in hydrophobic contacts with the protein, as shown in Fig S3 The Ca atoms of residues in site I not exhibit more than ˚ A deviations as compared with the substrate-free form of the protein Pantoate bound at site II The structure of nPS distinctly shows the presence of a second molecule of pantoate bound at site II in one of the monomers, albeit at full occupancy The pantoate in site II (Fig 5) has the same orientation as the pantoate molecule in site I In contrast to the pantoate bound to site I, this molecule has fewer direct interactions with protein residues This pantoate molecule is anchored to the protein by a hydrogen bond between the O2 atom of pantoate and the Nf atom of the side chain of Lys39 However, there are a large number of water-mediated networked interactions that stabilize the pantoate at site II The backbone carbonyl oxygen of Val175 is hydrogen-bonded to the O2 and O3 atoms of pantoate via a water molecule This water molecule also hydrogen bonds backbone nitrogen atoms of Glu150 and Lys151 to the O2 and O3 atoms of FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS K.S Chakrabarti et al Structure and binding studies of nPS C A Site II Site I N N Site I B Gln61 Gln61 Asp152 Gln155 Asp152 Gln155 Lys151 Lys151 Glu150 Glu150 Tyr71 Tyr71 Phe56 Phe56 Val175 Val175 Thr29 Thr29 Ser54 Ser54 His37 His37 Lys39 Lys39 Fig (A) Crystal structure of nPS bound to three molecules of pantoate The subunits are shown in ochre and blue, respectively Major secondary structural elements are numbered sequentially, where a is used to denote an a-helix and b is used to denote a b-strand The dimer is formed through association of b5 from each subunit (circled region) The pantoate molecules bound at site I and site II (ATP-binding site) are shown in green (carbon atoms) and red (oxygen atoms), respectively (B) Stereo view of the electron density map of the active site of nPS At this high resolution, the electron densities that define pantoate in site I and site II are clearly visible The atoms of the pantoate molecule are labeled The electron density clearly defines the backbones and side chains of the residues that line the active site pantoate The Nd atom of Asp152 is hydrogen-bonded via one water molecule to the O3 atom of pantoate, and via two water molecules to the O4 atom of pantoate The Ne atom of His37 is hydrogen-bonded to the O4 atom of pantoate via a water molecule, which is in turn hydrogen-bonded to a network of water molecules that are hydrogen-bonded to the O1 atom of pantoate in site I The O4 atom of pantoate in site II and the O1 atom of pantoate in site I are hydrogen-bonded via a water molecule Structural stabilization of ligands by networks of water-mediated hydrogen bonds is often observed in the active sites of enzymes [34–36] The pantoate molecule also has important hydrophobic interactions with the aliphatic carbons of the Lys39 side chain (Fig S4) Residues EKD(15–152) and Val175 are conserved in the case of the E coli and M tuberculosis enzymes, and the corresponding residues in the A thaliana protein are KKD(150–152) and Ser175, respectively Given the fact that the main types of interaction between pantoate and the residues in site II are water-mediated, it is almost certain that these interactions will be conserved in A thaliana PS as well An interesting feature of pantoate binding at site II is that it takes the position of the adenine ring in the ATP-binding site, as shown in Fig The oxygen atoms of the pantoate are spatially coincident with the nitrogen atoms of the adenine ring The O1, O2 and O4 atoms of pantoate occupy the positions of the FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS 703 Structure and binding studies of nPS Gln155 K.S Chakrabarti et al Asp152 Gln155 Lys151 Gln61 Asp152 Lys151 Gln61 Glu150 Glu150 Tyr71 Tyr71 Val175 Phe56 Val175 Phe56 Ser54 Thr29 His37 Ser54 Lys39 Thr29 His37 Lys39 Fig Protein–ligand and water-mediated protein–ligand hydrogen bond interactions (broken lines) that stablize the pantoate molecules in site I and site II of nPS Oxygen atoms of pantoate and water are colored magenta and ochre, respectively Direct protein–ligand hydrogen bonds are colored magenta, and water-mediated hydrogen bonds are colored red Several networks of water-mediated hydrogen bonds can be observed Fig An expanded view of the superposed substrate-binding sites of nPS (green) and M tuberculosis PS (magenta) In nPS, the pantoate molecule takes the position of the adenine ring of ATP The carbon and oxygen atoms of pantoate are shown in yellow and red, respectively The carbon and oxygen atoms of ATP are shown in red, the nitrogen atoms in blue, and the phosphorus atoms in orange (see text for details) N6, N1 and N9 atoms, respectively, of the adenine moiety of ATP The pantoate is bound in the sequence Thr29–Leu33, which forms the loop connecting strand (Ala25–Thr29) to helix II (Asp35–Arg47), and the sequence Gly149–Phe153 in the loop that connects strand (Ile145–Gly149) to helix X (Phe153–Met166) Attempts at cocrystallization or soaking of the nPS crystals with ATP and ⁄ or b-alanine were unsuccessful These results were consistent with the solution behavior of the protein Comparison of PS structures Comparison of monomer chains of nPS show that there is no significant difference in B-factors for the residues that lie in the canonical pantoate-binding site (site I) However, Arg64–Leu75 show higher B-factors in the B-chain than in the A-chain These residues 704 form the ‘gate loop’, which is thought to play a significant role in channeling the substrate into the active site in the M tuberculosis protein The deviations of backbone atoms in this region are small Significant changes are observed for residues that line the site II pantoate-binding pocket The residues that are shifted ˚ by more than A upon pantoate binding are listed in Table Figure shows a superposition of chain A of E coli PS (1IHO.pdb) on the A-chain and B-chain of nPS in the region of the HXGH (34–37) motif (where X = Asp for E coli PS and Glu for M tuberculosis and A thaliana PS) As compared with the substratefree form, the imidazole rings of His34 in the A-chain ˚ ˚ and B-chain of nPS move by  A and 5.5 A, whereas the backbone Ca atom moves by only ˚ ˚  0.9 A and  0.5 A, respectively The backbone Ca ˚ atom of Asp35 moves by as much as 1.62 A and FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS K.S Chakrabarti et al Structure and binding studies of nPS ˚ Table The residues that show > A displacement upon binding pantoate at both site I and site II Residue ˚ Displacement (A) Arg18 His34 Asp35 Gly36 Arg64 Pro65 Glu66 Asp67 Arg70 Tyr71 Pro72 Thr74 Leu75 Gln76 Glu77 Lys96 Glu97 Tyr99 Pro100 1.06 1.10 1.75 1.74 1.89 1.32 1.57 1.45 1.10 1.02 1.06 1.17 1.13 1.41 1.19 1.38 1.27 1.07 1.57 in the A-chain and B-chain of nPS, and in the case of His37, the v1 torsion angle changes from 63.34° to )67.80° and )52.11° for the A-chain and B-chain of nPS, respectively The imidazole ring of His34 points inwards in the substrate-free form of E coli PS as well as in the ATP-bound form of M tuberculosis PS Thus, this movement of the imidazole ring in the case of His34 is necessary to accommodate the pantoate molecule in the ATP-binding site (Fig 8) Binding of pantoate and ATP to nPS The crystal structure of nPS has shown clearly the bound form of pantoate in the ATP-binding site The enzymology of substrate binding has been studied in detail in the case of PS from M tuberculosis, which has enzyme kinetic parameters comparable to those of E coli PS [7] E coli nPS is saturated at an ATP concentration of 10 mm This strongly suggests that the KD for ATP in the case of nPS is similar that that observed for M tuberculosis PS, for which it has been shown to be 1.8 mm The binding of the pantoate to the ATP-binding site must be weaker, which is not surprising, given that ATP binding at this site will be thermodynamically more favored [11–13], because of more extensive hydrogen bond and hydrophobic interaction networks, which stabilize the ATP in the ATPbinding pocket Evidence that the ATP replaces the pantoate from site II is given by the pattern of changes in the resonance positions for the backbone atoms of His34 in NMR spectra In the presence of ATP, the His34 resonance reverts back to the position of the Fig Superimposition of the A-chain of nPS (green), the B-chain of nPS (cyan), and the substrate-free protein (1IHO.pdb; magenta) The carbon and oxygen atoms of pantoate are shown in green and red, respectively The imidazole ring of His34 in the pantoate-bound A-chain nPS is forced to move away from the pantoate when compared to the free form of E coli PS ˚ 0.34 A in the A-chain and B-chain of nPS Finally, for ˚ His37, the A-chain Ca atom is displaced by 0.72 A ˚ and the B-chain Ca atom deviates by 0.23 A, concomitant with a very minor deviation in the positions of the imidazole rings Comparison of side chain torsion angles for these residues between the free and the liganded forms shows that the v1 torsion angle for His34 changes from )64° in the former to 164° and )169° in the A-chain and Bchain of nPS, respectively The v1 torsion angle for Asp35 changes from )75.20° to )171.65° and )89.25° Fig (A) Superimposition of the A-chain of nPS (green), the PS from M tuberculosis in ATP-bound form (1N2B.pdb; blue), and the substrate-free PS from E coli (1IHO.pdb; magenta ⁄ blue) As seen for the ATP-bound form of M tuberculosis PS, the His34 imidazole ring moves in to interact with the oxygen of the b-phosphate group of ATP On the other hand, in nPS, the imidazole ring of His34 moves out to accommodate the incoming pantoate FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS 705 Structure and binding studies of nPS K.S Chakrabarti et al unbound protein Addition of ATP to the pantoatebound form of the protein displaces the pantoate from the ATP-binding site The bound ATP is stabilized by a hydrogen bond between the imidazole side chain of His34 and the a-phosphate group of ATP [11,12] This is most probably accompanied by a reversion of the structural changes induced by the pantoate binding at the ATP-binding site (see above) In light of this, it is not unreasonable to expect that the substrate-binding properties shown for E coli PS may be invoked to explain the observed biochemical properties of the plant PSs regarding the substrate inhibition by pantoate and the alleviation of this inhibition by high concentrations of ATP Discussion PSs from all known sources are dimeric, although the contribution of this dimerization to the properties of the enzyme is not clear Kinetic and structural studies of E coli PS [3,8,13] and M tuberculosis PS [7,11,12] suggest that active sites of the dimer are independent of each other, and that reactions occur at both sites simultaneously In plant PSs, dimerization has been shown to have a very different effect on the kinetic properties of the enzyme [9] A study of the sequence (Fig 9) and the modeled structure of A thaliana PS (Fig S5 and Table S1) shows that it has a large loop at the dimer interface and fewer energetically favorable N-terminal and C-terminal interdomain interactions, such as the hydrophobic, hydrogen bond and ionic interactions, than E coli PS Thus A thaliana PS is expected to be structurally more open than E coli PS Importantly, the residues at the active site are highly conserved, and thus the nature of the interactions between enzyme and substrate are expected to be identical M tuberculosis PS has a significantly higher number of such energetically favorable interactions that stabilize the ‘closed’ conformation For PS from L japonicas, the reported dissociation constants for the first and second molecules of pantoate are 0.042 mm and 5.33 mm, respectively; that is, the second pantoate molecule binds with 100-fold lower affinity than the first Thus, the substrate inhibition by pantoate is consistent with the finding that pantoate binds to the ATPase site as well, and is displaced from this site at equimolar concentrations of ATP On the basis of kinetic studies, an enzymatic scheme has been proposed [9] in which the second molecule of pantoate binds to the pantoyl adenylate-bound form of the protein, at a site other than the active site, which then has a regulatory role in the kinetics of the reaction Using high-resolution solution NMR methods, we have shown that the N-terminal domain of E coli PS is capable of binding both pantoate and ATP, at equivalent sites in the dimer, which is the first step of the bicatalytic–unicatalytic–unicatalytic–bicatalytic mechanism X-ray crystallographic studies have unambiguously shown that this domain binds two molecules of one of the substrates, i.e pantoate It is important to note that, at the level of sensitivity and resolution available for these studies, pantoate was not found to bind to any other site on the protein, leading us to conclude that there is no additional regulatory Fig The sequence alignment showing the high degree of homology between the E coli, A thaliana (A th) and M tuberculosis (M tb) PS The alignment was made using CLUSTALW2 Asterisks indicate strictly conserved positions Colons and periods indicate full conservation of strong and weak groups, respectively The C-terminal domain is highlighted with cyan, and the insertion sequence corresponding to Lys108 to Arg139 in the A thaliana sequence is highlighted in green 706 FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS K.S Chakrabarti et al site on the N-terminal domain of PS [9] A kinetic scheme to explain this observation is shown in Fig 10 Such competitive inhibition by one of the substrates of a reaction has also been observed in the case of adenylosuccinate synthetase, one of whose substrates, IMP, binds to the GTP-binding site and inhibits the reaction [37–39] The binding of pantoate to the ATP-binding site also reveals the origin of the negative cooperativity with b-alanine [9] at high pantoate concentrations The model of A thaliana PS (Fig S5) shows that there is a 24 residue insertion between Thr105 and His106 of the E coli sequence [9] The HETWIRVER(131–139) motif, which is part of the 24 residue insertion, and is present in all plant PSs and absent in bacterial PSs, has been shown to be important for the observed allosteric behavior [9] of plant PSs This insertion sequence is rich in Gly residues N-terminal to the HETWIRVER(131–139) motif, suggesting that this loop may adopt an extended conformation in A thaliana PS Mutations in or deletion of this stretch of residues at the dimer interface are known to affect the catalytic properties of A thaliana PS [9], as a consequence of disruption of long-range allosteric interactions Gly102, which is N-terminal to the insertion and completely conserved in E coli, M tuberculosis and A thaliana, is virtually unaffected by substrate binding (Fig 2) His106 in E coli PS, which is located at the end of the insertion sequence in A thaliana PS, is also unaffected by substrate binding His106 is involved in intersubunit interactions via its side chain, and is in a similar position to Glu132 in A thaliana PS Val111 and Gly113, on the other hand, are both affected by both pantoate and ATP These residues have their positional equivalents in Val137 and Arg139 in A thaliana PS, as shown in Fig S5 As the structure of A thaliana PS is not known, the structural basis of the HETWIRVER(131–139) insertion sequence Fig 10 A model for substrate inhibition under conditions of high pantoate concentration In this model, a second pantoate molecule binds to the enzyme–substrate complex to form a catalytically inert E.Pt.Pt* species, where Pt is a pantoate bound to the canonical pantoate-binding site, and Pt* is a second molecule of pantoate bound to the ATP-binding site ATP at high concentrations can displace Pt*, and the reaction goes to completion Structure and binding studies of nPS in establishing long-range allosteric interactions remains to be elucidated Interestingly, residues in the dimer interface and those in the vicinity exhibit high flexibility Preliminary analysis of 15N relaxation data shows that residues 118–122 have a higher degree of flexibility than other ordered regions of the protein Unfortunately, missing electron density for residues 113–124 prevents any structural interpretations from being made An important outcome of this study is the potential use of bipantoate molecules [40] as inhibitors of this enzyme, with the aim of developing of antibacterial and ⁄ or herbicidal agents [41,42] The endto-end distance between pantoate molecules in site I ˚ and site II is  A Therefore, bipantoate-based inhibitors, tethered by a flexible linker, could be attractive candidates for inhibition of pantothenate synthetase Experimental procedures The isotopically enriched chemicals, i.e., 15NH4Cl, 13 C6H12O6, 13C62H51H7O6, 12C62H51H7O6, 2-ketovalerate (13C5, 3-2H), 2-ketobutyrate (13C5, 3,3-2H), and deuterated dodecylphosphocholine, were purchased from Cambridge Isotopes Limited (USA) or Isotec 2H2O (100%), d-pantolactone, ATP (disodium), b-alanine and pantothenate were purchased from Sigma The plasmid containing the tobacco etch virus (TEV)– maltose-binding protein construct was a kind gift from B V Geisbrecht (University of Missouri, KS, USA) [43] The plasmid containing the full-length E coli PS-hexahistidine tag construct was a kind gift from C Abell and coworkers (University of Cambridge, UK) Cloning of nPS Specific primers were designed (forward primer, 5¢-GAC CAGCTTCATGTGGCCATCGTGCAGGTT-3¢; reverse primer, 5¢-CACGATGGCCACATGAAGCT-3¢) to PCRamplify the coding regions of these domains in the panC gene from E coli genomic DNA [44] The amplified product was ligated into an appropriately digested pet21a plasmid vector between Nde1 and EcoR1 restriction sites, to obtain the clone of the N-terminal domain of PS She gene starts with an unusual codon, GTG The C-terminal domain of PS was cloned as a cytb5-based fusion protein The gene corresponding to the C-terminal domain of PS was cloned downstream of the cytb5 gene, between BamH1 and EcoR1 restriction sites [45] Protein expression and purification All proteins were expressed using E coli BL21(DE3) as host strain, and purified using the protocol described below FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS 707 Structure and binding studies of nPS K.S Chakrabarti et al Purification of the N-terminal domain of PS The cell lysate was loaded onto a Q-Sepharose column pre-equilibrated with buffer (20 mm Tris, pH 8.0, 0.01% azide) The protein was collected in the flow-through, and the collected fractions, after being checking by denaturing SDS ⁄ PAGE, were pooled together The protein was passed through an S-100 16 ⁄ 60 sephacryl (Pharmacia) gel filtration column for final purification The protein was later dialyzed into appropriate buffer (20 mm sodium dihydrogen phosphate ⁄ disodium hydrogen phosphate, 20 mm NaCl, mm dithiothreitol, 0.01% sodium azide, pH 6.8) and concentrated to  0.7 mm Purification of the C-terminal domain of PS The cell lysate was passed through a DEAE anion exchange column The protein was eluted with a linear gradient of NaCl (0–500 mm), and the fractions containing the protein were pooled together after being checking by denaturing SDS ⁄ PAGE The protein was passed through an immobilized metal ion affinity chromatography (Ni2+) column The protein, which binds weakly to the column, was eluted with a linear gradient of imidazole (0–500 mm) TEV protease digestion Cleavage of target proteins from the fusion host was achieved following dialysis of the fusion proteins into the TEV protease cleavage buffer (50 mm Tris, pH 8.0, 100 mm NaCl, mm dithiothreitol) The protein and TEV protease were added in a : molar ratio to the digestion reaction, which was continued for 16 h at 22 °C TEV protease was purified in the laboratory, using the method described by Geisbrecht et al [43] Separation of the C-terminal domain of PS from the fusion host The cleaved C-terminal domain of the PS was separated from the fusion host as well as the TEV protease by immobilized metal ion affinity chromatography (Ni2+) chromatography Apo-cytb5 and TEV protease bind to the column, and the protein of interest is collected in the flow-through The protein was dialyzed against 20 mm phosphate buffer (pH 6.8) containing 20 mm NaCl, mm dithiothreitol, and 0.01% sodium azide column Protein was eluted from the metal affinity column with a linearly increasing gradient of imidazole The PS was further purified in the ATP-bound form by passage through a Sepharose blue column that is crosslinked with cibacron blue dye [46], and then eluting the protein with a buffer containing ATP Crystallization and data collection For protein crystallization, the purified protein was exchanged in a dilute buffer of 20 mm Tris (pH 8.0) containing 50 mm d-pantoate [47] The concentration of the protein was  15 mg ⁄ mL Crystallization experiments were performed using the hanging-drop vapor diffusion method In each drop, lL of protein was mixed with lL of the reservoir solution The best crystals were obtained using 8–15% poly(ethylene glycol) 4000, 40–150 mm NaCl, and 100 mm acetate buffer (pH 5.0), at 20 °C The crystal with ˚ 50 mm pantoate diffracted to 1.7 A Images were indexed, and reflections were integrated using the program mosflm [48], and scaled and merged with scala [49] For data collection, the crystals with 50 mm pantoate were flash-frozen in a cryostream of N2 gas at 100 K Diffraction data were collected on a Mar FRD generator with a Bruker detector Data reduction and scaling were performed with the programs mosflm and scala (ccp4 [30]) Data-processing statistics are given in Table The pantoate-bound enzyme crystallized in the space group P21212 Both datasets collected showed two molecules per asymmetric unit Molecular replacement, model building, and refinement The crystal structure of the N-terminal domain of the enzyme was determined by the molecular replacement method, using the program phaser [29] The N-terminal domain (residues 1–176) of subunit A of E coli PS (1IHO.pdb) was used as a model for molecular replacement Preparation of isotopically enriched samples of nPS Uniformly 2H, 13C, 15N enriched ILV - methyl protonated samples of nPS were prepared following protocols described previously [50,51] All perdeuterated samples of nPS were subjected to unfolding–refolding The levels of isotopic enrichment for the different samples described above were ascertained using electrospray MS Purification of the full-length PS The cell lysate containing the full-length PS was passed over a Q-Sepharose column The protein was eluted with a linearly increasing gradient of NaCl Fractions containing PS were pooled, and passed over a Ni2+-nitrilotriacetic acid 708 Samples for NMR spectroscopy All of the NMR samples (0.6–0.8 mm) were prepared in 20 mm phosphate buffer, 20 mm NaCl, mm dithiothreitol, and 0.01% NaN3 (pH 6.8) FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS K.S Chakrabarti et al Structure and binding studies of nPS NMR spectroscopy Acknowledgements NMR spectra were acquired on a Bruker-Avance spectrometer operating at a proton frequency of 700 MHz, equipped with a mm triple-resonance cryoprobe fitted with a single (z-axis) pulsed field gradient accessory The 15N relaxation datasets [52] were acquired on a Varian INOVA 600 MHz spectrometer equipped with a cryogenically cooled tripleresonance (z-axis) pulsed field gradient probe All NMR spectra were acquired at 303 K NMR data were processed using nmrpipe ⁄ nmrdraw [53], and assigned using ansig [54,55] The NMR and MS facilities at the Indian Institute of Science are funded from grants by DBT and DST K S Chakrabarti is a recipient of a CSIR Senior Research Fellowship The authors thank K V Srinivas for help with preparing the clone of nPS, A Arora, CDRI, Lucknow, for help in collecting the relaxation data, and N Srinivasan and S Yamunadevi for their help with modeling and analyzing the A thaliana PS protein References NMR titration studies The stock solutions of substrates (100 mm) were prepared by dissolving weighed amounts of substrates in water The d-pantolactone solution was hydrolyzed to d-pantoate as described previously [47] The substrates were added to protein solutions (0.5 mm) to final concentrations of 100 lm, 500 lm, mm, mm and 10 mm in buffers containing 90% H2O ⁄ 10% D2O Chemical shift mapping The changes in chemical shifts as a concentration were monitored by TROSY experiments with a digital per point for the 1H dimension and in the 15N dimension Shifts were equation function of substrate performing 1H–15N resolution of  Hz  16.63 Hz per point calculated using the D ẳ ẵDd2 ỵ ð0:1DdN Þ2 Š1=2 HN where D is the cumulative chemical shift deviation, DdHN is the change in chemical shift of the proton, and DdN is the change in the chemical shift of the nitrogen [56] Modeling and comparison of the structures of PSs dali [57] was used to superimpose the crystal structures of PSs from E coli and M tuberculosis (1IHO.pdb and 1MOP.pdb) for structural alignment The sequence alignment of PSs from E coli, M tuberculosis and A thaliana was achieved using clustalw2 [58] Finally, modeller [59] was used to model the A thaliana PS structure, using the structures of PSs from E coli and M tuberculosis Structural analyses Analysis of the sites of substrate binding from NMR data, and structural comparison of the E coli, M tuberculosis and A thaliana PSs with nPS, was 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Sanders C (1996) Mapping the protein universe Science 273, 595–693 58 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R et al (2007) ClustalW2 and ClustalX version Bioinformatics 23, 2947–2948 59 Eswar N, Marti-Renom MA, Webb B, Madhusudan MS, Eramian D, Shen M, Pieper U & Sali A (2007) Comparative Protein Structure Modeling with MODELLER Curr Protoc Protein Sci Nov; Chapter 2: Unit 2.9 60 Humphrey W, Dalke A & Schulten K (1996) VMD – Visual Molecular Dynamics J Mol Graph 14, 33–38 61 DeLano WL (2002) The PyMOL Molecular Graphics System http://www.pymol.org Supporting information The following supplementary material is available: Fig S1 Sequential connectivity walk along the protein backbone for Glu5–Glu20 of nPS Fig S2 Secondary structure distribution of nPS in solution along with the sequence, short-range NOE pattern, and secondary chemical shifts of 13Ca and 13 Cb atoms Fig S3 Two-dimensional projection showing the pantoate–protein interactions in the canonical pantoatebinding site (site I) Fig S4 Two-dimensional projection showing the pantoate–protein interactions in the ATP-binding site (site II) Fig S5 Model structure of pantothenate synthetase from Arabidopsis (green), constructed using modeller, superimposed on the crystal structures of PS from E coli (1IHO.pdb; blue) and M tuberculosis (1MOP.pdb; red) Table S1 E coli residues (black) and aligned residues in A thaliana (pink) Table S2 M tuberculosis residues (black) and aligned residues in A thaliana (pink) Table S3 List of atom–atom interactions across the dimer interface FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS 711 Structure and binding studies of nPS K.S Chakrabarti et al This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and 712 may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS ... robust synthesis of pantothenate from low amounts of pantoate [9] Here, we report the results of the structural studies of the pantoate- bound form of the N-terminal domain of E coli PS (nPS) The structure... [13] Details of the structural basis for the biochemically observed inhibition of plant PS by pantoate, as well as of the alleviation of this inhibition by another substrate of the enzyme, ATP,... back to the position of the Fig Superimposition of the A-chain of nPS (green), the B-chain of nPS (cyan), and the substrate-free protein (1IHO.pdb; magenta) The carbon and oxygen atoms of pantoate