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The Structures of Thermoplasma volcanium Phosphoribosyl Pyrophosphate Synthetase Bound to Ribose-5-Phosphate and ATP Analogs
J. Mol. Biol. (2011) 413, 844–856
doi:10.1016/j.jmb.2011.09.007 Contents lists available at www.sciencedirect.com
Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b
The Structures of Thermoplasma volcanium Phosphoribosyl Pyrophosphate Synthetase Bound to Ribose-5-Phosphate and ATP Analogs Maia M. Cherney, Leonid T. Cherney I , Craig R. Garen and Michael N. G. James⁎ Group in Protein Structure and Function, Department of Biochemistry, School of Molecular and Systems Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Received 11 June 2011; received in revised form 31 August 2011; accepted 4 September 2011 Available online 21 September 2011 Edited by M. Guss Keywords: X-ray crystallography; phosphoribosyl pyrophosphate synthetase; complex with substrate analogs; Thermoplasma volcanium
Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P21. The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP–Mg 2+ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively. © 2011 Elsevier Ltd. All rights reserved.
Introduction *Corresponding author. E-mail address: [email protected]. I Present address: L. T. Cherney, Department of Chemistry and Center for Research on Biomolecular Interactions, Faculty of Science and Engineering, York University, Toronto, Ontario, Canada M3J 1P3. Abbreviations used: PRPP, phosphoribosyl pyrophosphate; R5P, ribose-5-phosphate; Tv, Thermoplasma volcanium; mATP, β,γ-methylene ATP; PEG, polyethylene glycol; Mj, Methanocaldococcus jannaschii; Bs, Bacillus subtilis; PDB, Protein Data Bank; MBP, maltose binding protein.
Phosphoribosyl pyrophosphate (PRPP) is an important metabolite that plays a central role in many life processes, namely, the biosynthesis of purine and pyrimidine nucleotides, the biosynthesis of the coenzyme NAD +, and the biosynthesis of the amino acids histidine and tryptophan; 1,2 it is a substrate for many enzymes. 3 It is produced in a transfer reaction of the intact β,γ-diphosphate group from ATP to the anomeric oxygen atom (O1) of ribose-5-phosphate (R5P): R5P + ATP → PRPP + AMP by the PRPP synthetase 4 (E.C. 2.7.6.1). Three classes of PRPP synthetases have been described. 5,6
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Phosphoribosyl Pyrophosphate Synthetase Structures
Class I enzymes occur in a diverse range of bacteria and mammals. They are characterized by their dependence on phosphate and Mg 2+ ions for activity; they can be allosterically regulated by ADP and, in some cases, by GDP. Three class I PRPP synthetases from Bacillus subtilis (Bs), 7 Burkholderia pseudomallei, 8 and human 9 have had their structures determined. They are two-domain monomeric molecules having similar folds and a significant sequence identity (about 50%). They adopt a hexameric quaternary structure. The six protomers that comprise the hexamers represent a trimer of dimers that are related by 32 point group symmetry; they form a propeller in which the N-terminal domains are oriented inwards toward the center of the hexamer, and the C-terminal domains are turned toward the outside. The allosteric site is situated not far from the active site, at the junction of three subunits; two of the subunits belong in the same dimer, and the third subunit comes from an adjacent dimer. Class II PRPP synthetases are found in plants. 10 They are independent of phosphate for activity and are not allosterically inhibited by purine nucleoside
845 diphosphates. Class II enzymes have the lowest sequence similarity scores to the class I and class III enzymes as outlined below (less than 20%). Recently, a third class of PRPP synthetases has been proposed. This class is represented by the enzyme from Methanocaldococcus jannaschii (Mj). 6 It is activated by phosphate and Mg 2+ ions but is independent of allosteric regulation. The Mj enzyme forms a tetramer in which two dimers are related by a non-crystallographic 2-fold axis. The interfaces that are present in the allosteric sites of the class I enzymes are not preserved in the Mj enzyme. The Mj PRPP synthetase has a low sequence similarity with the known class I enzymes (about 25%).The sequence alignment of the Tv PRPP synthetase and the four enzymes mentioned above having known structures is shown in Fig. S1 (see Supplementary Information). The previously reported structures of the PRPP synthetases did not fully explain the mechanism of activity, as the catalytically important areas of the active sites, particularly the catalytically important hairpin, were disordered. No productive complexes were previously obtained. Here, we report four crystal
Table 1. Crystal parameters and data collection statistics for Tv PRPP synthetase Complex crystal
Data collection Space group Cell dimensions a, b, c (Å) α, β, γ (°) No. of molecules per asymmetric unit Temperature (K) Detector Wavelength (Å) Resolution (Å) Multiplicity I/σ(I) Completeness (%) Rsymb Refinement Resolution No. of unique reflections Rwork/Rfreec No. of atomsd Protein Ions/ligands Water Mean B-factors (Å2) Protein Ions/ligands Water r.m.s.d. Bond lengths (Å) Bond angles (°) a
ADP/SO4 PDB code 3LRT
ADP/SO4 PDB code 3NAG
mATP/SO4 PDB code 3LPN
ADP–Mg2+/R5P PDB code 3MBI
P21
P21
P21
P21
38.6, 141.6, 50.1 90.0, 96.2, 90.0 2 100 MAR225 0.97950 50.0–1.53 (1.58–1.53)a 3.4 (2.6)a 12.4 (1.6)a 89.6 (58.1)a 0.08 (0.42)a
38.6, 141.6, 49.6 90, 95.9, 90 2 100 MAR225 0.9795 50–1.75 (1.81–1.75)a 3.6 (3.4)a 14.0 (1.4)a 93.9 (80.8)a 0.09 (0.84)a
38.6, 141.2, 50.0 90.0, 96.0, 90.0 2 100 MAR225 0.97950 50.0–1.80 (1.86–1.80)a 3.8 (3.6)a 11.7 (2.0)a 99.9 (100.0)a 0.11 (0.59)a
76.6, 61.8, 127.9 90.0, 103.9, 90.0 4 100 MAR225 0.97950 50.0–1.80 (1.86–1.80)a 3.9 (3.8)a 11.9 (1.8)a 97.8 (96.4)a 0.12 (0.71)a
24.6–1.53 66,070 18.6/21.9 5093 4576 20/54 443
31.9–1.75 49,953 17.6/21.7 5029 4517 31/27 454
29.1–1.8 49,076 17.1/21.5 5108 4525 25/62 496
25.5–1.8 105,818 17.6/21.9 10,470 9075 19/122 1254
22 34/29 32
27 43/40 35
27 38/29 36
19 17/19 29
0.006 1.106
0.007 1.065
0.006 1.079
0.016 1.059
Values for the highest-resolution shell are given in parentheses. Rsym = ∑h∑i(|Ii(h) − 〈I(h)〉|)/∑h∑iIi(h), where Ii(h) is the ith intensity measurement, and 〈I(h)〉 is the weighted mean of all measurements of I(h). c Rwork and Rfree = ∑h(|Fo(h)| − |Fc(h)|)/|Fo(h)| for reflections in the working and test sets (5% of the data). d Number of non-hydrogen atoms present in the asymmetric unit. b
846 structures of Tv PRPP synthetase bound to several ATP analogs and to R5P in which the ribose adopts the five-membered furanose ring conformation. Based on these structures, a proposal for the mechanism of the PRPP synthetase activity has been suggested.
Results Structure determination The Tv PRPP synthetase open reading frame Tvn0197 was amplified by polymerase chain reaction (PCR), and the enzyme was overproduced in Escherichia coli and purified to homogeneity. Analytical gel-filtration experiments were carried out at two different protein concentrations, 1 mg/ml and 10 mg/ml; they showed a major peak corresponding to a molecular mass of 55 kDa that is slightly less than the theoretical molecular mass of the dimer (64.3 kDa). Two crystal forms, both in the space group P21, of the Tv PRPP synthetase were grown under two different conditions. One crystal form (form I) was obtained from a high-salt and low-pH (2 M ammonium sulfate, pH 4.0) condition and had
Phosphoribosyl Pyrophosphate Synthetase Structures
two enzyme molecules in the asymmetric unit. The other form (form II) was produced from low-salt and neutral-pH conditions [30% polyethylene glycol (PEG) 600, pH 7.5] and had four molecules in the asymmetric unit. The protein preparation that was used to produce the crystals contained one of the substrates, ATP, that was added during the first purification step in the crude extract. Consequently, the protein preparation and the resulting crystals (forms I and II) contained ATP or more likely ADP, a product of ATP hydrolysis by another enzyme in the crude extract. Additionally, endogenous phosphate and Mg 2+ ions, as well as R5P, were observed to be present in the enzyme in crystal form II. The form I crystals also were soaked with β,γ-methylene ATP (mATP) and PRPP (separately). PRPP has not been observed in any of the crystal structures; however, its effect on ADP binding has been observed. ADP ligand has been displaced from its binding site in one molecule of the dimer. These several crystal structures were solved by the molecular replacement method using the structure of the Mj PRPP synthetase monomer as the search model. Crystallographic data collection information and refinement statistics are presented in Table 1.
Fig. 1. Structure of the Tv PRPP synthetase in complex with ADP in an open conformation (crystal form I). The asymmetric unit contains two molecules (yellow and pink) that form a biological dimer. The dimer interface is formed predominantly by residues on helices α2 and α3, the β4-strand, and loop segments of each subunit. The β10-strand adopts an open conformation; β10 includes two catalytic residues Lys184 and Arg186. ADP (shown in sticks) is situated in the ATP binding site.
Phosphoribosyl Pyrophosphate Synthetase Structures
Overall structure of the Tv PRPP synthetase The overall structure of the Tv PRPP synthetase is shown in Fig. 1. It is very similar to the known PRPP synthetase structures from class I 5–7 and class III 9 (with r.m.s.d. values in the range of 1.1–1.9 Å for ∼250 C α atom pairs), although the sequence homology among them is not very high; the highest score (29% sequence identity) is for the Mj enzyme. Each subunit comprises two very similarly folded domains that are related by an approximate non-crystallographic 2-fold axis. Superposition of the N-terminal and C-terminal domains of one subunit is shown in Fig. S2 (Supplementary Information). They had only 11% of sequence identity. Both domains can be classified as having an open α/β fold with a central six-stranded parallel β-sheet surrounded by four helices from the same domain and, in the case of the N-terminal domain, by one additional helix from the C-terminal domain. Each domain also contains a β-hairpin (β3–β4 in the N-domain and β10–β11 in the C-domain) that plays an important role in the enzymatic function of the PRPP synthetases. Both hairpins are structurally equivalent. The β-hairpin loop of the N-terminal domain (the loop between strands β3 and β4) forms a part of the active site of the adjoining molecule in the dimer. The β-hairpin of the C-terminal domain (β10 and β11) (the catalytic β-hairpin, residues 179–187) can adopt two different conformations of the Tv PRPP synthetase: open and closed. The form I crystals have PRPP synthetase in the open conformation, and the form II crystals adopt the closed conformation. That this β-hairpin plays an important role in the catalysis of Bs PRPP synthetase was shown by kinetic analysis of the substitutions of the corresponding residues Lys184 and Arg186 (Tv numbering) for Ala that are located on the β-strand 10. These substitutions caused approximately 30,000 and 24,000 times reduction in the maximal velocity, Vmax, respectively, in Bs. 11 Oligomeric state There are two Tv PRPP synthetase molecules in the asymmetric unit of the form I crystals and four Tv PRPP synthetase molecules in the asymmetric unit of the form II crystals. The dimer in the asymmetric unit (form I) has an extensive solventinaccessible interface between monomers of 1945 Å 2 per subunit (Fig. 1). The interface consists mainly of two α-helices (α2 and α3), the strand β4, and a loop segment 86–102 of each subunit. Interactions across the dimer interface include predominantly hydrophobic contacts (the solvation free-energy gain upon formation of the interface, ΔG i = − 17 kcal/mol), as well as 34 hydrogen bonds and 15 salt bridges (Table S1 and Supplementary Information; PISA server). 12 Consequently, the interface has a significant positive ΔGdiss value (22 kcal/mol), which means that the Gibbs free energy would increase by this amount on
847 the breakup of the dimer interface. As a result, dimer dissociation would be strongly energetically unfavorable. Tv PRPP synthetase is a biological dimer, and it does not form higher symmetry oligomers (hexamers or tetramers) as described for the class I and class III PRPP synthetases. Comparison of Tv PRPP synthetase with other PRPP synthetases Superposition of the Tv PRPP synthetase subunit with the corresponding subunits of the Mj and Bs enzymes showed r.m.s.d. values of 1.3 Å (for 259 C α atom pairs) and 1.85 Å (for 260 C α atom pairs), respectively. The Tv PRPP synthetase polypeptide has 286 amino acids and is more closely related to the Mj enzyme with a sequence identity of 29%; it has lower sequence identities with the class I PRPP synthetases (∼ 23%) and the class II PRPP synthetases (e.g., from Spinacia oleracea isozymes 3 and 4, 18% and 12%, respectively) (data not shown). Superposition of the Tv PRPP synthetase dimer on the Mj dimer using a single subunit (A) reveals that there is a major difference between the two dimers; it results in the deviation of the B subunits by 6°. Consequently, superposition of two Tv dimers on the Mj tetramer shows that the Tv dimers do not come close enough to form the tetrameric interfaces. There is also a large difference in the protein sequences that can be seen in the sequence alignment (Fig. S1 and Supplementary Information). The differences become even greater with Bs PRPP synthetase; the Tv enzyme does not form hexamers, likely because it lacks the final C-terminal helix that is involved in oligomerization of the class I enzymes, and the allosteric ADP binding sites that form at the interfaces of the hexamer are not possible with the Tv enzyme. One of the important differences between the present structures of Tv PRPP synthetase and the structures of the other PRPP synthetases lies in the orientation of the catalytic β-hairpin that is disordered in both the Mj (residues 185–196) and the Bs (residues 196–208) structures. In Tv PRPP synthetase, the corresponding sequence for the catalytic β-hairpin (residues 179–187) has well-defined associated electron density. Complex with ADP As it was pointed out above, ATP was added to the lysis buffer at the beginning of purification. Thus, the final purified protein contained bound ATP or, more likely, judging from the resulting electron density, ADP, a product of ATP hydrolysis presumably by an ATPase in the crude extract. The protein was crystallized from ammonium sulfate at low pH, and the resultant crystals had two molecules in the asymmetric unit [crystal form I,
848 Fig. 1; Protein Data Bank (PDB) code 3lrt]. Each molecule contained ADP in the ATP binding site and a sulfate ion in the R5P binding site. The two binding sites and the catalytic residues Lys184 and Arg186 located on the strand β10 together comprise the active site of the Tv PRPP synthetase. In crystal form I, each subunit has an open conformation with the active site fully accessible for substrate binding. The active-site residues Lys184 and Arg186 that
Phosphoribosyl Pyrophosphate Synthetase Structures
were previously identified in Bs by kinetic activity studies of variants are far from the substrates. Thus, the average distance between the O3B atom of ADP and the N ζ atom of Lys184 is ∼ 14–15 Å. Similarly, the average distance between the O3B atom of ADP and the N η2 of Arg186 is about 15–16 Å. This conformation would allow the substrates to enter the active site, but it would not support the catalytic reaction.
Fig. 2. (a) Binding of ADP in the ATP binding site (crystal form I). (b) Binding of mATP in the ATP binding site (crystal form I). The ATP binding site is situated in the cleft between two domains of one subunit (yellow); it is also restricted on the other side by the loop residues (Loop L) of the adjacent subunit of the dimer (cyan). The phosphate binding site of R5P involving main-chain and side-chain interactions of residues 214–218 is occupied by a sulfate ion. Hydrogen bonding and electrostatic interactions are shown in broken lines. The |Fo| − |Fc|, αc electron density omit map is contoured at 2 σ level in the regions of ADP or mATP. Water molecules are depicted as red spheres.
Phosphoribosyl Pyrophosphate Synthetase Structures
There are two independent active sites in the Tv PRPP synthetase dimer (Fig. 1). Each site is situated in the cleft between the two domains of one subunit (Fig. 2a); it is also limited by a hairpin Loop L (the hairpin of the N-terminal domain involving the strands β3 and β4) of the twofold related subunit of the dimer. A network of hydrogen bonds and electrostatic interactions forms between the enzyme and ADP (Table S2 and Supplementary Information). Tyr96 and His93 interact with the β-phosphate group of ADP (atoms O1B and O2B). Gln92 makes a hydrogen bond to the O2′ hydroxyl group of the ribose moiety. The adenine ring of ADP is sandwiched between Arg91 and Phe32; the latter residue belongs to the β3–β4 hairpin loop of the adjacent subunit of the dimer. Asp34 and Glu36, also from the adjacent subunit, form hydrogen bonds with nitrogen atoms of the adenine ring. Several contacts are also made through water molecules (Fig. 2a and b). The proposed R5P binding site is occupied by a sulfate ion that was present in the crystallization mother liquor; it mimics the phosphate group of R5P (Fig. 2a). The phosphate-binding residues are identical with those seen in the Mj enzyme. The sulfate ion is situated near the phosphate-binding segment that includes a loop (residues 214–216) and the Nterminus of helix α6 with a highly conserved Gly217–Thr218 motif (Fig. S1 and Supplementary Information). The sulfate ion forms hydrogen bonds to four residues in the phosphate-binding segment, mainly to main-chain nitrogen atoms and to the hydroxyl groups of the threonine residues. In addition, the sulfate ion makes a salt bridge (distance, 3.7 Å) with the guanidinium group of Arg91. Several hydrogen bonds are made to water molecules. One of these water molecules also contacts the O γ atom of Ser214. Additional sulfate ions were modeled elsewhere on the surface of the enzyme. Complex with one ADP molecule per dimer When the original form I crystals were soaked overnight with PRPP, the resulting structure (PDB code 3nag) showed the presence of neither PRPP nor R5P. Instead, one of the ADP molecules of the dimer was displaced from its position in the ATP binding site; the site remained empty, and it collapsed. The loop (“flexible” loop, residues 91–96) changed its position and became notably disordered, with poor density for both the main-chain and the sidechain residues. The movement of this loop was also observed in Bs, human, and Mj PRPP synthetases. 5–7,9 The other subunit had ADP bound, and there were no changes in the position or in the degree of order of the loop formed by residues 91–96. Evidently, the conformation of the flexible loop is determined by the presence of ADP. Noteworthy, ADP here has a bound magnesium ion that coordinates three oxygen
849 atoms of phosphate groups of ADP and two water molecules. The sixth position in the octahedral Mg 2+ coordination is occupied by the N ɛ2 atom of the imidazole ring of His124; the distance between the N ɛ2 atom and the Mg 2+ ion is 3.7 Å. The coordination is similar to that observed in the complex with ADP and R5P (see below). The sulfate ions occupied the usual positions in the R5P binding site in both subunits. The α-phosphate group (O2A atom) of ADP (in the PRPP-soaked crystal) makes a salt bridge with the guanidinium group of Arg40 that belongs to the symmetry-related dimer (symmetry operation x, y, z − 1 or x, y, z + 1). The distances between the O2A atom of ADP and atoms N η2 and N η1 of Arg40 are 3.4 Å and 3.7 Å, respectively. In the case of the original ADP complex crystals, these distances were around 4 Å. Complex with mATP The original form I crystals containing ADP were soaked overnight with mATP. As a result, ADP was substituted by mATP in both subunits of the dimer (Fig. 2b; PDB code 3lpn). The complex with mATP retained the open conformation of the enzyme subunits; the catalytic β10-strand that contains residues Lys184 and Arg186 is situated far from the substrates. The positions of the adenine and ribose rings did not change; they make all the same interactions with the PRPP synthetase as they do in the ADP complex (Table S2 and Supplementary Information). The main differences are observed in the positions of the phosphate groups. His93 in this complex makes hydrogen bonds with two phosphate groups (β and γ; atoms O1B and O3G, respectively). Also, His124 makes a hydrogen bond (2.8 Å) with the γ-phosphate group of mATP (atom O1G). Tyr96 interacts with the β-phosphate group of mATP in the same way as it does with ADP. Interestingly, the carboxylate of Asp125 that is positioned at the N-terminus of a 310 helix is close to the γ-phosphate group. The charge on the carboxylate of Asp125 is partly neutralized through a hydrogen bond with a main-chain amide of Lys127. The distance between O δ1 of Asp125 and O3 γ of mATP is 2.4 Å. The α-phosphate group (O1A atom) of mATP makes similar electrostatic interactions with the guanidinium group of Arg40 of the symmetry-related dimer as that of ADP. The distances between the O1A atom of mATP and atoms N η2 and N η1 of Arg40 are 2.9 Å and 3.7 Å, respectively. The sulfate ions are present in the R5P binding site of both subunits. Complex with ADP and R5P Crystal form II was grown from low-salt conditions (i.e., no sulfate ions); it has two biological
850
Phosphoribosyl Pyrophosphate Synthetase Structures
Fig. 3. Structure of the Tv PRPP synthetase in crystal form II. The asymmetric unit contains four molecules representing two biological dimers. Each molecule is colored individually. Ligands, ADP and R5P, are shown as sticks in molecule D (yellow).
dimers (totally, four protein molecules) in the asymmetric unit (PDB code 3mbi). ADP ligands each having a bound Mg 2+ ion are present in all of the four protein molecules, but only one R5P ligand is bound to the tetramer in the asymmetric unit, specifically, subunit D. As Mg 2+ ions and R5P were
not added at any stage during the protein preparation, they have likely come from the E. coli expression system and have remained bound during the purification steps. The four protein molecules of the asymmetric unit do not form a biological tetramer as seen in the structure of Mj PRPP
Fig. 4. Superposition of the Tv PRPP synthetase dimers in two different conformations: open (green shades for A and B molecules, crystal form I) and closed (magenta shades for B and D molecules, crystal form II). The N-terminal domains that form the dimer interface superpose well, whereas the C-terminal domains differ considerably. The catalytic β-hairpin (strands β10 and β11) bends over toward the ligands (shown in sticks) forming a closed conformation for the enzyme. The positions of catalytic residues Lys184 and Arg186 are marked as K and R.
Phosphoribosyl Pyrophosphate Synthetase Structures
synthetase (Fig. 3). Individual monomers of form I crystals were superimposed with the monomers of form II crystals resulting in r.m.s.d. values in the range of 2.3–2.4 Å over 280–285 C α atom pairs for each superposition. The N-terminal domains of the form I and form II monomers superpose very well; the largest deviation occurs between the secondary structural elements of the C-terminal domains, particularly, in the region of the flexible catalytic β-hairpin formed by strands β10 and β11 (Fig. 4). In the closed conformation, strand β10 splits into two strands β10a and β10b. The β10–β11 hairpin bends over by approximately 45° toward the bound
851 ligands forming the closed conformation for the enzyme. This movement places the catalytic residues Lys184 and Arg186 within hydrogen-bonding distance to the β-phosphate group of ADP and likely to the β-phosphate group of ATP when it is bound here (Table S3 and Supplementary Information). In the closed conformation, the active-site region containing the reacting substrates in the transition state is shielded from solvent. The surface representation of the active site shows that the ligands are situated in deep pockets (Fig. 5a). The closed conformation was also reported for the crystal structure of the Bs PRPP synthetase complexed with AMP, R5P, and the
Fig. 5. Surface representation of the active site of the Tv PRPP synthetase in the closed conformation. (a) The ligands, R5P and ADP (shown in sticks), are buried in the deep surface pockets and are shielded from the bulk solvent. (b) An ATP model is aligned with ADP. The γ-phosphate group of ATP (green phosphorus atom) is modeled in the orientation directed toward the N-terminus of the joint 310–α5 helix (residue Gly163). One of the oxygen atoms of the γ-phosphate group would likely replace a water molecule (shown beside it) in the coordination sphere of Mg 2+.
852
Phosphoribosyl Pyrophosphate Synthetase Structures
Fig. 6. A stereo view of the interactions of Tv PRPP synthetase with ADP and R5P. Residues of subunit D (yellow sticks) and subunit C (cyan sticks) form the active site. Hydrogen bonds and electrostatic interactions of substrates ADP and R5P with active-site residues directly and via water molecules (red spheres) are shown in broken lines. The O1 atom (the nucleophile) of R5P is in an appropriate position and distance from β-phosphorous atom of ADP to carry out the nucleophilic attack.
transition state analog AlF3. 11,13 It was shown that the equivalent Bs catalytic residues, Lys197 and Arg199, interacted with two of the fluoride atoms that are analogous to the oxygen atoms of the βphosphate group in the trigonal bipyramidal transition state. 11,14,15 R5P was modeled only in molecule D of the unit cell. The R5P molecule has a well-defined electron density for the phosphate group (with occupancy of 1.0) but not so well defined electron density for the ribose moiety (with occupancy of 0.6). Only the
phosphate groups have been included in the model for the other three molecules A, B, and C in the asymmetric unit, the rest of the density was modeled as waters. The ribose moiety (in molecule D) is in a five-membered ring conformation (the furanose form) with the anomeric hydroxyl in the α-configuration. This is different from the ring-opened or linear conformation of the ribose seen in the Mj PRPP synthetase complex. 9 R5P is bound in the regular R5P binding site with its phosphate group occupying the same position as the sulfate ions occupy in the
Fig. 7. Magnesium binding to ADP. The Mg 2+ ion is shown as a green sphere. It coordinates five oxygen ligands, two of them are from the pyrophosphate group of ADP and three are from water molecules. The sixth position is occupied by the N ɛ2 atom of the imidazole ring of His124. The distance Mg–N ɛ2 is 2.7 Å, whereas the average Mg–O distance is 2.1 Å.
Phosphoribosyl Pyrophosphate Synthetase Structures
853
Fig. 8. Superposition of the ligands bound in the ATP binding site. The labeled conformations ADP-I, ADP-II, and mATP represent ADP in the form I crystal structure (open conformation), ADP in the form II crystal structure (closed conformation), and mATP in the form I crystal structure (open conformation), respectively. ADP-II has the conformation that is necessary for ATP to adopt for the active nucleophilic attack on the β-phosphorous atom.
form I crystals. Besides the interactions with the phosphate-binding segment (residues 214–218), R5P makes a network of hydrogen bonds with the catalytic residues Lys184 and Arg186 and the three aspartate residues Asp161, Asp210, and Asp211 (Fig. 6). These three Asp residues are highly conserved among PRPP synthetases of the three classes (Fig. S1 and Supplementary Information). ADP is positioned in the regular ATP binding site with the adenine ring and the ribose ring adopting the same positions as found in the crystals of other complexes of the crystal form I; the adenine ring is sandwiched between side chains of Arg91 of each subunit and Phe32 of the neighboring subunit. The ribose moiety leans against the aromatic ring of Tyr96. Consequently, they make all the same interactions with the PRPP synthetase as in the structures of complexes with ADP and mATP. However, the α,β-pyrophosphate group of ADP adopts a different conformation in the ternary structure with R5P. The pyrophosphate group has a bound Mg 2+ ion that coordinates five oxygen atoms, three of them are from water molecules and the remaining two oxygen atoms belong to the pyrophosphate group of ADP. The sixth coordination position is occupied by the N ɛ2 atom of the imidazole ring of His124 (Fig. 7). The distance from Mg to N ɛ2 is 2.7 Å. The Mg 2+ coordination in this complex is similar to that in the form I crystal soaked with PRPP, although the β-phosphate group has a different orientation. In this complex, the β-phosphate group is in close proximity to the ribose moiety of R5P, particularly to its anomeric oxygen atom (O1) that is the nucleophile proposed to attack the β-phosphorus atom (PB). The O1 atom of R5P makes hydrogen bonds with three oxygen atoms of the β-phosphate group of ADP. These hydrogen bonds increase the polarity of the O1 atom of R5P and, consequently, its nucleophilicity. The anomeric oxygen atom of R5P is ideally positioned for
nucleophilic attack on the β-phosphorous atom; it is at the right distance from PB (2.7 Å) and in the right location. Any other conformation of R5P, such as the linear form or the β-D-ribofuranose form, would not have had the properly positioned O1 atom for the in-line nucleophilic attack. Superposition of the ligands bound in the ATP binding site of the Tv PRPP synthetase showed that only in the closed conformation of the enzyme does the β-phosphate group of ADP adopt the correct orientation (Fig. 8). In the open conformation, the ATP analogs adopted unproductive conformations for the β-phosphate.
Discussion Three classes of PRPP synthetases have been described so far, two of them are also structurally characterized. The oligomeric state of the enzyme plays an important role in the enzymatic mechanism. Kinetic and structural analyses revealed that, in the hexameric (class I) PRPP synthetases, ADP and phosphate ions compete for binding to a regulatory site formed at the junction of three subunits of the hexamer. Such an allosteric site does not exist in the class III enzymes, 9 as these molecules assemble into tetramers. As a result, the class III enzymes are regulated only by competitive inhibition. The tetrameric organization does not produce any other allosteric sites affecting enzymatic activity. Thus, the tetrameric assembly should not be a factor in designation of a protein to class III. The Tv PRPP synthetase oligomerization pattern is different from the tetrameric organization of the Mj PRPP synthetase and from the hexameric organization of the Bs PRPP synthetase. On the basis of the closest sequence similarity and the absence of the allosteric sites, the Tv enzyme could be best fit into the class III of PRPP synthetases. The four molecules in the asymmetric
854 unit of the crystal form II (closed conformation) do not assemble in the proposed biologically relevant tetramer of Mj PRPP synthetase (Fig. 3). Only a biological dimer is possible in this conformation. In fact, the analytical gel filtration carried out on a substrate-free enzyme at two very different concentrations (1 mg/ml and 10 mg/ml) only showed the presence of dimers. Possible catalytic mechanism The structures presented here provide the basis for a catalytic mechanism of the enzyme activity. The binding of R5P and ATP in the active site stabilizes the closed conformation of the protein by a network of interactions of the catalytic residues Lys184 and Arg186 with both substrates, particularly with the oxygen atoms of the β-phosphate group (Table S3 and Supplementary Information). The residues Lys184 and Arg186 also place the β-phosphorus atom in the perfect position for a nucleophilic attack by the O1 hydroxyl group of R5P. The pyrophosphate group conformation and position is also stabilized by a Mg 2+ ion that coordinates two of the oxygen atoms of the nucleotide's α-phosphate and β-phosphate (and three water molecules). The coordination environment of Mg 2+ also includes the N ɛ2 atom of His124 that, in turn, affects the position of the metal ion. The orientation of the His124 ring is mainly determined by the hydrogen-bonding interactions with Asp211, a highly conserved residue (Figs. 7 and 9). The γ-phosphate group of ATP that was modeled in the active site (Fig. 5b) could possibly interact with the N-terminus (Gly163) of the joint 310–α5 helix due to the dipole moment on the helix. One of the oxygen atoms of the γ-phosphate could possibly replace one of the coordinated waters
Phosphoribosyl Pyrophosphate Synthetase Structures
by Mg 2+. The in-line nucleophilic SN2 attack of the activated anomeric hydroxyl group of R5P on the β-phosphorus atom proceeds via a transition state with a pentacoordinate, trigonal bipyramidal β-phosphate (Fig. 9). Similar pentacoordinate transitional states were proposed for many enzymatic mechanisms involving the transfer of phosphates 16 and their derivatives. For instance, the pentacoordinate transition state of the α-phosphate group was proposed for the reactions of nucleotidyl transfer onto the 3′ end of a DNA primer by polymerases 17 and of the γ-phosphate in the reactions of GTP hydrolysis. 13,14,18 In the present mechanism, a β-phosphate oxygen atom (likely, O2B) plays the role of the general base in abstracting the proton from the anomeric α-hydroxyl group of the ribose ring. The distance between atoms O1 and O2B is 2.5 Å. The transition state complex is likely stabilized by the network of hydrogen bonds between the β-phosphate oxygen atoms and the catalytic residues Lys184 and Arg186. The catalytic β-hairpin acting as a lid blocks solvent access to the active site and thus shields the intermediate complex (Figs. 4 and 5). The SN2 nucleophilic attack on the β-phosphorus atom of ATP leads to inversion of this phosphate group configuration and formation of the α-pyrophosphate group of PRPP. The reaction also results in the formation of AMP. Inversion in the αphosphate group of PRPP would increase its electrostatic repulsion from the α-phosphate group of AMP. As a result, the interactions that existed between the β-phosphate group of ATP and catalytic residues (Lys184 and Arg186) would likely be lost with PRPP, causing the catalytic β-hairpin to open the active site. That would compel the products PRPP and AMP to leave their binding
Fig. 9. Possible catalytic mechanism for PRPP synthetase. The active site features the transition state with a pentacoordinate, trigonal bipyramidal β-phosphate resulting from the in-line SN2 nucleophilic attack by the anomeric α-oxygen of R5P (red) onto the β-phosphorus atom of ATP. Several factors facilitate this reaction, particularly, (1) the two catalytic residues, Arg186 and Lys184, that form a network of hydrogen bonds to both substrates (see Table S2 and Supplementary Information) especially to the βphosphate oxygen atoms; (2) the metal ion (Mg 2+) chelating two or possibly three phosphate groups of ATP; and (3) proton transfer from the anomeric α-hydroxyl group of R5P to the β-phosphate O2B atom.
Phosphoribosyl Pyrophosphate Synthetase Structures
sites and to make these binding sites available for the next R5P and ATP molecules.
Materials and Methods
855 quent matrix-assisted laser desorption/ionization–time of flight mass spectrometry analysis (Institute of Biomolecular Design, University of Alberta) revealed peptides consistent with Tvn0197. Analytical gel filtration
Cloning, expression, and purification The open reading frame Tvn0197 from Tv strain GSS1 encoding residues 1–286 of PRPP synthetase was amplified from genomic DNA template (American Type Culture Collection) using PCR. A PCR primer pair was designed for directional cloning (boldface) of the insert into the Gateway cloning system (Invitrogen). The primer sequences were Tvn0197F, 5′-GGGACAAGTTTGTACAAAAAAGCAGGCTCCGAAAACCTGTATTTTCAGGGTGGTTCGGGTGGTTCCGGTATGAAGATCATAGCTCTACG-3′, and Tvn0197R, 5′-GGGACCACTTTGTACAAGAAAGCTGGGTCTCAGGCATCAATATCCC-3′. A tobacco etch mosaic virus cleavage site followed by a Gly-Ser-Gly spacer peptide (italicized) is encoded by the forward primer preceding the native start codon for removal of the N-terminal affinity tag. The resulting PCR product was inserted into the pDONR-221 vector (Invitrogen) for DNA amplification, and then subsequently transferred to an expression vector (pVP16) that an amino-terminal maltose binding protein (MBP) tag immediately preceded by a histidine octapeptide. The expression plasmid pVP16-Tvn0197 was confirmed by restriction endonuclease analysis and DNA sequencing (DNA Core Facility, University of Alberta). The His8-MBP-Tvn0197 fusion protein was expressed in E. coli BL21(DE3) pLysS cells (Novagen). Incubation of the transformed cells in 2 l of LB+100 μg/ml ampicillin, 34 μg/ ml chloramphenicol, and 0.2% (w/v) glucose at 310 K was continued until an OD of 600 nm reached 0.8. Fusion protein expression was induced by adding isopropyl-β,-D-thiogalactopyranoside to a final concentration of 0.5 mM, and then the incubation temperature was raised to 317 K overnight. The cells were harvested by centrifugation then resuspended in buffer A [20 mM Tris–HCl (pH 7.5), 200 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM NaN3, 1 mM ATP, and 10 mM β-mercaptoethanol] supplemented with 200 μM PMSF, 625 μg/ml hen egg white lysozyme, and one Complete ethylenediaminetetraacetic-acid-free protease inhibitor tablet (Roche). For fusion protein purification, the cells were lysed by freeze–thaw and then subjected to ultrasonication in the resuspension buffer. The lysate was cleared by centrifugation (30 min, 20,000g), and the supernatant was loaded onto a 5-ml MBP trap FF column (GE Healthcare) preequilibrated with buffer A. The loaded sample was washed with buffer A, and then the bound protein was eluted using buffer A+ 10 mM maltose. The MBP tag and the N-terminal recombination site were removed by proteolysis at 277 K overnight using recombinant tobacco etch virus protease. Tvn0197 was isolated by passing the proteolytic reaction over an IMAC column charged with Ni 2+. The cleaved enzyme flowed through this column, while the His-tagged MBP remained bound and was pooled then concentrated to 7.5 mg/ml using an Amicon Ultra concentrator (10-kDa molecular mass cutoff; Millipore). The results of each step were monitored by 12% SDSPAGE from which a final concentrated protein sample was excised. Resulting in-gel trypsinization and subse-
The molecular mass of the PRPP synthetase was determined by analytical gel filtration using a Superdex 200 HR 10/30 column. A set of protein standards was run in phosphate-buffered saline buffer. PRPP synthetase samples at 1 mg/ml and 10 mg/ml protein concentrations were run under the same conditions. Peak data of the standards were used to perform linear regression analysis, and a corresponding standard curve was obtained. The molecular mass of the Tv PRPP synthetase was determined from the peak data using the standard curve. Crystallization and data collection Sitting-drop vapor diffusion techniques in a 96-well format were used to screen for crystallization of 7.5 mg/ml Tv PRPP synthetase. Crystals grew under a number of conditions in 24–48 h at room temperature from the Index Screen (Hampton Research). Two of these conditions were refined in hanging drops to produce diffraction-quality crystals: (form I) 0.1 M citric acid (pH 4.0) and 2.3 M NH4SO4 and (form II) 0.1 M Hepes (pH 7.5), 30% PEG 600, 10% 2-methyl-2,4-pentanediol, and 0.1% lauryl dimethylamine N-oxide. For data collection, crystals from the ammonium sulfate condition were rinsed in a cryoprotectant containing 30% glycerol in mother liquor and then flash cooled in liquid nitrogen. Crystals from the PEG conditions did not need cryoprotection. The data sets were collected on beamline 08ID-1 at the Canadian Light Source (Saskatoon) using a temperature of 100 K and a MAR225 detector. The raw data were processed with the HKL-2000 suite. 19 Structure solution and analysis Molecular replacements were performed with the program suite BALBES. 20 Refinement of the coordinates, the atomic temperature factors, and the ligand occupancies was carried out using the Phenix package and a maximum likelihood target. 21 Model rebuilding was performed using Coot. 22 The secondary structure and stereochemistry of the protein were analyzed by PROCHECK. 23 Sequence alignment was performed using the program ClustalW, 24 and the corresponding Fig. S1 (Supplementary Data) was generated with the program ESPript. 25 Various values of buried surface area and ΔGdiss were calculated with the European Molecular Biology Laboratory–European Bioinformatics Institute PISA server.12 r.m.s.d. values between analogous structures were calculated with Coot. 16 Figures 2–9 and Fig. S2 (Supplementary Data) were generated with PyMOL. 26 Accession codes Coordinates and structure factors have been deposited and released in the PDB with accession numbers 3lrt, 3nag, 3lpn, and 3mbi.
856 Supplementary materials related to this article can be found online at doi:10.1016/j.jmb.2011.09.007
Acknowledgements We are very grateful to Pawel Grochulski for his help in the data collection on beamline 08ID-1 at the Canadian Light Source (Saskatoon). This work was partially supported by the Canadian Institute for Health Research and the Alberta Heritage Foundation for Medical Research.
References 1. Hove-Jensen, B. (1988). Mutation in the phosphoribosylpyrophosphate synthetase gene (prs) that results in simultaneous requirements for purine and pyrimidine nucleosides, nicotinamide nucleotide, histidine, and tryptophan in Escherichia coli. J. Bacteriol. 170, 1148–1152. 2. Hove-Jensen, B. (1989). Phosphoribosylpyrophosphate (PRPP)-less mutants of Escherichia coli. Mol. Microbiol. 3, 1487–1492. 3. Jensen, K. F. (1983). Metabolism of 5-phosphoribosyl 1-pyrophosphate (PRPP) in Escherichia coli and Salmonella typhimurium. In Metabolism of Nucleotides, Nucleosides and Nucleobases (Munch-Petersen, A., ed.), pp. 1–25, Academic Press, London, UK. 4. Khorana, H. G., Fernandes, J. F. & Kornberg, A. (1958). Pyrophosphorylation of ribose 5-phosphate in the enzymatic synthesis of 5-phosphorylribose 1-pyrophosphate. J. Biol. Chem. 230, 941–948. 5. Krath, B. N. & Hove-Jensen, B. (2001). Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure. Protein Sci. 10, 2317–2324. 6. Kadziola, A., Jepsen, C. H., Johansson, E., McGuire, J., Larsen, S. & Hove-Jensen, B. (2005). Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii. J. Mol. Biol. 354, 815–828. 7. Eriksen, T. A., Kadziola, A., Bentsen, A. K., Harlow, K. W. & Larsen, S. (2000). Structural basis for the function of Bacillus subtilis phosphoribosylpyrophosphate synthetase. Nat. Struct. Biol. 7, 303–305. 8. Seattle Structural Genomics Center for Infectious Disease (SSGCID). 2.3 A crystal structure of ribosephosphate pyrophosphokinase from Burkholderia pseudomallei. DOI:10.2210/pdb3dah/pdb. 9. Li, S., Lu, Y., Peng, B. & Ding, J. (2007). Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site. Biochem. J. 401, 39–47.
Phosphoribosyl Pyrophosphate Synthetase Structures 10. Krath, B. N., Eriksen, T. A., Poulsen, T. S. & HoveJensen, B. (1999). Cloning and sequencing of cDNAs specifying a novel class of phosphoribosyl diphosphate synthase in Arabidopsis thaliana. Biochim. Biophys. Acta, 1430, 403–408. 11. Hove-Jensen, B., Bentsen, A. K. K. & Harlow, K. W. (2005). Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase. Alanine-scanning mutagenesis of the flexible catalytic loop. FEBS J. 272, 3631–3639. 12. Krissinel, E. & Henrick, K. (2007). Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797. 13. Nygaard, F. B. (2001). The molecular mechanism of catalysis and allosteric regulation in the phosphoribosyldiphosphate synthase from Bacillus subtilis. Thesis, University of Copenhagen, Denmark. 14. Sondek, J. S., Lambright, D. G., Noel, J. P., Hamm, H. E. & Sigler, P. B. (1994). GTPase mechanism of G proteins from the 1.7-Å crystal structure of transducin α–GDP AIF −4. Nature, 372, 276–279. 15. Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G. & Sprang, S. R. (1994). Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis. Science, 265, 1405–1412. 16. Cleland, W. W. & Hengge, A. C. (2006). Enzymatic mechanisms of phosphate and sulfate transfer. Chem. Rev. 106, 3252–3278. 17. Brautigam, C. A. & Steitz, T. A. (1998). Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. Curr. Opin. Struct. Biol. 8, 54–63. 18. Sprang, S. R. (1997). G proteins, effectors and GAPs: structure and mechanism. Curr. Opin. Struct. Biol. 7, 849–856. 19. Otwinowski, Z. & Minor, W. (1997). Processing of Xray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. 20. Long, F., Vagin, A., Young, P. & Murshudov, G. N. (2008). BALBES: a molecular replacement pipeline. Acta Crystallogr., Sect. D: Biol. Crystallogr. 64, 125–132. 21. Afonine, P. V., Grosse-Kunstleve, R. W. & Adams, P. D. (2005). The Phenix refinement framework. CCP4 Newsletter, 42; contribution 8. 22. Emsley, P. & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126–2132. 23. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291. 24. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H. et al. (2007). ClustalW and ClustalX version 2. Bioinformatics, 23, 2947–2948. 25. Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. (1999). ESPript: multiple sequence alignments in PostScript. Bioinformatics, 15, 305–308. 26. DeLano, W. L. (2002). The PyMOL Molecular Graphics System. http://pymol.sourceforge.net/.
doi:10.1016/j.jmb.2011.09.007 Contents lists available at www.sciencedirect.com
Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b
The Structures of Thermoplasma volcanium Phosphoribosyl Pyrophosphate Synthetase Bound to Ribose-5-Phosphate and ATP Analogs Maia M. Cherney, Leonid T. Cherney I , Craig R. Garen and Michael N. G. James⁎ Group in Protein Structure and Function, Department of Biochemistry, School of Molecular and Systems Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Received 11 June 2011; received in revised form 31 August 2011; accepted 4 September 2011 Available online 21 September 2011 Edited by M. Guss Keywords: X-ray crystallography; phosphoribosyl pyrophosphate synthetase; complex with substrate analogs; Thermoplasma volcanium
Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P21. The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP–Mg 2+ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively. © 2011 Elsevier Ltd. All rights reserved.
Introduction *Corresponding author. E-mail address: [email protected]. I Present address: L. T. Cherney, Department of Chemistry and Center for Research on Biomolecular Interactions, Faculty of Science and Engineering, York University, Toronto, Ontario, Canada M3J 1P3. Abbreviations used: PRPP, phosphoribosyl pyrophosphate; R5P, ribose-5-phosphate; Tv, Thermoplasma volcanium; mATP, β,γ-methylene ATP; PEG, polyethylene glycol; Mj, Methanocaldococcus jannaschii; Bs, Bacillus subtilis; PDB, Protein Data Bank; MBP, maltose binding protein.
Phosphoribosyl pyrophosphate (PRPP) is an important metabolite that plays a central role in many life processes, namely, the biosynthesis of purine and pyrimidine nucleotides, the biosynthesis of the coenzyme NAD +, and the biosynthesis of the amino acids histidine and tryptophan; 1,2 it is a substrate for many enzymes. 3 It is produced in a transfer reaction of the intact β,γ-diphosphate group from ATP to the anomeric oxygen atom (O1) of ribose-5-phosphate (R5P): R5P + ATP → PRPP + AMP by the PRPP synthetase 4 (E.C. 2.7.6.1). Three classes of PRPP synthetases have been described. 5,6
0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.
Phosphoribosyl Pyrophosphate Synthetase Structures
Class I enzymes occur in a diverse range of bacteria and mammals. They are characterized by their dependence on phosphate and Mg 2+ ions for activity; they can be allosterically regulated by ADP and, in some cases, by GDP. Three class I PRPP synthetases from Bacillus subtilis (Bs), 7 Burkholderia pseudomallei, 8 and human 9 have had their structures determined. They are two-domain monomeric molecules having similar folds and a significant sequence identity (about 50%). They adopt a hexameric quaternary structure. The six protomers that comprise the hexamers represent a trimer of dimers that are related by 32 point group symmetry; they form a propeller in which the N-terminal domains are oriented inwards toward the center of the hexamer, and the C-terminal domains are turned toward the outside. The allosteric site is situated not far from the active site, at the junction of three subunits; two of the subunits belong in the same dimer, and the third subunit comes from an adjacent dimer. Class II PRPP synthetases are found in plants. 10 They are independent of phosphate for activity and are not allosterically inhibited by purine nucleoside
845 diphosphates. Class II enzymes have the lowest sequence similarity scores to the class I and class III enzymes as outlined below (less than 20%). Recently, a third class of PRPP synthetases has been proposed. This class is represented by the enzyme from Methanocaldococcus jannaschii (Mj). 6 It is activated by phosphate and Mg 2+ ions but is independent of allosteric regulation. The Mj enzyme forms a tetramer in which two dimers are related by a non-crystallographic 2-fold axis. The interfaces that are present in the allosteric sites of the class I enzymes are not preserved in the Mj enzyme. The Mj PRPP synthetase has a low sequence similarity with the known class I enzymes (about 25%).The sequence alignment of the Tv PRPP synthetase and the four enzymes mentioned above having known structures is shown in Fig. S1 (see Supplementary Information). The previously reported structures of the PRPP synthetases did not fully explain the mechanism of activity, as the catalytically important areas of the active sites, particularly the catalytically important hairpin, were disordered. No productive complexes were previously obtained. Here, we report four crystal
Table 1. Crystal parameters and data collection statistics for Tv PRPP synthetase Complex crystal
Data collection Space group Cell dimensions a, b, c (Å) α, β, γ (°) No. of molecules per asymmetric unit Temperature (K) Detector Wavelength (Å) Resolution (Å) Multiplicity I/σ(I) Completeness (%) Rsymb Refinement Resolution No. of unique reflections Rwork/Rfreec No. of atomsd Protein Ions/ligands Water Mean B-factors (Å2) Protein Ions/ligands Water r.m.s.d. Bond lengths (Å) Bond angles (°) a
ADP/SO4 PDB code 3LRT
ADP/SO4 PDB code 3NAG
mATP/SO4 PDB code 3LPN
ADP–Mg2+/R5P PDB code 3MBI
P21
P21
P21
P21
38.6, 141.6, 50.1 90.0, 96.2, 90.0 2 100 MAR225 0.97950 50.0–1.53 (1.58–1.53)a 3.4 (2.6)a 12.4 (1.6)a 89.6 (58.1)a 0.08 (0.42)a
38.6, 141.6, 49.6 90, 95.9, 90 2 100 MAR225 0.9795 50–1.75 (1.81–1.75)a 3.6 (3.4)a 14.0 (1.4)a 93.9 (80.8)a 0.09 (0.84)a
38.6, 141.2, 50.0 90.0, 96.0, 90.0 2 100 MAR225 0.97950 50.0–1.80 (1.86–1.80)a 3.8 (3.6)a 11.7 (2.0)a 99.9 (100.0)a 0.11 (0.59)a
76.6, 61.8, 127.9 90.0, 103.9, 90.0 4 100 MAR225 0.97950 50.0–1.80 (1.86–1.80)a 3.9 (3.8)a 11.9 (1.8)a 97.8 (96.4)a 0.12 (0.71)a
24.6–1.53 66,070 18.6/21.9 5093 4576 20/54 443
31.9–1.75 49,953 17.6/21.7 5029 4517 31/27 454
29.1–1.8 49,076 17.1/21.5 5108 4525 25/62 496
25.5–1.8 105,818 17.6/21.9 10,470 9075 19/122 1254
22 34/29 32
27 43/40 35
27 38/29 36
19 17/19 29
0.006 1.106
0.007 1.065
0.006 1.079
0.016 1.059
Values for the highest-resolution shell are given in parentheses. Rsym = ∑h∑i(|Ii(h) − 〈I(h)〉|)/∑h∑iIi(h), where Ii(h) is the ith intensity measurement, and 〈I(h)〉 is the weighted mean of all measurements of I(h). c Rwork and Rfree = ∑h(|Fo(h)| − |Fc(h)|)/|Fo(h)| for reflections in the working and test sets (5% of the data). d Number of non-hydrogen atoms present in the asymmetric unit. b
846 structures of Tv PRPP synthetase bound to several ATP analogs and to R5P in which the ribose adopts the five-membered furanose ring conformation. Based on these structures, a proposal for the mechanism of the PRPP synthetase activity has been suggested.
Results Structure determination The Tv PRPP synthetase open reading frame Tvn0197 was amplified by polymerase chain reaction (PCR), and the enzyme was overproduced in Escherichia coli and purified to homogeneity. Analytical gel-filtration experiments were carried out at two different protein concentrations, 1 mg/ml and 10 mg/ml; they showed a major peak corresponding to a molecular mass of 55 kDa that is slightly less than the theoretical molecular mass of the dimer (64.3 kDa). Two crystal forms, both in the space group P21, of the Tv PRPP synthetase were grown under two different conditions. One crystal form (form I) was obtained from a high-salt and low-pH (2 M ammonium sulfate, pH 4.0) condition and had
Phosphoribosyl Pyrophosphate Synthetase Structures
two enzyme molecules in the asymmetric unit. The other form (form II) was produced from low-salt and neutral-pH conditions [30% polyethylene glycol (PEG) 600, pH 7.5] and had four molecules in the asymmetric unit. The protein preparation that was used to produce the crystals contained one of the substrates, ATP, that was added during the first purification step in the crude extract. Consequently, the protein preparation and the resulting crystals (forms I and II) contained ATP or more likely ADP, a product of ATP hydrolysis by another enzyme in the crude extract. Additionally, endogenous phosphate and Mg 2+ ions, as well as R5P, were observed to be present in the enzyme in crystal form II. The form I crystals also were soaked with β,γ-methylene ATP (mATP) and PRPP (separately). PRPP has not been observed in any of the crystal structures; however, its effect on ADP binding has been observed. ADP ligand has been displaced from its binding site in one molecule of the dimer. These several crystal structures were solved by the molecular replacement method using the structure of the Mj PRPP synthetase monomer as the search model. Crystallographic data collection information and refinement statistics are presented in Table 1.
Fig. 1. Structure of the Tv PRPP synthetase in complex with ADP in an open conformation (crystal form I). The asymmetric unit contains two molecules (yellow and pink) that form a biological dimer. The dimer interface is formed predominantly by residues on helices α2 and α3, the β4-strand, and loop segments of each subunit. The β10-strand adopts an open conformation; β10 includes two catalytic residues Lys184 and Arg186. ADP (shown in sticks) is situated in the ATP binding site.
Phosphoribosyl Pyrophosphate Synthetase Structures
Overall structure of the Tv PRPP synthetase The overall structure of the Tv PRPP synthetase is shown in Fig. 1. It is very similar to the known PRPP synthetase structures from class I 5–7 and class III 9 (with r.m.s.d. values in the range of 1.1–1.9 Å for ∼250 C α atom pairs), although the sequence homology among them is not very high; the highest score (29% sequence identity) is for the Mj enzyme. Each subunit comprises two very similarly folded domains that are related by an approximate non-crystallographic 2-fold axis. Superposition of the N-terminal and C-terminal domains of one subunit is shown in Fig. S2 (Supplementary Information). They had only 11% of sequence identity. Both domains can be classified as having an open α/β fold with a central six-stranded parallel β-sheet surrounded by four helices from the same domain and, in the case of the N-terminal domain, by one additional helix from the C-terminal domain. Each domain also contains a β-hairpin (β3–β4 in the N-domain and β10–β11 in the C-domain) that plays an important role in the enzymatic function of the PRPP synthetases. Both hairpins are structurally equivalent. The β-hairpin loop of the N-terminal domain (the loop between strands β3 and β4) forms a part of the active site of the adjoining molecule in the dimer. The β-hairpin of the C-terminal domain (β10 and β11) (the catalytic β-hairpin, residues 179–187) can adopt two different conformations of the Tv PRPP synthetase: open and closed. The form I crystals have PRPP synthetase in the open conformation, and the form II crystals adopt the closed conformation. That this β-hairpin plays an important role in the catalysis of Bs PRPP synthetase was shown by kinetic analysis of the substitutions of the corresponding residues Lys184 and Arg186 (Tv numbering) for Ala that are located on the β-strand 10. These substitutions caused approximately 30,000 and 24,000 times reduction in the maximal velocity, Vmax, respectively, in Bs. 11 Oligomeric state There are two Tv PRPP synthetase molecules in the asymmetric unit of the form I crystals and four Tv PRPP synthetase molecules in the asymmetric unit of the form II crystals. The dimer in the asymmetric unit (form I) has an extensive solventinaccessible interface between monomers of 1945 Å 2 per subunit (Fig. 1). The interface consists mainly of two α-helices (α2 and α3), the strand β4, and a loop segment 86–102 of each subunit. Interactions across the dimer interface include predominantly hydrophobic contacts (the solvation free-energy gain upon formation of the interface, ΔG i = − 17 kcal/mol), as well as 34 hydrogen bonds and 15 salt bridges (Table S1 and Supplementary Information; PISA server). 12 Consequently, the interface has a significant positive ΔGdiss value (22 kcal/mol), which means that the Gibbs free energy would increase by this amount on
847 the breakup of the dimer interface. As a result, dimer dissociation would be strongly energetically unfavorable. Tv PRPP synthetase is a biological dimer, and it does not form higher symmetry oligomers (hexamers or tetramers) as described for the class I and class III PRPP synthetases. Comparison of Tv PRPP synthetase with other PRPP synthetases Superposition of the Tv PRPP synthetase subunit with the corresponding subunits of the Mj and Bs enzymes showed r.m.s.d. values of 1.3 Å (for 259 C α atom pairs) and 1.85 Å (for 260 C α atom pairs), respectively. The Tv PRPP synthetase polypeptide has 286 amino acids and is more closely related to the Mj enzyme with a sequence identity of 29%; it has lower sequence identities with the class I PRPP synthetases (∼ 23%) and the class II PRPP synthetases (e.g., from Spinacia oleracea isozymes 3 and 4, 18% and 12%, respectively) (data not shown). Superposition of the Tv PRPP synthetase dimer on the Mj dimer using a single subunit (A) reveals that there is a major difference between the two dimers; it results in the deviation of the B subunits by 6°. Consequently, superposition of two Tv dimers on the Mj tetramer shows that the Tv dimers do not come close enough to form the tetrameric interfaces. There is also a large difference in the protein sequences that can be seen in the sequence alignment (Fig. S1 and Supplementary Information). The differences become even greater with Bs PRPP synthetase; the Tv enzyme does not form hexamers, likely because it lacks the final C-terminal helix that is involved in oligomerization of the class I enzymes, and the allosteric ADP binding sites that form at the interfaces of the hexamer are not possible with the Tv enzyme. One of the important differences between the present structures of Tv PRPP synthetase and the structures of the other PRPP synthetases lies in the orientation of the catalytic β-hairpin that is disordered in both the Mj (residues 185–196) and the Bs (residues 196–208) structures. In Tv PRPP synthetase, the corresponding sequence for the catalytic β-hairpin (residues 179–187) has well-defined associated electron density. Complex with ADP As it was pointed out above, ATP was added to the lysis buffer at the beginning of purification. Thus, the final purified protein contained bound ATP or, more likely, judging from the resulting electron density, ADP, a product of ATP hydrolysis presumably by an ATPase in the crude extract. The protein was crystallized from ammonium sulfate at low pH, and the resultant crystals had two molecules in the asymmetric unit [crystal form I,
848 Fig. 1; Protein Data Bank (PDB) code 3lrt]. Each molecule contained ADP in the ATP binding site and a sulfate ion in the R5P binding site. The two binding sites and the catalytic residues Lys184 and Arg186 located on the strand β10 together comprise the active site of the Tv PRPP synthetase. In crystal form I, each subunit has an open conformation with the active site fully accessible for substrate binding. The active-site residues Lys184 and Arg186 that
Phosphoribosyl Pyrophosphate Synthetase Structures
were previously identified in Bs by kinetic activity studies of variants are far from the substrates. Thus, the average distance between the O3B atom of ADP and the N ζ atom of Lys184 is ∼ 14–15 Å. Similarly, the average distance between the O3B atom of ADP and the N η2 of Arg186 is about 15–16 Å. This conformation would allow the substrates to enter the active site, but it would not support the catalytic reaction.
Fig. 2. (a) Binding of ADP in the ATP binding site (crystal form I). (b) Binding of mATP in the ATP binding site (crystal form I). The ATP binding site is situated in the cleft between two domains of one subunit (yellow); it is also restricted on the other side by the loop residues (Loop L) of the adjacent subunit of the dimer (cyan). The phosphate binding site of R5P involving main-chain and side-chain interactions of residues 214–218 is occupied by a sulfate ion. Hydrogen bonding and electrostatic interactions are shown in broken lines. The |Fo| − |Fc|, αc electron density omit map is contoured at 2 σ level in the regions of ADP or mATP. Water molecules are depicted as red spheres.
Phosphoribosyl Pyrophosphate Synthetase Structures
There are two independent active sites in the Tv PRPP synthetase dimer (Fig. 1). Each site is situated in the cleft between the two domains of one subunit (Fig. 2a); it is also limited by a hairpin Loop L (the hairpin of the N-terminal domain involving the strands β3 and β4) of the twofold related subunit of the dimer. A network of hydrogen bonds and electrostatic interactions forms between the enzyme and ADP (Table S2 and Supplementary Information). Tyr96 and His93 interact with the β-phosphate group of ADP (atoms O1B and O2B). Gln92 makes a hydrogen bond to the O2′ hydroxyl group of the ribose moiety. The adenine ring of ADP is sandwiched between Arg91 and Phe32; the latter residue belongs to the β3–β4 hairpin loop of the adjacent subunit of the dimer. Asp34 and Glu36, also from the adjacent subunit, form hydrogen bonds with nitrogen atoms of the adenine ring. Several contacts are also made through water molecules (Fig. 2a and b). The proposed R5P binding site is occupied by a sulfate ion that was present in the crystallization mother liquor; it mimics the phosphate group of R5P (Fig. 2a). The phosphate-binding residues are identical with those seen in the Mj enzyme. The sulfate ion is situated near the phosphate-binding segment that includes a loop (residues 214–216) and the Nterminus of helix α6 with a highly conserved Gly217–Thr218 motif (Fig. S1 and Supplementary Information). The sulfate ion forms hydrogen bonds to four residues in the phosphate-binding segment, mainly to main-chain nitrogen atoms and to the hydroxyl groups of the threonine residues. In addition, the sulfate ion makes a salt bridge (distance, 3.7 Å) with the guanidinium group of Arg91. Several hydrogen bonds are made to water molecules. One of these water molecules also contacts the O γ atom of Ser214. Additional sulfate ions were modeled elsewhere on the surface of the enzyme. Complex with one ADP molecule per dimer When the original form I crystals were soaked overnight with PRPP, the resulting structure (PDB code 3nag) showed the presence of neither PRPP nor R5P. Instead, one of the ADP molecules of the dimer was displaced from its position in the ATP binding site; the site remained empty, and it collapsed. The loop (“flexible” loop, residues 91–96) changed its position and became notably disordered, with poor density for both the main-chain and the sidechain residues. The movement of this loop was also observed in Bs, human, and Mj PRPP synthetases. 5–7,9 The other subunit had ADP bound, and there were no changes in the position or in the degree of order of the loop formed by residues 91–96. Evidently, the conformation of the flexible loop is determined by the presence of ADP. Noteworthy, ADP here has a bound magnesium ion that coordinates three oxygen
849 atoms of phosphate groups of ADP and two water molecules. The sixth position in the octahedral Mg 2+ coordination is occupied by the N ɛ2 atom of the imidazole ring of His124; the distance between the N ɛ2 atom and the Mg 2+ ion is 3.7 Å. The coordination is similar to that observed in the complex with ADP and R5P (see below). The sulfate ions occupied the usual positions in the R5P binding site in both subunits. The α-phosphate group (O2A atom) of ADP (in the PRPP-soaked crystal) makes a salt bridge with the guanidinium group of Arg40 that belongs to the symmetry-related dimer (symmetry operation x, y, z − 1 or x, y, z + 1). The distances between the O2A atom of ADP and atoms N η2 and N η1 of Arg40 are 3.4 Å and 3.7 Å, respectively. In the case of the original ADP complex crystals, these distances were around 4 Å. Complex with mATP The original form I crystals containing ADP were soaked overnight with mATP. As a result, ADP was substituted by mATP in both subunits of the dimer (Fig. 2b; PDB code 3lpn). The complex with mATP retained the open conformation of the enzyme subunits; the catalytic β10-strand that contains residues Lys184 and Arg186 is situated far from the substrates. The positions of the adenine and ribose rings did not change; they make all the same interactions with the PRPP synthetase as they do in the ADP complex (Table S2 and Supplementary Information). The main differences are observed in the positions of the phosphate groups. His93 in this complex makes hydrogen bonds with two phosphate groups (β and γ; atoms O1B and O3G, respectively). Also, His124 makes a hydrogen bond (2.8 Å) with the γ-phosphate group of mATP (atom O1G). Tyr96 interacts with the β-phosphate group of mATP in the same way as it does with ADP. Interestingly, the carboxylate of Asp125 that is positioned at the N-terminus of a 310 helix is close to the γ-phosphate group. The charge on the carboxylate of Asp125 is partly neutralized through a hydrogen bond with a main-chain amide of Lys127. The distance between O δ1 of Asp125 and O3 γ of mATP is 2.4 Å. The α-phosphate group (O1A atom) of mATP makes similar electrostatic interactions with the guanidinium group of Arg40 of the symmetry-related dimer as that of ADP. The distances between the O1A atom of mATP and atoms N η2 and N η1 of Arg40 are 2.9 Å and 3.7 Å, respectively. The sulfate ions are present in the R5P binding site of both subunits. Complex with ADP and R5P Crystal form II was grown from low-salt conditions (i.e., no sulfate ions); it has two biological
850
Phosphoribosyl Pyrophosphate Synthetase Structures
Fig. 3. Structure of the Tv PRPP synthetase in crystal form II. The asymmetric unit contains four molecules representing two biological dimers. Each molecule is colored individually. Ligands, ADP and R5P, are shown as sticks in molecule D (yellow).
dimers (totally, four protein molecules) in the asymmetric unit (PDB code 3mbi). ADP ligands each having a bound Mg 2+ ion are present in all of the four protein molecules, but only one R5P ligand is bound to the tetramer in the asymmetric unit, specifically, subunit D. As Mg 2+ ions and R5P were
not added at any stage during the protein preparation, they have likely come from the E. coli expression system and have remained bound during the purification steps. The four protein molecules of the asymmetric unit do not form a biological tetramer as seen in the structure of Mj PRPP
Fig. 4. Superposition of the Tv PRPP synthetase dimers in two different conformations: open (green shades for A and B molecules, crystal form I) and closed (magenta shades for B and D molecules, crystal form II). The N-terminal domains that form the dimer interface superpose well, whereas the C-terminal domains differ considerably. The catalytic β-hairpin (strands β10 and β11) bends over toward the ligands (shown in sticks) forming a closed conformation for the enzyme. The positions of catalytic residues Lys184 and Arg186 are marked as K and R.
Phosphoribosyl Pyrophosphate Synthetase Structures
synthetase (Fig. 3). Individual monomers of form I crystals were superimposed with the monomers of form II crystals resulting in r.m.s.d. values in the range of 2.3–2.4 Å over 280–285 C α atom pairs for each superposition. The N-terminal domains of the form I and form II monomers superpose very well; the largest deviation occurs between the secondary structural elements of the C-terminal domains, particularly, in the region of the flexible catalytic β-hairpin formed by strands β10 and β11 (Fig. 4). In the closed conformation, strand β10 splits into two strands β10a and β10b. The β10–β11 hairpin bends over by approximately 45° toward the bound
851 ligands forming the closed conformation for the enzyme. This movement places the catalytic residues Lys184 and Arg186 within hydrogen-bonding distance to the β-phosphate group of ADP and likely to the β-phosphate group of ATP when it is bound here (Table S3 and Supplementary Information). In the closed conformation, the active-site region containing the reacting substrates in the transition state is shielded from solvent. The surface representation of the active site shows that the ligands are situated in deep pockets (Fig. 5a). The closed conformation was also reported for the crystal structure of the Bs PRPP synthetase complexed with AMP, R5P, and the
Fig. 5. Surface representation of the active site of the Tv PRPP synthetase in the closed conformation. (a) The ligands, R5P and ADP (shown in sticks), are buried in the deep surface pockets and are shielded from the bulk solvent. (b) An ATP model is aligned with ADP. The γ-phosphate group of ATP (green phosphorus atom) is modeled in the orientation directed toward the N-terminus of the joint 310–α5 helix (residue Gly163). One of the oxygen atoms of the γ-phosphate group would likely replace a water molecule (shown beside it) in the coordination sphere of Mg 2+.
852
Phosphoribosyl Pyrophosphate Synthetase Structures
Fig. 6. A stereo view of the interactions of Tv PRPP synthetase with ADP and R5P. Residues of subunit D (yellow sticks) and subunit C (cyan sticks) form the active site. Hydrogen bonds and electrostatic interactions of substrates ADP and R5P with active-site residues directly and via water molecules (red spheres) are shown in broken lines. The O1 atom (the nucleophile) of R5P is in an appropriate position and distance from β-phosphorous atom of ADP to carry out the nucleophilic attack.
transition state analog AlF3. 11,13 It was shown that the equivalent Bs catalytic residues, Lys197 and Arg199, interacted with two of the fluoride atoms that are analogous to the oxygen atoms of the βphosphate group in the trigonal bipyramidal transition state. 11,14,15 R5P was modeled only in molecule D of the unit cell. The R5P molecule has a well-defined electron density for the phosphate group (with occupancy of 1.0) but not so well defined electron density for the ribose moiety (with occupancy of 0.6). Only the
phosphate groups have been included in the model for the other three molecules A, B, and C in the asymmetric unit, the rest of the density was modeled as waters. The ribose moiety (in molecule D) is in a five-membered ring conformation (the furanose form) with the anomeric hydroxyl in the α-configuration. This is different from the ring-opened or linear conformation of the ribose seen in the Mj PRPP synthetase complex. 9 R5P is bound in the regular R5P binding site with its phosphate group occupying the same position as the sulfate ions occupy in the
Fig. 7. Magnesium binding to ADP. The Mg 2+ ion is shown as a green sphere. It coordinates five oxygen ligands, two of them are from the pyrophosphate group of ADP and three are from water molecules. The sixth position is occupied by the N ɛ2 atom of the imidazole ring of His124. The distance Mg–N ɛ2 is 2.7 Å, whereas the average Mg–O distance is 2.1 Å.
Phosphoribosyl Pyrophosphate Synthetase Structures
853
Fig. 8. Superposition of the ligands bound in the ATP binding site. The labeled conformations ADP-I, ADP-II, and mATP represent ADP in the form I crystal structure (open conformation), ADP in the form II crystal structure (closed conformation), and mATP in the form I crystal structure (open conformation), respectively. ADP-II has the conformation that is necessary for ATP to adopt for the active nucleophilic attack on the β-phosphorous atom.
form I crystals. Besides the interactions with the phosphate-binding segment (residues 214–218), R5P makes a network of hydrogen bonds with the catalytic residues Lys184 and Arg186 and the three aspartate residues Asp161, Asp210, and Asp211 (Fig. 6). These three Asp residues are highly conserved among PRPP synthetases of the three classes (Fig. S1 and Supplementary Information). ADP is positioned in the regular ATP binding site with the adenine ring and the ribose ring adopting the same positions as found in the crystals of other complexes of the crystal form I; the adenine ring is sandwiched between side chains of Arg91 of each subunit and Phe32 of the neighboring subunit. The ribose moiety leans against the aromatic ring of Tyr96. Consequently, they make all the same interactions with the PRPP synthetase as in the structures of complexes with ADP and mATP. However, the α,β-pyrophosphate group of ADP adopts a different conformation in the ternary structure with R5P. The pyrophosphate group has a bound Mg 2+ ion that coordinates five oxygen atoms, three of them are from water molecules and the remaining two oxygen atoms belong to the pyrophosphate group of ADP. The sixth coordination position is occupied by the N ɛ2 atom of the imidazole ring of His124 (Fig. 7). The distance from Mg to N ɛ2 is 2.7 Å. The Mg 2+ coordination in this complex is similar to that in the form I crystal soaked with PRPP, although the β-phosphate group has a different orientation. In this complex, the β-phosphate group is in close proximity to the ribose moiety of R5P, particularly to its anomeric oxygen atom (O1) that is the nucleophile proposed to attack the β-phosphorus atom (PB). The O1 atom of R5P makes hydrogen bonds with three oxygen atoms of the β-phosphate group of ADP. These hydrogen bonds increase the polarity of the O1 atom of R5P and, consequently, its nucleophilicity. The anomeric oxygen atom of R5P is ideally positioned for
nucleophilic attack on the β-phosphorous atom; it is at the right distance from PB (2.7 Å) and in the right location. Any other conformation of R5P, such as the linear form or the β-D-ribofuranose form, would not have had the properly positioned O1 atom for the in-line nucleophilic attack. Superposition of the ligands bound in the ATP binding site of the Tv PRPP synthetase showed that only in the closed conformation of the enzyme does the β-phosphate group of ADP adopt the correct orientation (Fig. 8). In the open conformation, the ATP analogs adopted unproductive conformations for the β-phosphate.
Discussion Three classes of PRPP synthetases have been described so far, two of them are also structurally characterized. The oligomeric state of the enzyme plays an important role in the enzymatic mechanism. Kinetic and structural analyses revealed that, in the hexameric (class I) PRPP synthetases, ADP and phosphate ions compete for binding to a regulatory site formed at the junction of three subunits of the hexamer. Such an allosteric site does not exist in the class III enzymes, 9 as these molecules assemble into tetramers. As a result, the class III enzymes are regulated only by competitive inhibition. The tetrameric organization does not produce any other allosteric sites affecting enzymatic activity. Thus, the tetrameric assembly should not be a factor in designation of a protein to class III. The Tv PRPP synthetase oligomerization pattern is different from the tetrameric organization of the Mj PRPP synthetase and from the hexameric organization of the Bs PRPP synthetase. On the basis of the closest sequence similarity and the absence of the allosteric sites, the Tv enzyme could be best fit into the class III of PRPP synthetases. The four molecules in the asymmetric
854 unit of the crystal form II (closed conformation) do not assemble in the proposed biologically relevant tetramer of Mj PRPP synthetase (Fig. 3). Only a biological dimer is possible in this conformation. In fact, the analytical gel filtration carried out on a substrate-free enzyme at two very different concentrations (1 mg/ml and 10 mg/ml) only showed the presence of dimers. Possible catalytic mechanism The structures presented here provide the basis for a catalytic mechanism of the enzyme activity. The binding of R5P and ATP in the active site stabilizes the closed conformation of the protein by a network of interactions of the catalytic residues Lys184 and Arg186 with both substrates, particularly with the oxygen atoms of the β-phosphate group (Table S3 and Supplementary Information). The residues Lys184 and Arg186 also place the β-phosphorus atom in the perfect position for a nucleophilic attack by the O1 hydroxyl group of R5P. The pyrophosphate group conformation and position is also stabilized by a Mg 2+ ion that coordinates two of the oxygen atoms of the nucleotide's α-phosphate and β-phosphate (and three water molecules). The coordination environment of Mg 2+ also includes the N ɛ2 atom of His124 that, in turn, affects the position of the metal ion. The orientation of the His124 ring is mainly determined by the hydrogen-bonding interactions with Asp211, a highly conserved residue (Figs. 7 and 9). The γ-phosphate group of ATP that was modeled in the active site (Fig. 5b) could possibly interact with the N-terminus (Gly163) of the joint 310–α5 helix due to the dipole moment on the helix. One of the oxygen atoms of the γ-phosphate could possibly replace one of the coordinated waters
Phosphoribosyl Pyrophosphate Synthetase Structures
by Mg 2+. The in-line nucleophilic SN2 attack of the activated anomeric hydroxyl group of R5P on the β-phosphorus atom proceeds via a transition state with a pentacoordinate, trigonal bipyramidal β-phosphate (Fig. 9). Similar pentacoordinate transitional states were proposed for many enzymatic mechanisms involving the transfer of phosphates 16 and their derivatives. For instance, the pentacoordinate transition state of the α-phosphate group was proposed for the reactions of nucleotidyl transfer onto the 3′ end of a DNA primer by polymerases 17 and of the γ-phosphate in the reactions of GTP hydrolysis. 13,14,18 In the present mechanism, a β-phosphate oxygen atom (likely, O2B) plays the role of the general base in abstracting the proton from the anomeric α-hydroxyl group of the ribose ring. The distance between atoms O1 and O2B is 2.5 Å. The transition state complex is likely stabilized by the network of hydrogen bonds between the β-phosphate oxygen atoms and the catalytic residues Lys184 and Arg186. The catalytic β-hairpin acting as a lid blocks solvent access to the active site and thus shields the intermediate complex (Figs. 4 and 5). The SN2 nucleophilic attack on the β-phosphorus atom of ATP leads to inversion of this phosphate group configuration and formation of the α-pyrophosphate group of PRPP. The reaction also results in the formation of AMP. Inversion in the αphosphate group of PRPP would increase its electrostatic repulsion from the α-phosphate group of AMP. As a result, the interactions that existed between the β-phosphate group of ATP and catalytic residues (Lys184 and Arg186) would likely be lost with PRPP, causing the catalytic β-hairpin to open the active site. That would compel the products PRPP and AMP to leave their binding
Fig. 9. Possible catalytic mechanism for PRPP synthetase. The active site features the transition state with a pentacoordinate, trigonal bipyramidal β-phosphate resulting from the in-line SN2 nucleophilic attack by the anomeric α-oxygen of R5P (red) onto the β-phosphorus atom of ATP. Several factors facilitate this reaction, particularly, (1) the two catalytic residues, Arg186 and Lys184, that form a network of hydrogen bonds to both substrates (see Table S2 and Supplementary Information) especially to the βphosphate oxygen atoms; (2) the metal ion (Mg 2+) chelating two or possibly three phosphate groups of ATP; and (3) proton transfer from the anomeric α-hydroxyl group of R5P to the β-phosphate O2B atom.
Phosphoribosyl Pyrophosphate Synthetase Structures
sites and to make these binding sites available for the next R5P and ATP molecules.
Materials and Methods
855 quent matrix-assisted laser desorption/ionization–time of flight mass spectrometry analysis (Institute of Biomolecular Design, University of Alberta) revealed peptides consistent with Tvn0197. Analytical gel filtration
Cloning, expression, and purification The open reading frame Tvn0197 from Tv strain GSS1 encoding residues 1–286 of PRPP synthetase was amplified from genomic DNA template (American Type Culture Collection) using PCR. A PCR primer pair was designed for directional cloning (boldface) of the insert into the Gateway cloning system (Invitrogen). The primer sequences were Tvn0197F, 5′-GGGACAAGTTTGTACAAAAAAGCAGGCTCCGAAAACCTGTATTTTCAGGGTGGTTCGGGTGGTTCCGGTATGAAGATCATAGCTCTACG-3′, and Tvn0197R, 5′-GGGACCACTTTGTACAAGAAAGCTGGGTCTCAGGCATCAATATCCC-3′. A tobacco etch mosaic virus cleavage site followed by a Gly-Ser-Gly spacer peptide (italicized) is encoded by the forward primer preceding the native start codon for removal of the N-terminal affinity tag. The resulting PCR product was inserted into the pDONR-221 vector (Invitrogen) for DNA amplification, and then subsequently transferred to an expression vector (pVP16) that an amino-terminal maltose binding protein (MBP) tag immediately preceded by a histidine octapeptide. The expression plasmid pVP16-Tvn0197 was confirmed by restriction endonuclease analysis and DNA sequencing (DNA Core Facility, University of Alberta). The His8-MBP-Tvn0197 fusion protein was expressed in E. coli BL21(DE3) pLysS cells (Novagen). Incubation of the transformed cells in 2 l of LB+100 μg/ml ampicillin, 34 μg/ ml chloramphenicol, and 0.2% (w/v) glucose at 310 K was continued until an OD of 600 nm reached 0.8. Fusion protein expression was induced by adding isopropyl-β,-D-thiogalactopyranoside to a final concentration of 0.5 mM, and then the incubation temperature was raised to 317 K overnight. The cells were harvested by centrifugation then resuspended in buffer A [20 mM Tris–HCl (pH 7.5), 200 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM NaN3, 1 mM ATP, and 10 mM β-mercaptoethanol] supplemented with 200 μM PMSF, 625 μg/ml hen egg white lysozyme, and one Complete ethylenediaminetetraacetic-acid-free protease inhibitor tablet (Roche). For fusion protein purification, the cells were lysed by freeze–thaw and then subjected to ultrasonication in the resuspension buffer. The lysate was cleared by centrifugation (30 min, 20,000g), and the supernatant was loaded onto a 5-ml MBP trap FF column (GE Healthcare) preequilibrated with buffer A. The loaded sample was washed with buffer A, and then the bound protein was eluted using buffer A+ 10 mM maltose. The MBP tag and the N-terminal recombination site were removed by proteolysis at 277 K overnight using recombinant tobacco etch virus protease. Tvn0197 was isolated by passing the proteolytic reaction over an IMAC column charged with Ni 2+. The cleaved enzyme flowed through this column, while the His-tagged MBP remained bound and was pooled then concentrated to 7.5 mg/ml using an Amicon Ultra concentrator (10-kDa molecular mass cutoff; Millipore). The results of each step were monitored by 12% SDSPAGE from which a final concentrated protein sample was excised. Resulting in-gel trypsinization and subse-
The molecular mass of the PRPP synthetase was determined by analytical gel filtration using a Superdex 200 HR 10/30 column. A set of protein standards was run in phosphate-buffered saline buffer. PRPP synthetase samples at 1 mg/ml and 10 mg/ml protein concentrations were run under the same conditions. Peak data of the standards were used to perform linear regression analysis, and a corresponding standard curve was obtained. The molecular mass of the Tv PRPP synthetase was determined from the peak data using the standard curve. Crystallization and data collection Sitting-drop vapor diffusion techniques in a 96-well format were used to screen for crystallization of 7.5 mg/ml Tv PRPP synthetase. Crystals grew under a number of conditions in 24–48 h at room temperature from the Index Screen (Hampton Research). Two of these conditions were refined in hanging drops to produce diffraction-quality crystals: (form I) 0.1 M citric acid (pH 4.0) and 2.3 M NH4SO4 and (form II) 0.1 M Hepes (pH 7.5), 30% PEG 600, 10% 2-methyl-2,4-pentanediol, and 0.1% lauryl dimethylamine N-oxide. For data collection, crystals from the ammonium sulfate condition were rinsed in a cryoprotectant containing 30% glycerol in mother liquor and then flash cooled in liquid nitrogen. Crystals from the PEG conditions did not need cryoprotection. The data sets were collected on beamline 08ID-1 at the Canadian Light Source (Saskatoon) using a temperature of 100 K and a MAR225 detector. The raw data were processed with the HKL-2000 suite. 19 Structure solution and analysis Molecular replacements were performed with the program suite BALBES. 20 Refinement of the coordinates, the atomic temperature factors, and the ligand occupancies was carried out using the Phenix package and a maximum likelihood target. 21 Model rebuilding was performed using Coot. 22 The secondary structure and stereochemistry of the protein were analyzed by PROCHECK. 23 Sequence alignment was performed using the program ClustalW, 24 and the corresponding Fig. S1 (Supplementary Data) was generated with the program ESPript. 25 Various values of buried surface area and ΔGdiss were calculated with the European Molecular Biology Laboratory–European Bioinformatics Institute PISA server.12 r.m.s.d. values between analogous structures were calculated with Coot. 16 Figures 2–9 and Fig. S2 (Supplementary Data) were generated with PyMOL. 26 Accession codes Coordinates and structure factors have been deposited and released in the PDB with accession numbers 3lrt, 3nag, 3lpn, and 3mbi.
856 Supplementary materials related to this article can be found online at doi:10.1016/j.jmb.2011.09.007
Acknowledgements We are very grateful to Pawel Grochulski for his help in the data collection on beamline 08ID-1 at the Canadian Light Source (Saskatoon). This work was partially supported by the Canadian Institute for Health Research and the Alberta Heritage Foundation for Medical Research.
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