The Crystal Structure of ATP-bound Phosphofructokinase from Trypanosoma brucei Reveals Conformational Transitions Different from those of Other Phosphofructokinases

J. Mol. Biol. (2009) 385, 1519–1533

doi:10.1016/j.jmb.2008.11.047

Available online at www.sciencedirect.com

The Crystal Structure of ATP-bound Phosphofructokinase from Trypanosoma brucei Reveals Conformational Transitions Different from those of Other Phosphofructokinases Iain W. McNae 1 , José Martinez-Oyanedel 1,2 , Jeffrey W. Keillor 3 , Paul A. M. Michels 4 , Linda A. Fothergill-Gilmore 1 and Malcolm D. Walkinshaw 1 ⁎ 1

Structural Biochemistry Group, Institute of Structural and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, Scotland 2

Departamento de Bioquímica y Biología Molecular, Universidad de Concepción, Casilla 160-C, Concepción, Chile

3

Département de Chimie, Université de Montréal, CP 6128, Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada

4

Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Université catholique de Louvain, TROP 74.39, Avenue Hippocrate 74, B-1200 Brussels, Belgium Received 14 September 2008; received in revised form 4 November 2008; accepted 11 November 2008 Available online 3 December 2008

Edited by G. Schulz

The crystal structure of the ATP-bound form of the tetrameric phosphofructokinase (PFK) from Trypanosoma brucei enables detailed comparisons to be made with the structures of the apoenzyme form of the same enzyme, as well as with those of bacterial ATP-dependent and PPi-dependent PFKs. The active site of T. brucei PFK (which is strictly ATP-dependent but belongs to the PPi-dependent family by sequence similarities) is a chimera of the two types of PFK. In particular, the active site of T. brucei PFK possesses amino acid residues and structural features characteristic of both types of PFK. Conformational changes upon ATP binding are observed that include the opening of the active site to accommodate the two substrates, MgATP and fructose 6-phosphate, and a dramatic ordering of the C-terminal helices, which act like reaching arms to hold the tetramer together. These conformational transitions are fundamentally different from those of other ATP-dependent PFKs. The substantial differences in structure and mechanism of T. brucei PFK compared with bacterial and mammalian PFKs give optimism for the discovery of species-specific drugs for the treatment of diseases caused by protist parasites of the trypanosomatid family. Crown Copyright © 2008 Published by Elsevier Ltd. All rights reserved.

Keywords: phosphofructokinase; trypanosomes; X-ray crystallography; allostery; eukaryote

Introduction

*Corresponding author. E-mail address: [email protected]. Abbreviations used: F6P, fructose 6-phosphate; F-1,6-BP, fructose 1,6-bisphosphate; F-2,6-BP, fructose 2,6bisphosphate; PFK, phosphofructokinase; PPi, inorganic pyrophosphate; TbPFK, T. brucei phosphofructokinase.

Phosphofructokinase (PFK) has a key role in the metabolism of most organisms and is regarded as an archetypal allosterically regulated metabolic enzyme. It was one of the first to be described in the context of the symmetry model proposed by Monod, Wyman and Changeux.1,2 PFK catalyzes the formation of fructose 1,6-bisphosphate (F-1,6-BP) at an early step in the glycolytic pathway, and in many

0022-2836/$ - see front matter. Crown Copyright © 2008 Published by Elsevier Ltd. All rights reserved.

1520 metabolic circumstances makes an important contribution to flux control. Mammals, plants, yeasts and many protists and bacteria have PFKs that use ATP as the phospho donor in an essentially irreversible reaction (EC 2.7.1.11).3–5 The activity of this ATP-dependent PFK in mammals and yeasts is regulated by a wide repertoire of allosteric effectors, and the enzyme is thereby able to achieve an energy-efficient balance between glucose catabolism and anabolism. In addition to ATP-dependent PFKs, plants and certain other protists and bacteria possess PFKs that use inorganic pyrophosphate (PPi) to catalyze a reaction that can be near equilibrium in vivo (EC 2.7.1.90).6,7 ATP-dependent and PPi-dependent PFKs share a common ancestor, but enzymes from present-day organisms display only a very low percentage amino acid sequence identity (∼ 20–30%). In the context of the structural work reported here, it is important to note that PFKs from trypanosomatids such as Trypanosoma brucei and Leishmania donovani are strictly ATP-dependent, although sequence comparisons clearly show them to be more similar to the PPi-dependent family.8,9 The evolution of PFK from a relatively simple ATP-dependent ancestor has been complex,10,11 and has involved gene duplication steps as well as changes of phospho donor and of allosteric effectors (Fig. 1). As a consequence, many strategies for the allosteric regulation of this metabolically important enzyme are observed among present-day organisms. At the simplest end of the scale among ATPdependent PFKs are the homo-tetrameric enzymes from the parasitic protists T. brucei and L. donovani, for which only a single heterotropic allosteric activator, AMP, is known.8,9 Intermediate are the bacterial homo-tetrameric PFKs that are activated allosterically by ADP (or GDP) and inhibited by PEP.12 The most complex are the hetero-octameric PFKs from Saccharomyces cerevisiae and other yeasts, for which more than 20 heterotropic allosteric activators and inhibitors have been described (see

T. brucei Phosphofructokinase Conformers

Fig. 1).13 In addition, most ATP-dependent PFKs share the property of homotropic allosteric activation by the substrate fructose 6-phosphate (F6P). The best studied PFKs are those from Escherichia coli and Bacillus stearothermophilus. Crystal structures have been elucidated in both T and R states,12,14,15 and some of the first applications of site-directed mutagenesis have helped to dissect the roles of key amino acid residues.16,17 In addition, the crystal structures of the ATP-dependent PFK from Lactobacillus bulgaricus18 and of the PPi-dependent PFK from Borrelia burgdorferi19 have also been solved. By contrast, eukaryotic PFKs have been recalcitrant to crystallization and X-ray analysis, and no highresolution structural information was available until the recently published apoenzyme structure of T. brucei PFK.20 We now report the crystal structure of T. brucei PFK with bound ATP, and show from a comparison of the holo- and apoenzyme structures that the conformational transitions are quite distinct from those experienced by bacterial, yeast and mammalian ATP-dependent PFKs. It appears that although the trypanosome enzyme is ATP dependent, it has nonetheless retained many features from the PPi-dependent PFK lineage. These observations give further encouragement to structure-based drug discovery approaches to identify parasite-specific inhibitors to treat trypanosomatid-borne human diseases such as sleeping sickness, Chagas disease and kala azar that affect millions of people worldwide.

Results and Discussion Overall structure and topology Full-length T. brucei PFK (487 residues) with an Nterminal extension of 19 residues including a His6 tag was expressed in Escherichia coli and purified by metal-affinity chromatography as described.21 Crystals of the holoenzyme diffracting to 2.7 Å´ were

Fig. 1. Evolution of PFK. A partial, simplified scheme is shown to highlight the position of T. brucei PFK (circled) relative to typical ATP- and PPi-dependent PFKs. An ancestral ATP-dependent PFK with single active-site and effector-site domains gave rise to the several independent lineages of present-day ATP- and PPi-dependent enzymes. The broken lines on the right of the scheme indicate that there is no single line of descent for PPi-dependent PFKs, and that multiple events and lateral gene transfers have occurred. The lengths of the lines do not correspond to evolutionary distance. The PFKs in yeasts and mammals have arisen from a gene duplication/fusion event that yielded double-size chains,47 and these subsequently underwent additional duplications to give the two chains in yeasts,13 and the three tissue-specific isoenzymes in mammals.5 The yeast PFKs acquired an additional 200 residues at the N-terminus (compared to the bacterial PFKs). The mammalian enzymes are about 40 residues longer at the C-terminus. The PPi-dependent lineages arose on multiple independent occasions11 from relatively modest changes at the active site such that nucleotides could no longer bind, and PPi became the new phospho donor; these changes appear frequently to have coincided with partial or complete loss of function of the effector site. In addition, the protein chain has acquired about 100 residues at the Nterminus and about 30 residues at the C-terminus, as well as insertions of functionally important loops.19 This type of PFK can be found in present-day bacteria, protists and plants, frequently in company with ATP-dependent PFK. When both types of PFK occur together, it is possible that one type has been acquired by horizontal gene transfer.11 In the case of PPidependent PFK of plants, the gene duplicated to give the two chains.6 The active site of the α chain is no longer functional for catalysis, but seems to act as a new type of effector site that responds to fructose 2,6-bisphosphate (F-2,6-BP). PFKs from trypanosomatids are a special case because they have reverted back to ATP dependence, while retaining many of the features of PPi-dependent enzymes.8,20 Key: (■) Active site, ATP-dependent; (□) active site, PPi-dependent; (●) effector site, activation by ADP / inhibition by PEP; ( ) effector site derived from ancestral effector site, inhibition by citrate and ATP; ○ effector site, rudimentary with partial or complete loss of function; (▴) effector site derived from active site, activation by F-2,6-BP and/or AMP/ADP.

1521

Fig. 1 (legend on previous page)

T. brucei Phosphofructokinase Conformers

1522 obtained and belong to the orthorhombic space group P212121 with a complete tetramer per asymmetric unit. The structure was determined by molecular replacement as described in Experimental Procedures. The overall subunit architecture is very similar to that described for the apoenzyme form of T. brucei PFK,20 but there are critical differences at the active site, near the C-terminus and in the subunit contacts. The holoenzyme, like the apoenzyme, is folded into three domains (Fig. 2) comprising the loosely packed domain A (residues 8–94 and 410–441 with

T. brucei Phosphofructokinase Conformers

three pairs of anti-parallel β strands and four small α helices), together with two compact domains B and C. In addition, there is a 36 residue, mostly helical, C-terminal extension (helix 17, labelled reaching arm in Fig. 2) that spans from one subunit to another. (Residue numbering starts with the initiation methionine of non-recombinant T. brucei PFK, and thus does not include the N-terminal tagged extension: see Supplementary Data Fig. S1). Among ATP-dependent PFKs, domain A appears to be unique to trypanosomatid PFKs, and possesses the so-called embracing arm (residues 62–81)20 that

Fig. 2. Structure of T. brucei PFK holoenzyme subunit with bound MgATP. A cartoon representation of a single subunit of the holoenzyme is shown in the foreground; the remainder of the tetramer is shown faded behind, each with the bound MgATP highlighted in green. The three domains are labeled and are color-coded in different shades of red. The crystallographic N- and C-termini are indicated, as are the three functionally important regions that are unique to trypanosomatid PFKs. ATP and Arg173 are shown as sticks, with Mg2+ as a yellow sphere. The labeling of secondary structure (numbers for helices, letters for strands) corresponds to that used earlier for the apoenzyme structure,20 except for helix 17, which was not visible in that structure. Three small loop regions (two in domain A and one in the 329-348 loop) that were disordered and not observed in the electron density map are in lighter shades.

T. brucei Phosphofructokinase Conformers

links with the corresponding arm of the adjacent subunit. The PPi-dependent PFK from B. burgdorferi also has a substantial N-terminal extension (70 residues), but it adopts a different fold, and cannot provide a similar function because of the enzyme's different quaternary structure.19 Domain B (residues 95–233 and 386–409) comprises a four-stranded parallel β sheet flanked by α helices, and contains the residues involved in ATP binding. Domain C (residues 234–385 and 442–453) is a five-stranded parallel β sheet flanked by α helices. It contains the large inserted 329–348 loop that is an important part of the active site. Among ATP-dependent PFKs, this loop is also unique to trypanosomatids, although the PPi-dependent PFK from B. burgdorferi has an extra domain of 82 residues in a similar position in the basic PFK topology. The B. burgdorferi domain comprises four helices followed by a β hairpin that sits over the active site and is important for conferring PPi specificity.19 The trypanosomatid PFK 329-348 loop and the B. burgdorferi PFK hairpin occupy similar positions adjacent to the active sites, although they provide different functions. A more detailed analysis of the structural similarities between the apoenzyme form of T. brucei PFK and B. burgdorferi PFK is given by Martinez-Oyanedel et al.20 In addition, an overlay of the ATP-bound PFK reported here with that of B. burgdorferi PFK is given in Supplementary Data Figs. S1b, and S2c and d. Of particular note in the holoenzyme structure is the presence of a further 32 residues at the C terminus (residues 454–485) that were not visible in the electron density for the apoenzyme. These residues comprise almost the entire C-terminal extension of 36 residues, with only the final two residues at the C-terminus not in electron density. This C-terminal region is mostly helical, and extends away from the monomeric subunit as shown in Fig. 2, but within the tetramer the C-terminal region reaches across the dimer interface and results in an increase in inter-dimer contacts. This C-terminal region is not present in E. coli or B. stearothermophilus ATP-dependent PFKs, and its structure has thus not been described. The PPi-dependent PFK from B. burgdorferi also possesses a C-terminal extension (34 residues), but there is no structural resemblance to that of T. brucei PFK, and the two Cterminal extensions fulfill different functions.19 It seems that the evolution of trypanosomatid PFKs via the PPi-dependent family (see Fig. 1) has involved the acquisition of substantial N- and Cterminal extensions, as well as the β hairpin part of the helical domain excursion from domain C. In effect, the trypanosomatid PFK appears to be a chimera of ATP-dependent and PPi-dependent PFKs. Active site of T. brucei PFK with bound ATP The active site (Fig. 3) is found at the boundary of domains B and C (see Fig. 2), with the ATP bound primarily to the B domain. Electron density at the active site (Fig. 3b) clearly indicates the presence of

1523 only the MgATP substrate, despite the fact that the crystals were grown in the presence of Mg and the products of the enzyme reaction, ADP and F-1,6-BP. There is no interpretable electron density for the F1,6-BP, although adjacent to the ATP-binding site an additional small peak of density is apparent, which could be consistent with a partially occupied formate ion (Fig. 3a). Indeed this residual electron density is in a similar position to one of the two sulphate ions found in the active site of the PPi-dependent PFK.19 It is possible, however, that the unassigned electron density could be interpreted as the low occupancy (b 20%) presence of F-1,6-BP with ADP. The lack of evidence for the ADP and F-1,6-BP products in the electron density can be explained by the presence of contaminating ATP in the ADP sample used for co-crystallization. ATP was shown to be present by mass spectrometric analysis of the ADP (data not shown). The presence of the γphospho group would obviate the binding of F-1,6BP, which typically binds to PFKs about 30-fold less tightly than nucleotides.22 The ATP-binding site is sandwiched between the N-terminal portion of helix 8 (residues 199–204) and the loop containing Arg173. In order to further an understanding of the structure and function of T. brucei PFK, a molecule of F6P was modeled at the active site in a position similar to that observed in E. coli and B. stearothermophilus PFKs (Fig. 3). The predominant interactions between the nucleotide and the protein are from the ribose and phospho groups. The α-, β- and γ- phospho groups interact with a Mg ion that is also coordinated by Asn343 from the large inserted 329-348 loop. The triphosphate group forms a total of 15 electrostatic or hydrogen bonds involving the seven sp2 oxygen atoms (Fig. 3). Of the six amino acids involved from the PFK protein, three are glycine residues (Gly198, Gly200 and Gly 107). The ribose O2 hydroxyl forms a hydrogen bond to the main chain oxygen of Gly174, while the O1 hydroxyl forms an interesting close (2.8 Å) interaction pointing perpendicularly into the plane of the ring nitrogen of Pro175. The adenine base forms no hydrogen bonds and is positioned parallel with helix 8, with one face forming van der Waals contacts with one side of the helix, in particular with Gly200, Gly204 and the side chain of Arg203. The binding pose adopted by the adenine group would be disallowed by any other (larger) amino acids than glycine at these positions in the helix. Analysis of the site of the modeled F6P (Fig. 3a) reveals that with some small changes in side chain positions the F6P would fit into the active site of the T. brucei PFK holoenzyme structure in a manner similar to that seen in the B. stearothermophilus structure containing F6P.14 Comparison of the active-site of T. brucei PFK with those of E. coli and B. stearothermophilus PFKs A best fit of the 306 Cα atoms of T. brucei PFK onto chain B of E. coli PFK (1PFK) gives an RMS deviation

1524 of 1.86 Å (Supplementary Data Fig. S2a). The resulting overlay of T. brucei ATP and E. coli ADP ligands is very close, with the Mg ions in the two structures separated by about 1 Å (Supplementary Data Fig. S2b). A survey of the Mg-ATP coordination geometries using the database tool MESPEUS23 identified 61 crystallographically independent ATPMg complexes refined at better than 2 Å resolution. Of these, 39 showed Mg coordinated to both the βand γ-phospho groups, while 13 structures showed Mg coordinated to all three α-, β- and γ-phospho groups as observed in T. brucei PFK. The side chain of Asn343 in the 329-348 loop (not present in other ATP-dependent PFKs) forms a fourth ligand, with the remaining two octahedral sites filled by water (although only one of these is clearly identified in this structure). The Mg ion associated with chain B of the E. coli PFK structure is located in the identical position and is coordinated to the α- and β-phospho

T. brucei Phosphofructokinase Conformers

groups of ADP, but with the remaining four coordination sites occupied by water molecules. Interestingly, chain A of E. coli PFK shows the Mg ion in a position some 4 Å removed, and stabilizing the products by bridging between the β-phospho group of ADP and the 1-phospho group of F-1,6-BP. The position of the Mg ion in T. brucei PFK and in chain A of E coli PFK is consistent with its role in enhancing the effective polarization of the transferable phospho group,24 while the second Mg position observed in 1PFK stabilizes the binding of the products. In general, the adenine ring of ATP makes fewer interactions in T. brucei PFK than it does in the B. stearothermophilus and E. coli enzymes. In particular, the T. brucei enzyme has a five residue deletion between the Arg173 loop and the N-terminus of helix 7 (Fig. 2), and is thereby lacking a basic residue that helps to stabilize the adenine ring in the

Fig. 3. Active site of T. brucei PFK. (a) General view of the active site, with the substrates ATP and F6P shown as sticks with yellow and green carbon atoms respectively. The ATP position was determined crystallographically, whereas the F6P position is modeled according to its position in B. stearothermophilus PFK.41 Potential hydrogen bonds are indicated by broken red lines. The identity of the small peak of electron density cannot be assigned unambiguously. Lys374 (indicated by the asterisk) is from the neighboring subunit across the inter-dimer interface. (b) Close-up of ATP-binding site with electron density for MgATP. Three water molecules are shown as red spheres. Amino acid residues are all from subunit 1, with those involved in hydrogen bonding labeled. The pale brown and green ribbons correspond to subunits 1 and 4, respectively.

T. brucei Phosphofructokinase Conformers

1525

Fig. 3 (legend on previous page)

bacterial PFKs (a sequence alignment is given in Supplementary Data Fig. S1). These differences are consistent with the slightly lower affinity of T. brucei PFK for ATP; Km 90 μM25 compared to Km 60 μM for E. coli PFK.26 The region that surrounds F6P and the transferable phospho group of ATP is generally well conserved, with Asp229 (corresponding to the catalytic base in bacterial ATP-dependent PFKs), Gly273, Met272 and Glu325 favorably placed to interact with the F6P. It is notable that the position of Asp229 in the closed-type subunit of the apoenzyme structure would preclude F6P binding,20 and a substantial movement of the Asp229-containing loop similar to that observed between the closed and open subunits of the apoenzyme structure from T. brucei would be needed. Therefore, one major effect of ATP binding is to favor the open form of the F6P binding site (the movements involved are demonstrated in animation files available in the Supplementary Data Figs. S3 and S4). The binding site for the 6-phospho group of the F6P comprises residues from the adjacent subunit across the inter-dimer interface as well as residues from the same subunit. There are significant differences in side chains when compared to bacterial PFKs, especially among the inter-subunit residues that are contributed by a small loop between helix 9 and strand i. In T. brucei PFK, these residues have the sequence 262AlaAsn-Tyr-Gly265, whereas the B. stearothermophilus

enzyme has a shorter loop with the sequence 160HisGlu-Arg162. These latter residues have a particular significance in bacterial PFK, where the glutamic acid and arginine residues swap positions on the transition from the inactive T-state (Glu blocking the 6-phospho group) to active R-state (Arg binding the 6-phospho group).12 Clearly, this type of switch is not present in the trypanosome enzyme. The other inter-subunit residue in the binding site is Lys374 (Fig. 3a) that has replaced Arg243 in the bacterial PFKs. Why is ATP and not PPi the phospho donor for T. brucei PFK? The evolution of PFK has been complex, with changes in phospho donor as well as gene duplications and changes in allosteric properties. T. brucei PFK has absolute specificity for ATP as phospho donor, but belongs to the PPi-dependent family by sequence comparisons (Fig. 1). Its evolution has apparently involved a change of phospho donor from ATP to PPi, and back to ATP again.8 It is thus of interest to examine its active site for remnants of its passage through the PPi-PFK lineage. The determination of the structure of the bacterial PPi-dependent PFK from B. burdgorferi19 gave a structural context to two active-site regions that had been highlighted by sequence alignments and site-directed mutagenesis to be important for phospho donor specificity.9,25,27 One of these regions

1526 corresponds to the N-terminal portion of helix 8 (Fig. 2), residues 197Gly-Gly-Asp-Gly200 in T. brucei PFK. The other region contains the catalytic Asp229, and has the sequence 225Pro-Lys-Thr-Ile-Asp-AsnAsp231 in T. brucei PFK. Sequence alignments show that the residue corresponding to Gly200 is always glycine in ATPdependent PFKs, but almost invariably an aspartic acid residue in PPi-dependent PFKs. It is clear from the structure of T. brucei PFK as well as from those of E. coli and B. stearothermophilus PFKs that only glycine in this position would allow the α-phospho group of ATP to bind. The point mutant of E. coli PFK Gly104Asp (equivalent to T. brucei PFK Gly200) had no activity,27 whereas the reciprocal mutant of the PPi-dependent PFK from Entamoeba histolytica, Asp175Gly, had an increased affinity for ATP and a modest activity with ATP as phospho donor (kcat 6.9 s− 1 compared to wild type kcat 341 s− 1 with PPi as phospho donor). These authors as well as Moore et al.19 from the crystal structural information concluded that there is a latent ATP-binding site present in PPi-dependent PFKs. The situation is more complex for the other highlighted region in which residue 226 is lysine in PPi-dependent PFKs, but glycine in most ATPdependent enzymes. Trypanosomatid PFKs such as those from T. brucei8 and L. donovani9 have lysine and, in this respect, resemble PP i -dependent enzymes. Site-directed mutagenesis of the lysine residues in the PPi-dependent PFKs from Propioni-

T. brucei Phosphofructokinase Conformers

bacterium freudenreichii (Lys148Met)28 and from E. histolytica (Lys201Gly)27 yielded mutant enzymes with reduced affinity for PPi and lower kcat values. These results indicated that the lysine residue was important for binding PPi, and this was indeed confirmed by the determination of the crystallographic structure of the B. burgdorferi enzyme.19 The site-directed mutant of T. brucei PFK Lys226Gly still retained activity with ATP although the binding affinity was slightly lower (Km 220 μM instead of 90 μM for the wild-type enzyme). Moreover, the binding affinity for F6P was much lower (S0.5 ∼ 5 mM instead of 0.59 mM for the wild-type enzyme) unless the allosteric activator was present (S0.5 for F6P 0.82 mM).25 The structure of the T. brucei PFK holoenzyme with bound ATP reported here shows that Lys226 forms an electrostatic interaction with the γ-phospho group of ATP, and is positioned close to the 1-OH of the modeled F6P (Fig. 3a and Supplementary Data Figs. S2b and S2d). Our structural studies now show that the lysine can be readily accommodated at the active site of an ATPdependent PFK, and that this residue should no longer be considered as diagnostic of whether a PFK is functionally dependent on ATP or PPi. ATP binding causes a change in the shape of the active site A notable feature of the T. brucei PFK apoenzyme structure20 is the presence of two distinct alternative

Fig. 4. Comparison of holoenzyme and apoenzyme active sites. The stereo image shows an overlay of the active sites of the apoenzyme (cyan) and holoenzyme (pink) structures. The figure is in an orientation similar to that in Fig. 3, and represents the A subunits. ATP from the holoenzyme is shown in stick representation with white carbons, and the Mg as a green sphere. Amino acid residues are labeled blue for the apoenzyme or red for the holoenzyme. The broken blue line shows a hydrogen bond between Arg173 and Ser341 in the apoenzyme, and the broken red line is a hydrogen bond between the backbone of Gly174 and Ser341 in the holoenzyme. The Arg173 loop, the inserted loop and the Asp229 loop can be seen to intrude into the ATP binding site in the apoenzyme structure.

T. brucei Phosphofructokinase Conformers

conformations in the region of the active site. In particular, three loops (the catalytic Asp229 loop, the Arg173 loop and the inserted 329-348 loop) adopted different conformations: one conformation (named APO-1) is relatively open but with the Arg173 loop precluding ATP binding, while in the second conformation (APO-2), all three loops move into the active site and block ATP binding (Fig. 4 and Supplementary Data Fig. S3). In the APO-2 conformer, the inserted 329-348 loop is found to partly obstruct the ATP-binding site, with the hydroxyl of Ser341 in particular blocking the position of the αphospho group by interacting with the side chain of Arg173. In the ATP-bound holoenzyme structure, this loop is moved out of the ATP-binding pocket, and Ser341 shifts position to interact with the backbone of Gly174. The Asp229-containing loop also shows significant movement. In the APO-2 conformer this loop is positioned in the ATP binding site, with Asn230 located to block the binding of the γ-phospho group. In the holoenzyme this Asp229containing loop is found away from the active site and is in a position to that found in the open-type subunit of the apoenzyme. In general, the holoenzyme shows greater similarity to the APO-1 type subunit. Both APO-1 and APO-2 type subunits show closed ATP-binding pockets, and would be unable to bind ATP, as the Arg173-loop is located significantly closer (1 to 2 Å) to helix 8, with Gly174 and Pro175 adopting positions that would preclude the binding of the ribose group of ATP. [The move-

1527 ments involved are demonstrated in an animation file (Fig. S3) in the Supplementary Data.] Comparison of the holoenzyme and apoenzyme subunit structures As well as changes within the active site, the binding of ATP results in wider-reaching changes to the subunit structure. Most obvious is the dramatic appearance of an additional 32 residues of the Cterminal region that were not visible within the electron density of the apoenzyme structure. In addition, movement is found when individual domains are compared. An overlay of the four crystallographically independent B domains of the holoenzyme structure using Cα atoms of residues 95–227 and 386–489 gives RMS deviations between 0.07 Å and 0.05 Å. A fit of the B domains of the holoenzyme onto the B domains of the two different apoenzyme chains using the same Cα atoms gives RMS deviations of 0.83 Å and 0.78 Å. Using this overlay to compare the full-length chains (of the closed APO-2 conformer) shows that the deviation between corresponding Cα atoms increases towards the bottom of the C domain to over 3 Å (Fig. 5). In the holoenzyme structure, the angle between the Cα atoms from Gly216 (sitting at the distal end of the B domain), Ala384 (at the linker between the B and C domains) and Arg386 (at the distal end of the C domain) is 131.2°, which compares to an angle of 128.1° for the equivalent atoms from the APO-2

Fig. 5. Comparison of holoenzyme and apoenzyme subunits. The overlay is of the B domain (residues 95–233 and 386–409) of the holoenzyme with the B domain from the closed-type subunit of the apoenzyme, with all C α atoms having RMSD = 0.968 Å´. The apoenzyme is colored blue and the holoenzyme is colored red. ATP is shown in the active site as sticks. The position of the hinge is indicated, and the arrow indicates the direction of hinge movement on ATP binding. The disordered loop regions not observed in the electron density are in lighter shades. Note that the C-terminal region is visible only in the holoenzyme structure.

1528

T. brucei Phosphofructokinase Conformers

closed conformer. These measurements suggest a hinge movement of around 3o between the B and C domains on transition from the apoenzyme to the holoenzyme. It is possible that the large movements of the 229 loop and the 329-348 loop may also be regulated by this inter-domain hinge, which acts to enlarge the active site in order to accommodate the ligands. This type of domain movement has not been observed for bacterial ATP-dependent PFKs. A superposition of T. brucei PFK holoenzyme and B. burgdorferi PFK subunit structures (Supplementary Data Figs. S2c and S2d) shows, however, that the T. brucei 329-348 inserted loop matches the β-hairpin loop of the B. burgdorferi PFK helical domain. Comparison of dimer interfaces of the holoenzyme and the apoenzyme quaternary structures The quaternary structure of T. brucei PFK is a homotetramer and, as in the case of other reported ATP-dependent PFK structures14,15,18,20 the holoenzyme form described here is a dimer of dimers (Fig. 6a in which the dark subunits, chains 1 and 2, correspond to one dimer, and the light subunits, chains 3 and 4, correspond to the other dimer). The major set of contacts is across the interface within each dimer where the B domain of one subunit interacts with the C domain of the neighboring subunit. In addition, a large loop (residues 62–81) within the novel N-terminal domain of T. brucei PFK forms a pair of “embracing” arms that links the two domains together across the major interface within the dimer (Fig. 6a), and serves to help to stabilize the dimer. There are 87 residues that are involved in noncovalent contacts of less than 4 Å between chain 1 and chain 2 in the holoenzyme structure (using all residues from 8 to 485), with a total buried surface area of 3007 Å 2 , while 86 residues make the corresponding dimer interface between chains 3 and 4 with a buried surface area of 2963 Å2.29 There are thus 173 residues within the tetramer that are involved in non-covalent dimer contacts, with a total buried surface area of 5970 Å2. There are 32 direct hydrogen bonds holding chains 1 and 2 together, 15 of which involve the embracing arms (residues 59–81) of the A-domain. Of these, seven are conserved in both the apoenzyme and holoenzyme structures. The inter-dimer interface (chains 1 and 2 contacting chains 3 and 4) is composed of 113 residues and has a buried surface area of 3791 Å2. There are 46 direct hydrogen bonds holding this interface together, 26 of which come from hydrogen bonds to the C-terminal helix 17. In the apoenzyme structure, the dimer interface is composed of 69 residues with a buried surface area of 2522 Å2 (total in tetramer 138 residues with a buried surface area of 5044 Å2), and the inter-dimer interface is composed of 87 residues with a buried surface of 2736 Å2. The large difference in both

Fig. 6. Quaternary structure of the holoenzyme tetramer. A surface representation of the PFK tetramer looking down the dimer 2-fold axis between the dimers formed by chains 1 and 2, and chains 3 and 4. The other dimer 2-fold direction (between chains 2 and 3) is indicated by the blue arrow. Each chain within the dimer is colored red and green, with dark and light shading corresponding to the different dimers. The “embracing” arms are indicated by green arrows, the “reaching” arms (helix 17) and their direction are indicated by white arrows. The positions of the putative effector sites are indicated by white ellipses and are covered by the reaching arms.

interfaces between the holoenzyme and apoenzyme structures is mainly due to the incorporation of the ordered C-terminal helix (helix 17) into both interfaces in the holoenzyme structure. This helix extends across the inter-dimer interface, as shown between the green subunits 2 and 4 in Fig. 6a. In a similar way, the C-terminal helical regions of the red subunits 1 and 3 interact with each other on the opposite face of the enzyme. This type of “reaching” arm structure that includes helix 17 has not been observed in other PFKs. The holoenzyme structure solved at 2.7 Å has allowed the identification of a number of water molecules in the tetramer interfaces that provide direct bridging hydrogen bonds (using a hydrogen bond length cut-off of 3.5 Å). There are ten water molecules in the interface between chains 1 and 2, 11 water molecules between chains 3 and 4, and 22 water molecules in the inter-dimer interface between chains 1 and 2, and chains 3 and 4. In the slightly higher resolution (2.4 Å) apoenzyme structure there are only four bridging water molecules between chains 1 and 2, and only ten in the inter-dimer interface. Within the inter-dimer interface, four water

T. brucei Phosphofructokinase Conformers

1529

molecule positions are conserved between the holoenzyme and the apoenzyme tetramer structures. These four water molecules are related to each other by the (non-crystallographic) 222 symmetry of the tetramers and form inter-chain bridging hydrogen bonds from Tyr375(NH) to Tyr380(OH). The significant reduction in the number of water molecules in the apoenzyme interfaces compared with the holoenzyme structure appears genuine, as both structures are solved to essentially the same resolution and the apoenzyme tetramer has marginally more water molecules associated with it than the holoenzyme (Table 1), although these are not in the interfaces. Domain movements in the tetramer An overlay of the 400 Cα atoms in each chain that are not in loop regions was used to fit the holoenzyme tetramer onto the apoenzyme structure resulting in an RMS fit for all 1600 atoms of 0.6 Å. To analyze differences in tetramer shape, we calculated centroid positions for each chain and for each domain in each chain. (Domain A is defined for these calculations to be residues 8–94 and 410–441, domain B by residues 95–226 and 386–409, and Table 1. Data collection, refinement and Ramachandran plot statistics for T. brucei PFK holoenzyme with bound MgATP A. Data collection and processing Space group Unit cell parameters ´ a (Å) ´ b (Å) ´ c (Å) α (deg) β (deg) γ (deg) ´ Resolution (Å) [Highest shell] Observations Unique observations Redundancy Completeness (%) Mean I/σ(I)[High shell] R merge (%) B. Refinement Total atoms Solvent atoms ´ Resolution range (Å) Reflections used in refinement Reflections used for Rfree Rcryst (%) Rfree (%) RMSD. from ideal ´ Bond lengths (Å) Bond angles (deg) ´ Mean protein B-factor (Å2) ´ Mean solvent B-factor (Å2) Ramachandran plot statistics Residues in most favored regions (%) Residues in additionally allowed regions (%) Residues in generously allowed regions (%) Residues in disallowed regions (%)

P212121 96.578 117.570 176.587 90 90 90 15.88–2.70 (2.8–2.70) 235588 (28,337) 54525 (7872) 4.3 (3.6) 97.9 (97.8) 9.2 (2.3) 13.8 (55.4) 14,788 430 15.8–2.7 54,434 2775 22.25 28.55 0.01 1.26 35.9 29.8 91.7 8.3 0.1 0.0

Numbers in parentheses correspond to the highest resolution shell.

domain C by residues 236–332, 346–385 and 442– 453). The maximum distance between corresponding chain centroids of the fitted holoenzyme and apoenzyme tetramer structures was 0.1 Å. The maximum distances between centroids of corresponding B domains and C domains were 0.3 Å and 0.4 Å, respectively. These small distances show that there is no significant translational domain movement between the apoenzyme and the holoenzyme tetramer structures. Viewing the animated pictures of the holoenzyme and the apoenzyme tetramers (Supplementary Data Figs. S3 and S4), however, it is evident that there are concerted movements between the two tetramers with corresponding helices displaced by up to 1 Å. The analysis of the differences between holoenzyme and apoenzyme tetramers is complicated by the presence of the two APO-1 and APO-2 conformers in the apoenzyme tetramer. As the centroids of the holoenzyme and apoenzyme domains do not move by more than 0.4 Å, the hinge bending movement of 3° between the B and C domains is best described as small B domain and C domain rotations around the their respective centroids. These small domain rotations result in a complex pattern of breathing movements (typically less than 1 Å) of helices and strands that reshape the surface of the tetramer and allow the reaching arms of the long C-terminal helices to fit into the deep groove lined principally by helix 4. Effector site of T. brucei PFK T. brucei and L. donovani PFKs are allosterically activated by AMP but not by structurally similar effectors that modulate activity of ATP-dependent PFKs in other organisms, such as the allosteric activator ADP.8,9,30,31 Furthermore, trypanosomatid PFKs have no known allosteric inhibitors, in contrast to most ATP-dependent PFKs. Comparison between the E. coli and T. brucei PFK structures provides an explanation for the specificity for AMP over ADP and a model of AMP bound in the putative allosteric site of T. brucei PFK is shown in Fig. 7. The replacement of Arg154 and Arg25 of the bacterial enzyme (E. coli numbering) with Tyr256 and Leu121 eliminates many of the ionic interactions that would be formed with the β-phospho group of ADP. In addition, an inserted residue (Gln289) indirectly precludes the binding of the β-phospho group. It has been suggested via modeling studies that the side chain of this residue may intrude into the βphospho binding site.9 However, in the structure described here, the side chain is pointing away from the binding site. Instead, the insertion causes the backbone carbonyl of Ala288 to intrude into the binding site and therefore to preclude the binding of ADP. The binding of ATP to the active site has little direct effect upon the putative effector site of the enzyme. However, the C-terminal region clearly has a role in effector binding, with the reaching arm (helix 17) forming a lid over the putative effector site (Fig. 6).

1530

T. brucei Phosphofructokinase Conformers

Fig. 7. T. brucei holoenzyme PFK putative effector site structure (white carbons), with MgADP modeled in the position found for E. coli PFK.

The PPi-dependent PFK from B. burgdorferi has a C-terminal extension comparable in length and starting point to the T. brucei PFK C-terminal extension.19,20 However, the quaternary structure of B. burgdorferi PFK is a homodimer, and the enzyme is apparently not regulated by allosteric effectors. It has been suggested that the location of the C-terminal extension in its structure would “negate the formation of the allosteric effector site seen in eubacterial ATP-dependent PFKs”.19 Biological significance of different effector responses What is the biological significance of the different effector responses of AMP-regulated trypanosomatid PFKs compared with the wider range of allosteric activators and inhibitors in other PFKs? Trypanosomatid PFK is compartmentalized together with six other glycolytic enzymes, within the glycosome, a peroxisome-like organelle. 32,33 It is thus in a different cellular compartment from the enzymes that produce many of the metabolites (PEP, F-2,6-BP,

citrate) that regulate PFK activity in other organisms. Glycosomes do, of course, contain ADP but, as discussed earlier, trypanosomatid PFK is not able to accommodate this metabolite at its effector site. AMP is also available in the glycosome, and is produced primarily by adenylate kinase as well as by other enzymes catalyzing AMP-dependent reactions that are linked to glucose catabolism, such as pyruvate, phosphate dikinase and enzymes of purine salvage and pyrimidine biosynthesis.34 Computer simulation using a model based on kinetic data of all the enzymes involved35,36 has indicated that the glycosomal AMP concentration (in contrast to cytosolic AMP) increases at high concentrations of glucose. This would cause an extra activation of PFK under conditions of high substrate supply. However, the biological role of this regulation is enigmatic without further experimental data. The computer simulation has shown that the compartmentation of the first seven enzymes of glycolysis results in a special ATP/ADP ratio inside glycosomes, different from that in the cytosol of the trypanosomes.35,36 When the ATP/ADP ratio in the

T. brucei Phosphofructokinase Conformers

cytosol is low (and the requirement for glycolysis is high), the ATP/ADP ratio in the glycosome is high. In this situation, the high concentration of ATP would cause the PFK structure to switch from a lowaffinity T state to a state with higher affinity for F6P. Thus, the PFK activity appears to be potentially upregulated by this ATP/ADP ratio, independent of the net ATP production in the cytosol, and this control compensates for the lack of activity regulation by effectors and product.36 At present there is no information about whether a high cytosolic ATP/ ADP ratio or a high concentration of metabolites such as PEP can exert an influence on the glycolytic flux through the glycosome. The other side of the control of glycolytic flux is inhibition. In non-trypanosomatid organisms, feedback inhibition of PFK and hexokinase is essential to prevent the autocatalytic activation of pathways with a turbo-design such as glycolysis, in which ATP is invested at the first step while a net production takes place only at the end.37 Without such regulation, the flux through the ATP-consuming enzymes would be boosted above the capacity of the enzymes downstream, and intermediate metabolites would accumulate to extreme levels. In the case of trypanosomatids it seems that important regulation of glycolysis by cytosolic factors occurs at the level of the cytosolic pyruvate kinase. Thus, trypanosomes can be contrasted with other organisms such as E. coli, S. cerevisiae and humans that have developed PFKs with different activity-regulation mechanisms to enable their cells, which have a fundamentally different organization of their glycolytic pathways, to each deal properly with a different availability of the substrate and/or a different need for the products of the process. Trypanosomatid ATP-dependent PFKs have unique allosteric mechanism It has been shown by kinetic studies that the mechanism of T. brucei PFK is ordered, with ATP binding first to the enzyme.38 Circular dichroism and fluorescence spectroscopy together with kinetic measurements have demonstrated that the binding of ATP caused conformational changes and increased the binding affinity for F6P.25 The binding of AMP, the only known allosteric effector of T. brucei PFK,8,30,31 also caused conformational changes, although to a lesser extent. The binding of F6P similarly activated the enzyme,9,25,31 but did not give additional changes in the circular dichroism spectrum. The structures of the apoenzyme and ATP-bound holoenzyme forms of T. brucei PFK are entirely compatible with these observations. Thus, the binding of ATP can be seen to cause substantial conformational changes such that the active site can accommodate both ATP and F6P, and the effector site can bind AMP. Steady-state kinetic measurements have shown that B. stearothermophilus PFK has a sequential random mechanism39 in contrast to the ordered mechanism of T. brucei PFK in which ATP must bind first. The bacterial enzyme is allosterically inhibited

1531 by PEP, and this inhibition is reversed by the activators ADP and GDP.40 Both enzymes bind the substrate F6P in a cooperative manner. A comparison of the structures of B. stearothermophilus PFK in the inhibited T state (with bound 2phosphoglycollate, an analogue of PEP) and in the R state (with bound MgADP and F6P) shows that the conformational differences12,15,41,42 are quite distinct from those experienced by T. brucei PFK. In the bacterial enzyme, the two dimers rotate by about 7° with respect to each other (Supplementary Data Fig. S5). There is no change in the packing of the subunits within the dimers, but there are major changes to the packing between dimers, both to accommodate and to trigger the allosteric transition. This has the consequence that Glu161 from the neighboring subunit, which extends across the inter-dimer interface and blocks F6P binding in the T state, is replaced by Arg162, which contributes to binding the 6-phospho group of F6P.12 T. brucei PFK has a completely different sequence in this region (263Asn-Tyr-Gly265 , see Supplementary Data Fig. S1) and would be unable to have the same mechanism to control F6P binding. The transition from apo enzyme to ATP-bound holoenzyme in T. brucei PFK may indicate steps in the conformational transitions: the T-state apoenzyme has disordered loops and a closed active site. Upon ATP binding, a number of loops change conformation and become more ordered, opening up the active site; there is a small hinge bending between domains B and C; and there is a dramatic ordering of the C-terminal helices, which act like reaching arms to glue the tetramer together. The apoenzyme to holoenzyme transition of T. brucei PFK fits very well with recent reports suggesting that even small changes in flexibility and conformational state may regulate allosteric effects.43,44 Finally, our structural analysis supports the idea that T. brucei PFK is a chimera of PPi-dependent and ATP-dependent PFKs, and is thus unique among ATP-dependent PFKs.

Experimental Procedures Expression, purification, characterization and crystallization T. brucei PFK was expressed, purified and characterized as described.20,21 Briefly, the enzyme was expressed in E. coli with an N-terminal His tag that was used for a single-step purification by metal-affinity chromatography on a Talon column (Clontech). Crystallization was achieved by the hanging-drop, vapor-diffusion method at 17 °C with a well solution of 2.3–2.7 M sodium formate in 0.1 M sodium acetate buffer pH 4.3–4.6. The hanging drop (3 μl) contained 1.5 μl of protein solution at 3–4 mg/ml in 20 mM triethanolamine/HCl buffer pH 8.0 with 50 mM F-1,6-BP, 10 mM Na2ADP and 10 mM MgCl2 plus 1.5 μl of well solution.21 (All reagents were from Sigma, with the exception of F-1,6-BP and Na2ADP that were from Roche.) Crystals grew in three days and were transferred to cryoprotectant solution containing mother liquor with 25% (v/v) glycerol before data collection. No attempt was made to remove the His tag before crystallization.

1532 Structure determination The X-ray diffraction data were recorded at station 9.6 in SRS, Daresbury at 100 K, on an ADSC Q4 CCD detector using a φ scan with a step size of 1.00°. Data were indexed and scaled using MOSFLM and SCALA from the CCP4 suite. Data collection statistics for the orthorhombic crystal are given in Table 1. A molecular replacement solution was found using the program PHASER. The input search model was the A subunit from the apoenzyme structure (PDB code 2hig). Refinement was performed using Phenix refine45 with the application of NCS restraints. Each monomer was defined as a single NCS unit with the default restraint level of 0.1 Å´ RMSD for main chain and side chain atoms. The resultant R and Rfree values were 1.5 % lower with the use of NCS restraints. Any manual corrections were performed using COOT.46 A total of 430 water molecules were identified. The initial criterion for adding water molecules was an Fo – Fc difference map peak of greater than 3.2 σ. After refinement, water molecules were examined individually to ensure they were involved in at least one physically meaningful hydrogen bond and their 2Fo – Fc electron density peak was greater than 1 σ. In the final refined structure, the average B-factor for all water molecules is 29.8 Å2 and, on average, each water molecule is involved in 1.9 hydrogen bonds. Protein Data Bank accession number Atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ with the accession code 3F5M.

Acknowledgements We thank the Edinburgh Protein Production Facility for use of the facilities. We thank the Wellcome Trust for an International Travelling Fellowship to J. M.O. and a short-term travel grant to J.W.K. We are grateful to Dr L. Worrall and Dr E. Blackburn for the mass spectrometric analyses of ATP, and to the staff at the ESRF and SRS, Daresbury synchrotrons for their assistance. This work was supported by the European Commission under its INCO-DEV program and the UK Medical Research Council.

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2008.11.047

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