Advertisement

Structural comparisons of phosphoenolpyruvate carboxykinases reveal the evolutionary trajectories of these phosphodiester energy conversion enzymes

  • Yoko Chiba
    Correspondence
    To whom correspondence should be addressed: RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan. Tel.: 81-48-467-9372.
    Footnotes
    Affiliations
    Department of Subsurface Geobiological Analysis and Research, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15, Natsushima-cho, Yokosuka-city, Kanagawa, 237-0061, Japan
    Search for articles by this author
  • Takuya Miyakawa
    Footnotes
    Affiliations
    Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan
    Search for articles by this author
  • Yasuhiro Shimane
    Affiliations
    Super-Cutting-Edge Grand and Advanced Research Program, Institute for Extra-Cutting-Edge Science and Technology Avant-Garde, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15, Natsushima-cho, Yokosuka-city, Kanagawa, 237-0061, Japan
    Search for articles by this author
  • Ken Takai
    Affiliations
    Super-Cutting-Edge Grand and Advanced Research Program, Institute for Extra-Cutting-Edge Science and Technology Avant-Garde, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15, Natsushima-cho, Yokosuka-city, Kanagawa, 237-0061, Japan
    Search for articles by this author
  • Masaru Tanokura
    Affiliations
    Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan
    Search for articles by this author
  • Tomoyoshi Nozaki
    Affiliations
    Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
    Search for articles by this author
  • Author Footnotes
    1 Both authors contributed equally to this work.
Open AccessPublished:October 28, 2019DOI:https://doi.org/10.1074/jbc.RA119.010920
      Inorganic pyrophosphate (PPi) consists of two phosphate molecules and can act as an energy and phosphate donor in cellular reactions, similar to ATP. Several kinases use PPi as a substrate, and these kinases have recently been suggested to have evolved from ATP-dependent functional homologs, which have significant amino acid sequence similarity to PPi-utilizing enzymes. In contrast, phosphoenolpyruvate carboxykinase (PEPCK) can be divided into three types according to the phosphate donor (ATP, GTP, or PPi), and the amino acid sequence similarity of these PEPCKs is too low to confirm that they share a common ancestor. Here we solved the crystal structure of a PPi-PEPCK homolog from the bacterium Actinomyces israelii at 2.6 Å resolution and compared it with previously reported structures from ATP- and GTP-specific PEPCKs to assess the degrees of similarities and divergences among these PEPCKs. These comparisons revealed that they share a tertiary structure with significant value and that amino acid residues directly contributing to substrate recognition, except for those that recognize purine moieties, are conserved. Furthermore, the order of secondary structural elements between PPi-, ATP-, and GTP-specific PEPCKs was strictly conserved. The structure-based comparisons of the three PEPCK types provide key insights into the structural basis of PPi specificity and suggest that all of these PEPCKs are derived from a common ancestor.

      Introduction

      Inorganic pyrophosphate (PPi)
      The abbreviations used are: PPi
      inorganic pyrophosphate
      PFK
      phosphofructokinase
      ACK
      acetate kinase
      PEPCK
      phosphoenolpyruvate carboxykinase
      Ai
      Actinomyces israelii
      Pf
      Propionibacterium freudenreichii subsp. shermanii
      Eh
      Entamoeba histolytica
      RMSD
      root mean square deviation
      FHA
      forkhead-associated
      LUCA
      last universal common ancestor.
      is the simplest compound containing a high-energy phosphate bond (
      • Ma B.
      • Meredith C.
      • Schaefer III, H.F.
      Pyrophosphate structures and reactions: evaluation of electrostatic effects on the pyrophosphates with and without alkali cations.
      ) between two Pi molecules. PPi can act as an energy and phosphate donor, similar to nucleoside di- or triphosphates, including ATP, in cellular reactions (
      • Baltscheffsky M.
      • Baltscheffsky H.
      Inorganic pyrophosphate and inorganic pyrophosphatases.
      ,
      • Kornberg A.
      Inorganic polyphosphate: toward making a forgotten polymer unforgettable.
      ,
      • Kulaev I.S.
      • Vagabov V.M.
      Polyphosphate metabolism in micro-organisms.
      ). Several enzymes selectively utilize PPi over ATP and other nucleotides to catalyze similar reactions as ATP-dependent functional homologs.
      PPi-utilizing enzymes are potentially useful for metabolic engineering. In contrast to ATP, reactions involving PPi are reversible in vivo, with only a few exceptions (
      • Reeves R.
      How useful is energy in inorganic pyrophosphate.
      ,
      • Mertens E.
      Pyrophosphate-dependent phosphofructokinase, an anaerobic glycolytic enzyme?.
      ,
      • Dang T.
      • Ingram-Smith C.
      Investigation of pyrophosphate versus ATP substrate selection in the Entamoeba histolytica acetate kinase.
      ) because the energy released by cleavage of PPi is smaller than that of ATP or GTP (
      • Kulaev I.S.
      • Vagabov V.M.
      Polyphosphate metabolism in micro-organisms.
      ,
      • Thauer R.K.
      • Jungermann K.
      • Decker K.
      Energy conservation in chemotrophic anaerobic bacteria.
      ). In addition, utilization of PPi requires less cellular energy because PPi is generated as a byproduct of many in vivo reactions hydrolyzing nucleotide triphosphate (
      • Kulaev I.S.
      • Vagabov V.M.
      Polyphosphate metabolism in micro-organisms.
      ), whereas energy is required for ATP synthesis. A number of organisms, particularly anaerobic fermenting microbes, utilize PPi-dependent enzymes instead of ATP-dependent functional homologs for glycolysis and closely related metabolic reactions as a strategy to increase net ATP production (
      • Coombs G.H.
      • Müller M.
      Energy metabolism in anaerobic protozoa.
      ). Therefore, introducing or substituting PPi-dependent enzymes in place of ATP-dependent functional homologs is an attractive approach to alter metabolic flux or improve cellular energy efficiency. Furthermore, use of PPi-dependent enzymes is an attractive possibility for industrial production of phosphorylated compounds because PPi is 1000 times cheaper than ATP (
      • Nagata R.
      • Fujihashi M.
      • Sato T.
      • Atomi H.
      • Miki K.
      Identification of a pyrophosphate-dependent kinase and its donor selectivity determinants.
      ).
      The functional and structural study of PPi-utilizing enzymes is expected to provide important insights into the evolution of cellular energy currency. Because of its simple structure, PPi has been proposed to be the evolutionary precursor of ATP (
      • Kornberg A.
      Inorganic polyphosphate: toward making a forgotten polymer unforgettable.
      ,
      • Liu C.-L.
      • Hart N.
      • Peck Jr., H.D.
      Inorganic pyrophosphate: energy source for sulfate-reducing bacteria of the genus Desulfotomaculum.
      ). In addition, adoption of PPi-using enzymes by ancestral organisms with poor ATP-producing ability may have been energetically favorable. Therefore, understanding the evolutionary origin and relationship of PPi-dependent enzymes with ATP-dependent functional homologs is of interest for the evolutionary study of metabolism.
      The evolutionary relationship between PPi-dependent kinases and ATP-dependent functional homologs has been discussed in several studies. The best-studied enzyme to date is phosphofructokinase (PFK), and ATP-dependent (EC 2.7.1.11) and PPi-dependent PFKs (EC 2.7.1.90) have been shown to have a common ancestor (
      • Mertens E.
      Pyrophosphate-dependent phosphofructokinase, an anaerobic glycolytic enzyme?.
      ,
      • Carlisle S.M.
      • Blakeley S.D.
      • Hemmingsen S.M.
      • Trevanion S.J.
      • Hiyoshi T.
      • Kruger N.J.
      • Dennis D.T.
      Pyrophosphate-dependent phosphofructokinase. Conservation of protein sequence between the α- and β-subunits and with the ATP-dependent phosphofructokinase.
      ,
      • Bapteste E.
      • Moreira D.
      • Philippe H.
      Rampant horizontal gene transfer and phospho-donor change in the evolution of the phosphofructokinase.
      ). The specificity of these proteins for ATP or PPi is fundamentally determined by a single amino acid residue in the active site (
      • Chi A.
      • Kemp R.G.
      The primordial high energy compound: ATP or inorganic pyrophosphate?.
      ). The evolutionary relationship between ATP- and PPi-PFK is controversial (
      • Bapteste E.
      • Moreira D.
      • Philippe H.
      Rampant horizontal gene transfer and phospho-donor change in the evolution of the phosphofructokinase.
      ,
      • Alves A.M.
      • Meijer W.G.
      • Vrijbloed J.W.
      • Dijkhuizen L.
      Characterization and phylogeny of the pfp gene of Amycolatopsis methanolica encoding PPi-dependent phosphofructokinase.
      ,
      • Müller M.
      • Lee J.A.
      • Gordon P.
      • Gaasterland T.
      • Sensen C.W.
      Presence of prokaryotic and eukaryotic species in all subgroups of the PPi-dependent group II phosphofructokinase protein family.
      ); however, the results of the most recent large-scale phylogenetic analysis suggest that PPi-PFK evolved from an ATP-utilizing ancestor and that changes in the phosphodonors have occurred multiple independent times (
      • Bapteste E.
      • Moreira D.
      • Philippe H.
      Rampant horizontal gene transfer and phospho-donor change in the evolution of the phosphofructokinase.
      ). In contrast, all biochemically characterized acetate kinases (ACKs) utilize ATP (EC 2.7.2.1) as the phosphate donor. The only exception is an ACK from the eukaryotic parasite Entamoeba histolytica that strictly recognizes PPi as the substrate (EC 2.7.2.12) (
      • Dang T.
      • Ingram-Smith C.
      Investigation of pyrophosphate versus ATP substrate selection in the Entamoeba histolytica acetate kinase.
      ,
      • Reeves R.E.
      • Guthrie J.D.
      Acetate kinase (pyrophosphate): a fourth pyrophosphate-dependent kinase from Entamoeba histolytica.
      ). PPi-ACK has clear homology to ATP-ACKs, and phylogenetic analysis indicated that PPi-ACK has not formed a separate clade from ATP-ACKs (
      • Ingram-Smith C.
      • Martin S.R.
      • Smith K.S.
      Acetate kinase: not just a bacterial enzyme.
      ,
      • Fowler M.L.
      • Ingram-Smith C.
      • Smith K.S.
      Novel pyrophosphate-forming acetate kinase from the protist Entamoeba histolytica.
      ), suggesting that PPi-ACK also arose from an ATP-ACK. In addition, a member of the ribokinase family of proteins, which are considered to be ATP- or ADP-dependent, was recently found to be PPi-dependent (
      • Nagata R.
      • Fujihashi M.
      • Sato T.
      • Atomi H.
      • Miki K.
      Identification of a pyrophosphate-dependent kinase and its donor selectivity determinants.
      ). In summary, these three PPi-dependent kinases and their functional ATP-utilizing homologs have clear amino acid sequence similarity, and the former appear to have evolved from ATP-dependent ancestors.
      The evolution of phosphoenolpyruvate carboxykinase (PEPCK) seems to have followed a different pathway from the kinases described above. Depending on the phosphate donor to oxaloacetate, PEPCK can be divided into three types: GTP-PEPCK (EC 4.1.1.32), ATP-PEPCK (EC 4.1.1.49), and PPi-PEPCK (EC 4.1.1.38). Although both ATP- and GTP-PEPCK show significant amino acid sequence identity within each type, no significant overall sequence homology is observed between the two groups of enzymes (
      • Matte A.
      • Tari L.W.
      • Goldie H.
      • Delbaere L.T.
      Structure and mechanism of phosphoenolpyruvate carboxykinase.
      ). In contrast, crystal structure–based studies have revealed that ATP- and GTP-PEPCK have highly similar tertiary structures (
      • Matte A.
      • Goldie H.
      • Sweet R.M.
      • Delbaere L.T.
      Crystal structure of Escherichia coli phosphoenolpyruvate carboxykinase: a new structural family with the P-loop nucleoside triphosphate hydrolase fold.
      ,
      • Dunten P.
      • Belunis C.
      • Crowther R.
      • Hollfelder K.
      • Kammlott U.
      • Levin W.
      • Michel H.
      • Ramsey G.B.
      • Swain A.
      • Weber D.
      • Wertheimer S.J.
      Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site.
      ). In addition, these enzymes possess “consensus motifs,” including a PEPCK-specific domain, which directly associates with PEP or oxaloacetate, and P-binding loop (also called kinase-1a) and kinase-2 motifs, which directly interact with the phosphate moiety of nucleoside triphosphates (
      • Matte A.
      • Tari L.W.
      • Goldie H.
      • Delbaere L.T.
      Structure and mechanism of phosphoenolpyruvate carboxykinase.
      ,
      • Matte A.
      • Goldie H.
      • Sweet R.M.
      • Delbaere L.T.
      Crystal structure of Escherichia coli phosphoenolpyruvate carboxykinase: a new structural family with the P-loop nucleoside triphosphate hydrolase fold.
      ). In contrast to ATP- and GTP-PEPCK, PPi-PEPCK has not been structurally characterized at the tertiary level. Although a PPi-PEPCK was biochemically characterized more than 50 years ago (
      • Siu P.M.
      • Wood H.G.
      • Stjernholm R.L.
      Fixation of CO2 by phosphoenolpyruvic carboxytransphosphorylase.
      • Siu P.M.
      • Wood H.G.
      Phosphoenolpyruvic carboxytransphosphorylase, a CO2 fixation enzyme from propionic acid bacteria.
      ,
      • Lochmüller H.
      • Wood H.G.
      • Davis J.J.
      Phosphoenolpyruvate carboxytransphosphorylase II: crystallization and properties.
      ,
      • Reeves R.E.
      Phosphopyruvate carboxylase from Entamoeba histolytica.
      • Haberland M.E.
      • Willard J.M.
      • Wood H.G.
      Phosphoenolpyruvate carboxytransphosphorylase: VI: catalytic and physical structures.
      ), the amino acid and gene sequence of the enzyme has recently been reported (
      • Chiba Y.
      • Kamikawa R.
      • Nakada-Tsukui K.
      • Saito-Nakano Y.
      • Nozaki T.
      Discovery of PPi-type phosphoenolpyruvate carboxykinase genes in eukaryotes and bacteria.
      ). PPi-PEPCK consists of more than 1100 amino acid residues and is approximately twice as long as ATP- and GTP PEPCK. PPi-PEPCK does not share a significant amino acid sequence identity with ATP- or GTP-PEPCK (E-value ≥ 1). It remains to be determined whether PPi-PEPCK has a similar tertiary structure as ATP- and GTP-PEPCK.
      In this study, we obtained and compared the crystal structure of a PPi-PEPCK homolog from Actinomyces israelii (AiPEPCK) with those of ATP- and GTP-PEPCK to evaluate the degree of homology between PPi-, ATP-, and GTP-PEPCK and to determine the structural basis of the PPi specificity of PPi-PEPCK.

      Results and discussion

      PPi-dependent activity and structure determination of PEPCK

      Crystallization was attempted using the biochemically characterized PPi-PEPCKs from Propionibacterium freudenreichii subsp. shermanii (PfPEPCK, WP_013160152.1) and E. histolytica (EhPEPCK1, XP_654765.1) (
      • Chiba Y.
      • Kamikawa R.
      • Nakada-Tsukui K.
      • Saito-Nakano Y.
      • Nozaki T.
      Discovery of PPi-type phosphoenolpyruvate carboxykinase genes in eukaryotes and bacteria.
      ) and PPi-PEPCK homologs from several bacteria. The crystals of PfPEPCK and the PPi-PEPCK homolog from A. israelii (AiPEPCK, WP_043560275.1), which show 61% and 43% amino acid identity with PfPEPCK and EhPEPCK1, respectively, were obtained and subjected to X-ray analysis. Quality diffraction data were collected only from a crystal of AiPEPCK labeled with selenomethionine. The data collection and refinement statistics are presented in Table 1.
      Table 1Data collection and refinement statistics
      Data collection
      BeamlinePF BL-17A
      Wavelength (Å)0.97887
      Space groupP3221
      Cell dimensions: a, b, c (Å)160.4, 160.4, 200.2
      Resolution (Å)48.1–2.60 (2.64–2.60)
      Values in parentheses indicate those of the highest-resolution shell.
      No. of unique reflections91847 (4505)
      Rmeas0.158 (1.243)
      Rpim0.049 (0.389)
      CC[ifrax,1/2]0.999 (0.837)
      Mean I/σ(I)19.2 (3.3)
      Completeness (%)99.9 (83.7)
      Multiplicity20.1 (19.8)
      Refinement
      Resolution (Å)48.1–2.60
      No. of reflections91796
      Rwork/Rfree0.196/0.241
      No. atoms
      Protein17120
      Solvent355
      Metal (Co)2
      B-factors (Å2)
      Protein55.2
      Solvent42.4
      Metal (Co)41.6
      RMSDs
      Bond lengths (Å)0.014
      Bond angles (°)1.756
      Ramachandran plot (%)
      Favoured region96.86
      Allowed region3.09
      Outliers0.05
      a Values in parentheses indicate those of the highest-resolution shell.
      PPi-dependent PEPCK activity was detected (2 × 10−2 μmol·mg of protein−1·min−1) from purified AiPEPCK, whereas ATP- and GTP-dependent activity was not detected. The activity was not increased when Mn was added to the reaction mixture instead of Co. The PPi-PEPCK activity was two orders of magnitude lower than that of PfPEPCK (
      • Chiba Y.
      • Kamikawa R.
      • Nakada-Tsukui K.
      • Saito-Nakano Y.
      • Nozaki T.
      Discovery of PPi-type phosphoenolpyruvate carboxykinase genes in eukaryotes and bacteria.
      ) under the same reaction conditions. The lower PPi-PEPCK activity of AiPEPCK may have been attributable to the purification and experimental conditions; for example, AiPEPCK might have lower oxygen tolerance than PfPEPCK, resulting in reduced activity following purification under aerobic conditions. Because AiPEPCK has high (61%) amino acid sequence identity to biochemically characterized PfPEPCK, and strictly conserved residues are likely critical for the PEPCK activity of PfPEPCK and EhPEPCK, as described below, we considered that at least the three-dimensional structure of monomeric AiPEPCK is highly similar to that of the PPi-PEPCKs from P. freudenreichii and E. histolytica. The structure of AiPEPCK was therefore used as the representative structure of PPi-PEPCKs.

      Overall structure of AiPEPCK

      The crystal structure of AiPEPCK was determined at 2.6 Å resolution. Two molecules in an asymmetric unit contained 1117 amino acid residues (A chain: 8–28, 39–431, 435–523, 542–755, and 758–1149) and 1118 residues (B chain: 11–28, 42–523, and 542–1149) (Fig. S1). A Co ion was observed in each molecule, a finding that was not unexpected, as AiPEPCK was crystallized in the presence of Co and Mg ions, which are both required for EhPEPCK activity (
      • Reeves R.E.
      Phosphopyruvate carboxylase from Entamoeba histolytica.
      ). The crystal structure of AiPEPCK was highly similar between the A and B chains (Cα RMSD of 0.141 Å); therefore, the AiPEPCK structure was described using the A chain, which had a lower B factor (49.0 Å2) than the B chain (61.4 Å2) (Fig. S1).
      The AiPEPCK structure is divided into a core structure that grasps a Co ion and four lobe structures (lobes 1 to 4) that envelope the core structure (Fig. 1, A and B). The core structure is composed of two globular α/β domains, called the N-terminal (85–346, 685–721, and 758–792) and C-terminal domains (347–359, 475–496, 649–684, and 817–1047).
      Figure thumbnail gr1
      Figure 1Structural determination of AiPEPCK. A, wall-eye stereo diagram of the overall structure of AiPEPCK. Secondary structural elements are labeled as follows: α, α-helix; β, β-strand; η, 310-helix. Helix α8 is not labeled because it overlaps with α5. B, order of domains and lobes in the amino acid sequence of AiPEPCK. N, C, and 1–4 in the boxes indicate the N- and C-terminal domains and lobes 1–4, respectively. The size of each box corresponds to the length of the amino acid sequence.
      N-terminal domain adopts a six-stranded β-sheet (S1, the order of strands is 1-2-3-31-32-30) and a seven-stranded β-sheet (S2, the order of strands is 6-7-8-10-9-4-5), and three α-helices (α4, α7, and α10) are sandwiched between these two β-sheets. Lobe 1 (1–84) exists in the N terminus of the N-terminal domain and is composed of three α-helices (α1, α2, and α3) and a 310-helix (η1). Helix α3 contacts S2 in the N-terminal domain. S2 also contacts four α-helices (α5, α6, α8, and α9) and two 310-helices (η2 and η3). Lobe 3 (722–757), which is inserted between β31 and β32 of the N-terminal domain, contains three α-helices (α17–α19). Lobe 3 is located on the opposite face of S1 from S2 together with a helix α16.
      The C-terminal domain has a twisted β-sheet composed of 10 β-strands (S3, the order of strands is 29-28/27-11-23-33-35-34-39-36) and a β-hairpin that is inserted between β36 and β39 (Fig. 1A). S3 is surrounded by seven α-helices (α22–α28) and four 310-helices (η4, η8 and η9). The C-terminal domain contacts lobe 2 (360–474, 497–648, and 793–816) and lobe 4 (1048–1149) in addition to the N-terminal domain. Lobe 2 seems to adopt a large domain with an α + β structure, unlike other smaller lobes, and is divided into three parts in the primary structure (Fig. 1B). The N-terminal part of lobe 2 is inserted between β11 and β23 of the C-terminal domain and forms a β-sandwich fold with two antiparallel β-sheets (S4, the order of strands is 12-22-21-20-16-15-14, where β16 and β20 are parallel; S5, the order of strands is 13-17-18-19) and a helix α11. S4 contacts a part of helix α21 (793–816) and a portion of lobe 2 and connects α20 of the N-terminal domain and β33 of the C-terminal domain. The other part of lobe 2 (497–648) is inserted between η4 and β27 of the C-terminal domain and forms four α-helices (α12–α15), three 310-helices (η5–η7), and a β-hairpin (β25 and β26) with an attached β-strand (β24). On the other hand, lobe 4 is extended from α28 of the C-terminal domain and adopts a helical structure composed of six α-helices (α29–α34) and a 310-helix (η13). Helices α29 and α30 contact helices α22, α23, and α28 of the C-terminal domain. Helices α31–α34 cover the loops connecting the β-strands of S1 in the N-terminal domain.

      Dimer formation with the contacts between lobes 2 and 3

      To evaluate the oligomeric state of AiPEPCK, we carried out size-exclusion chromatography. The result indicated that soluble AiPEPCK exists mainly as a homodimer (Fig. 2A). Dimer formation was further analyzed using the PISA server (
      • Krissinel E.
      • Henrick K.
      Inference of macromolecular assemblies from crystalline state.
      ), and each chain was predicted to form the same homodimer with an identical chain generated by symmetry operation (Fig. 2B). The contact surface area between two protomers was 1605 Å2 (A-A’ dimer) and 1599 (B-B’ dimer).
      Figure thumbnail gr2
      Figure 2Dimer formation of AiPEPCK. A, gel filtration chromatography using Superdex 200 Increase 10/300. AiPEPCK was estimated to form a homodimer because the relative molecular mass of monomeric AiPEPCK estimated from the amino acid sequence is 125,702. Elution volumes of standard (STD) proteins are shown at the top. mAU, ×10−3 absorbance unit. B, overall structure of homodimeric AiPEPCK estimated by the PISA server. PPi-PEPCK consists of a core structure of the N-terminal domain (N domain) and C-terminal domain (C domain) surrounded by lobes 1–4. A Co ion is located in the deep cleft between the N- and C-terminal domains. The notation ′ is added to all the labels for the A′ chain. C and D, detailed diagrams of the structures in the dashed boxes in B. The residues for dimer contacts are represented by stick models. Spheres and dashed lines show van der Waals contacts and hydrogen bonds, respectively. The notation ′ is added to all labels for the A′ chain.
      In the quaternary structure of AiPEPCK, lobes 2 and 3 are located on the dimer interface (Fig. 2B). These lobes seem to form handclasp-like interactions; lobe 2 contacts lobes 2′ and 3′ of another protomer, and lobe 3 is positioned in close proximity to lobe 2′. The strand β26 of lobe 2 forms three main-chain hydrogen bonds, which are observed in a parallel β-sheet, with the loop connecting η7′ and β25′ in lobe 2′ (Fig. 2C). This dimer interface is further reinforced by two hydrogen bonds between Ser640 and Asn607′ and a van der Waals contact between Trp636 and Gly618′. On the other hand, van der Waals contacts mainly contribute to the dimer interface between lobes 3 and 2′ (Fig. 2D), and Asp731 and Ser736 in lobe 3 also form hydrogen bonds with Gln425′ and Trp467′ in lobe 2′. These residues located on α17 and α18 of lobe 3 and on the loops between Ser4′ and Ser5′ in lobe 2′. There is no interaction among other lobes and the core structure (Fig. 2B). These structural findings suggest that lobes 2 and 3 are required for dimer formation of AiPEPCK.

      Structure comparison of PPi-PEPCK with ATP- and GTP-PEPCK

      A structural similarity search using the DALI server (
      • Holm L.
      • Laakso L.M.
      Dali server update.
      ) revealed that PPi-PEPCK has significant similarity (Z score ≥ 10) only to ATP- and GTP-PEPCK. The top hit was ATP-PEPCK from Escherichia coli (PDB code 1OS1-A; Z score, 21.6; RMSD, 3.6 Å; sequence identity, 13%), and the top hit among GTP-PEPCK was an enzyme from Rattus norvegicus (PDB code 5FH0-A; Z score, 17.5; RMSD, 3.5 Å; sequence identity, 10%). The structural superposition of PPi-, ATP-, and GTP-PEPCK revealed that they share a core structure consisting of the N- and C-terminal domains (Fig. S2), although PPi-PEPCK is ∼500 amino acid residues longer than ATP- and GTP-PEPCK by lobe structure (lobes 1–4, Figs. 2B). Notably, the order of the secondary structural elements present in the core structure of PPi-, ATP-, and GTP PEPCK was completely conserved in the primary structures of all enzymes (Fig. S3). A Co ion was located in the deep cleft between the two globular domains of PPi-PEPCK. ATP- and GTP-PEPCK adopt Ca and Mn ions at the same position, respectively (Fig. S2). In addition, the residues surrounding the metal ions (Lys331, Lys332, His352, Asp655, and Asp656 in AiPEPCK) are spatially conserved among three types of PEPCKs. The Mn ion at this position is required for enzymatic activity of ATP- and GTP-PEPCK (
      • Dunten P.
      • Belunis C.
      • Crowther R.
      • Hollfelder K.
      • Kammlott U.
      • Levin W.
      • Michel H.
      • Ramsey G.B.
      • Swain A.
      • Weber D.
      • Wertheimer S.J.
      Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site.
      ,
      • Tari L.W.
      • Matte A.
      • Goldie H.
      • Delbaere L.T.
      Mg2+–Mn2+ clusters in enzyme-catalyzed phosphoryl-transfer reactions.
      ), indicating that the cleft likely functions as the active site in PPi-PEPCK.
      The lobe structures are specific to PPi-PEPCK and are not contained in ATP- and GTP-PEPCKs. Amino acid sequence comparisons revealed that lobes 1–4 existed in all PPi-PEPCKs (Fig. S4). We further searched for reported structures similar to individual lobe structures using the DALI server by extracting them from the overall structure of AiPEPCK. As a result, there was no protein hit with query of lobe 1, whereas the overall structure of lobe 3 and the partial structures of lobes 2 and 4 matched structural elements of other proteins.
      The DALI results indicated that the overall structural similarity with lobe 2 was not found in any other proteins. However, the structure of lobe 2 was partially related to those of various types of proteins, such as a component of the bacterial type VII secretion apparatus EssC, a putative transcriptional regulator of the arabinosyltransferase EmbR, propionyl-CoA synthetase, the serine phosphatase MtX, adenylate cyclase-like protein CT664, and so on (Fig. S5). Among the structural elements of lobe 2, the β-sandwich fold of β-sheets S4 and S5 highly emerges in the protein structures found in the DALI analysis. The top hit was EssC from Staphylococcus aureus (PDB code 1WV3-A; Z score, 4.3; RMSD, 2.9 Å; sequence identity, 6%). This protein and CT644 have a forkhead-associated (FHA) domain that adopts a β-sandwich fold and functions as a phosphopeptide recognition module (
      • Mahajan A.
      • Yuan C.
      • Lee H.
      • Chen E.S.
      • Wu P.-Y.
      • Tsai M.-D.
      Structure and function of the phosphothreonine-specific FHA domain.
      ). Although the loops connecting β-sheets S4 and S5 are used for peptide recognition of lobe 3, similar to the FHA domain (Fig. 2D), lobe 2 shows no sequence similarity to the FHA domain, and lobe 3 has no phosphorylated reside. The structure of lobe 3 was similar to the structural element of NUP155 (1166–1195), a component of the human nuclear pore complex (PDB code 5IJN-E and 5IJO-E; Z score, 2.1; RMSD, 2.4 Å; sequence identity, 6%) (Fig. S6) and a large protein with 1391 residues. According to the structures determined by cryoelectron tomography (
      • Kosinski J.
      • Mosalaganti S.
      • von Appen A.
      • Teimer R.
      • DiGuilio A.L.
      • Wan W.
      • Bui K.H.
      • Hagen W.J.
      • Briggs J.A.
      • Glavy J.S.
      • Hurt E.
      • Beck M.
      Molecular architecture of the inner ring scaffold of the human nuclear pore complex.
      ), this structural element is located outside of the pore ring and seems not to interact with any other subunits. In addition, the residues on the dimer interface of lobe 3 are not conserved in the similar structural element of NUP155. The helices of lobe 2 (497–648) also emerge as a partial structure of the large globular protein, as shown in propionyl-CoA synthetase (Fig. S5). However, the structural elements of lobe 2–2′ interaction (Fig. 2C) are not conserved in any proteins, according to DALI results. These structural findings suggest that lobes 2 and 3 may be specific modules for dimer formation of PPi-PEPCK.
      Structures similar to a part of lobe 4 were found in various types of proteins with antiparallel helices consisting of two long α-helices (Fig. S7). Five protein structures with the highest Z scores are as follows: two molecules in the Rad50 dimer, a component of the Mre11 complex for the eukaryotic DNA damage response; PHYL1, a phyllody-inducing effector protein of phytoplasma; seryl-tRNA synthetase; and Sso2, a t-SNARE (target-membrane-associated–soluble N-ethylmaleimide fusion protein attachment protein SNAP receptor) protein that functions in intracellular membrane fusion. The top hit was one molecule in the Rad50 dimer (PDB code 1GOX-A; Z score, 6.2; RMSD, 5.1 Å; sequence identity, 6%). Their antiparallel helices match helices α31 and α33 of lobe 4, whereas their functions are highly divergent. Rad50 uses the antiparallel helices as an arm for assembly of the Mre11 complex (
      • Park Y.B.
      • Hohl M.
      • Padjasek M.
      • Jeong E.
      • Jin K.S.
      • Krel A.
      • Petrini J.H.
      • Cho Y.
      Eukaryotic Rad50 functions as a rod-shaped dimer.
      ). This structural element is required for tetramer formation and folding of a four-helix bundle in PHT1 and Sso2, respectively (
      • Iwabuchi N.
      • Maejima K.
      • Kitazawa Y.
      • Miyatake H.
      • Nishikawa M.
      • Tokuda R.
      • Koinuma H.
      • Miyazaki A.
      • Nijo T.
      • Oshima K.
      • Yamaji Y.
      • Namba S.
      Crystal structure of phyllogen, a phyllody-inducing effector protein of phytoplasma.
      ,
      • Yue P.
      • Zhang Y.
      • Mei K.
      • Wang S.
      • Lesigang J.
      • Zhu Y.
      • Dong G.
      • Guo W.
      Sec3 promotes the initial binary t-SNARE complex assembly and membrane fusion.
      ). On the other hand, seryl-tRNA synthetase interacts with tRNASer using parallel helices (
      • Biou V.
      • Yaremchuk A.
      • Tukalo M.
      • Cusack S.
      The 2.9 Å crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNASer.
      ). In AiPEPCK, helices α31 and α33 of lobe 4 contact the loops connecting the β-strands of S1 in the N-terminal domain. Therefore, the structural homology search could not identify the functional role and evolutionary origin of lobe 4.

      Conservation of residues important for PEPCK activity

      Standard multiple sequence alignment algorithms work well for amino acid sequences with high similarity but are not suitable for sequences with low similarity or largely different lengths. Here a structure-based alignment algorithm was employed to perform an amino acid sequence alignment of PPi-, ATP-, and GTP-PEPCK because they share less than 10% amino acid sequence similarity, and PPi-PEPCK is twice as long as ATP- and GTP-PEPCK. The structure-based alignment revealed that the catalytic residues in ATP- and GTP-PEPCK are strictly conserved in AiPEPCK and other PPi-PEPCKs (Figs. 3A and Fig. S3). Most of the conserved residues are located within three motifs found in ATP- and GTP-PEPCK: a PEPCK-specific domain, P-binding loop, and kinase 2 domain, which interact with PEP or oxaloacetate, nucleotide triphosphate, and a divalent cation, respectively (
      • Matte A.
      • Tari L.W.
      • Goldie H.
      • Delbaere L.T.
      Structure and mechanism of phosphoenolpyruvate carboxykinase.
      ,
      • Tari L.W.
      • Matte A.
      • Pugazhenthi U.
      • Goldie H.
      • Delbaere L.T.
      Snapshot of an enzyme reaction intermediate in the structure of the ATP–Mg2+–oxalate ternary complex of Escherichia coli PEP carboxykinase.
      ). This finding strongly suggests that PPi-PEPCK also contains these three motifs and utilizes a similar catalytic mechanism.
      Figure thumbnail gr3
      Figure 3Comparison of active sites. A, comparison of residues in the active sites of PPi-, ATP-, and GTP-PEPCK. Red arrows, residues that interact with PEP, oxaloacetate, or analogs; orange circles, residues that interact with the phosphogroup of ATP and/or GTP; pink circles, residues that interact with a divalent cation. His920 of AiPEPCK, which is suggested to contribute to PPi specificity, is highlighted by a yellow box. B and C, comparison of the active site structure between AiPEPCK (B, light blue) and ATP-binding ATP-PEPCK (PDB code 2PXZ, yellow) and AiPEPCK and GTP-binding GTP-PEPCK (C, PDB code 3DT7, light brown). Residues of PPi-PEPCK and ATP- or GTP-PEPCK are indicated by regular letters and letters in parentheses, respectively.
      All of the conserved catalytic residues (Arg87, Tyr235, Gly237, Lys244, Ser286, Asn403, and Arg405; rat cytosolic GTP-PEPCK numbering) in GTP and/or ATP-PEPCK that directly interact with PEP, oxaloacetate, or the analog oxalate (
      • Matte A.
      • Tari L.W.
      • Goldie H.
      • Delbaere L.T.
      Structure and mechanism of phosphoenolpyruvate carboxykinase.
      ,
      • Dunten P.
      • Belunis C.
      • Crowther R.
      • Hollfelder K.
      • Kammlott U.
      • Levin W.
      • Michel H.
      • Ramsey G.B.
      • Swain A.
      • Weber D.
      • Wertheimer S.J.
      Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site.
      ,
      • Sullivan S.M.
      • Holyoak T.
      Structures of rat cytosolic PEPCK: insight into the mechanism of phosphorylation and decarboxylation of oxaloacetic acid.
      ,
      • Carlson G.M.
      • Holyoak T.
      Structural insights into the mechanism of phosphoenolpyruvate carboxykinase catalysis.
      ) were conserved in AiPEPCK as Arg134, Tyr326, Gly328, Lys332, Ser486, Asn780, and Arg782, respectively (Fig. 3A, red triangles). The amino acid residues making hydrogen bonds to the triphosphate group of ATP in ATP-PEPCK (His232, Ser250, Gly251, Gly253, Lys254, Thr255, and Arg333) (
      • Delbaere L.T.
      • Sudom A.M.
      • Prasad L.
      • Leduc Y.
      • Goldie H.
      Structure/function studies of phosphoryl transfer by phosphoenolpyruvate carboxykinase.
      ) were also conserved in PPi-PEPCK as His352, Ser486, Gly487, Gly489, Lys490, Ser491, and Arg782, respectively (Fig. 3A, orange circles). The orientation of the side chains was also similar between ATP/GTP-PEPCK and PPi-PEPCK. In addition, all residues ligand to the Co ion (Lys332, His352, and Asp656) in PPi-PEPCK (Fig. 3A, pink circles) were conserved in ATP/GTP-PEPCK.

      Structural basis of PPi specificity

      PPi-PEPCK does not recognize ADP or GDP as a phosphate acceptor (
      • Siu P.M.
      • Wood H.G.
      Phosphoenolpyruvic carboxytransphosphorylase, a CO2 fixation enzyme from propionic acid bacteria.
      ). As expected, the amino acid residues in purine-binding regions were not conserved between ATP-PEPCK (449RISIKDT455) (
      • Matte A.
      • Tari L.W.
      • Goldie H.
      • Delbaere L.T.
      Structure and mechanism of phosphoenolpyruvate carboxykinase.
      ), GTP-PEPCK (516WFRKDKNGKFLWPGFGEN533) (
      • Dunten P.
      • Belunis C.
      • Crowther R.
      • Hollfelder K.
      • Kammlott U.
      • Levin W.
      • Michel H.
      • Ramsey G.B.
      • Swain A.
      • Weber D.
      • Wertheimer S.J.
      Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site.
      ,
      • Fukuda W.
      • Fukui T.
      • Atomi H.
      • Imanaka T.
      First characterization of an archaeal GTP-dependent phosphoenolpyruvate carboxykinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
      ), and PPi-PEPCK, with the exception of a lysine residue corresponding to Lys917 of AiPEPCK (Fig. 3A). Superposition of PPi-PEPCK and ATP-PEPCK revealed that the bulky side chain of His920 sterically hinders access of ATP to the active site in PPi-PEPCK (Fig. 3B). His920 in PPi-PEPCK corresponds to Phe530 in GTP-PEPCK, one of the two phenylalanine residues whose side chains sandwich the guanine base (
      • Dunten P.
      • Belunis C.
      • Crowther R.
      • Hollfelder K.
      • Kammlott U.
      • Levin W.
      • Michel H.
      • Ramsey G.B.
      • Swain A.
      • Weber D.
      • Wertheimer S.J.
      Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site.
      ). However, the structural superposition of PPi-PEPCK and GTP-PEPCK showed that the side chains of His920 and Phe530 are arranged quite differently and that His920 also appears to block access of GTP to the catalytic site (Fig. 3C). In addition, His920 of AiPEPCK is strictly conserved in PPi-PEPCKs from a variety of organisms (Fig. S4). Therefore, this bulky residue of PPi-PEPCK may contribute to the specificity of PPi as the small phosphate donor and phosphate as the acceptor.
      A P-binding loop and purine-recognizing helix, which is located immediately after a purine-binding region (Fig. 3A), of ATP- and GTP-PEPCK are flexible and close upon substrate binding (
      • Holyoak T.
      • Sullivan S.M.
      • Nowak T.
      Structural insights into the mechanism of PEPCK catalysis.
      ,
      • Cotelesage J.J.
      • Prasad L.
      • Zeikus J.G.
      • Laivenieks M.
      • Delbaere L.T.
      Crystal structure of Anaerobiospirillum succiniciproducens PEP carboxykinase reveals an important active site loop.
      ) (Fig. 4A). Active-site comparison of AiPEPCK, ATP-binding ATP-PEPCK, and apoATP-PEPCK revealed that the P-binding loop of apoAiPEPCK is in an open conformation, similar to that in apoATP-PEPCK, whereas the purine-recognizing helix of AiPEPCK is even more closed than the closed helix of ATP-binding ATP-PEPCK (Fig. 4B). The purine-recognizing helix of apoPPi-PEPCK may adopt a closed conformation because of the presence of the PPi-PEPCK-specific appendage structures of lobes 2 and 3. Lobe 2 directly contacts and hinders movement of the purine-recognizing helix to an open position. Dimer formation at the surface of lobes 2 and 3 (Fig. 2B) may further inhibit movement of the purine-recognizing helix. Notably, all of the experimentally confirmed GTP-PEPCKs and ATP-PEPCK from E. coli are monomers (
      • Matte A.
      • Goldie H.
      • Sweet R.M.
      • Delbaere L.T.
      Crystal structure of Escherichia coli phosphoenolpyruvate carboxykinase: a new structural family with the P-loop nucleoside triphosphate hydrolase fold.
      ). ATP-PEPCKs from other organisms form homomultimers (
      • Matte A.
      • Goldie H.
      • Sweet R.M.
      • Delbaere L.T.
      Crystal structure of Escherichia coli phosphoenolpyruvate carboxykinase: a new structural family with the P-loop nucleoside triphosphate hydrolase fold.
      ), although this conformation may not inhibit movement of the purine-recognizing helix because ATP-PEPCK from Trypanosoma cruzi (
      • Trapani S.
      • Linss J.
      • Goldenberg S.
      • Fischer H.
      • Craievich A.F.
      • Oliva G.
      Crystal structure of the dimeric phosphoenolpyruvate carboxykinase (PEPCK) from Trypanosoma cruzi at 2 Å resolution.
      ) forms a homodimer between the N-terminal domains, which does not inhibit movement of the purine-recognizing helix on the C-terminal domain. In addition, His920 of AiPEPCK is located within the N terminus of the purine-recognizing helix. Therefore, inflexibility of the purine-recognizing helix may fix the bulky residue at the active site and sterically inhibit access of large substrates such as ATP.
      Figure thumbnail gr4
      Figure 4A and B, comparison of the purine-recognizing helix and P-loop positions between ATP-PEPCK with (PDB code 2PXZ, yellow) and without (PDB code 1OEN, light green) ATP (A) and AiPEPCK (light blue) and ATP-PEPCK without ATP (B). The purine-recognizing helix of AiPEPCK is closed, as in the case of ATP-binding ATP-PEPCK, whereas the P-loop of PPi-PEPCK is open, as in the case of apoATP-PEPCK.
      In summary, dimer formation by the PPi-PEPCK-specific appendage structures of lobes 2 and 3 and the bulky residue corresponding to His920 of AiPEPCK may contribute to the PPi specificity of PPi-PEPCK by conserving the small size of the phosphate donor/acceptor–binding site. Most PPi-dependent kinases characterized to date are homologous to their ATP-dependent counterparts, and large amino acid residues occluding a part of the ATP-binding pocket contribute to the PPi specificity of PPi-dependent kinases (
      • Nagata R.
      • Fujihashi M.
      • Sato T.
      • Atomi H.
      • Miki K.
      Identification of a pyrophosphate-dependent kinase and its donor selectivity determinants.
      ,
      • Chi A.
      • Kemp R.G.
      The primordial high energy compound: ATP or inorganic pyrophosphate?.
      ). In contrast, to our knowledge, the existence of a large tertiary structure and/or formation of a quaternary structure that contributes to substrate specificity appears to be unique to PEPCK.

      Evolutionary relationship between three types of PEPCKs

      ATP-PEPCK and GTP-PEPCK share less than 20% overall amino acid sequence identity and also share several conserved motifs; it has been considered that these two types of PEPCKs likely resulted from convergent evolution (
      • Matte A.
      • Tari L.W.
      • Goldie H.
      • Delbaere L.T.
      Structure and mechanism of phosphoenolpyruvate carboxykinase.
      ,
      • Jurado L.A.
      • Machín I.
      • Urbina J.A.
      Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent): transition metal ion requirement for activity and sulfhydryl group reactivity.
      ). The amino acid sequence of PPi-PEPCK was also found to lack overall sequence similarity with ATP-PEPCK and GTP-PEPCK and is nearly twice as long as that of ATP-PEPCK and GTP-PEPCK (
      • Chiba Y.
      • Kamikawa R.
      • Nakada-Tsukui K.
      • Saito-Nakano Y.
      • Nozaki T.
      Discovery of PPi-type phosphoenolpyruvate carboxykinase genes in eukaryotes and bacteria.
      ), suggesting that PPi-PEPCK also does not share a common ancestor with ATP-PEPCK or GTP-PEPCK. Although an amino acid sequence–based search failed to detect conserved motifs in PPi-PEPCK, crystal structure analyses revealed that PPi-PEPCK conserves motifs common to ATP-PEPCK and GTP-PEPCK (Fig. 3A), and the three-dimensional structures of the three types of PEPCKs share statistically significant similarity. Furthermore, the order of the secondary structural elements was completely conserved in the primary structures, without exception (Fig. S3). These facts strongly suggest that PPi-, ATP-, and GTP-PEPCKs are not the result of convergent evolution but have a shared origin, as in the case of enzymes that share whole structures and the catalytic domains (
      • Nakatsu T.
      • Kato H.
      • Oda J.
      Crystal structure of asparagine synthetase reveals a close evolutionary relationship to class II aminoacyl-tRNA synthetase.
      • Perutz M.F.
      • Kendrew J.C.
      • Watson H.C.
      Structure and function of haemoglobin: II: some relations between polypeptide chain configuration and amino acid sequence.
      ,
      • Babbitt P.C.
      • Gerlt J.A.
      Understanding enzyme superfamilies chemistry as the fundamental determinant in the evolution of new catalytic activities.
      • Hanefeld U.
      • Gardossi L.
      • Magner E.
      Understanding enzyme immobilisation.
      ). If this assumption is true, then the timing of the divergence of the three types of PEPCKs and the function of the common ancestor remain to be determined.
      The phylogenic tree constructed using the structure-based alignment of core structures revealed that each type of PEPCK forms a single clade with significant bootstrap values (Fig. S8), indicating that the phosphate donor change did not occur multiple times, unlike in the case of PFK. It should be noted that the order of division cannot be discerned from this tree solely because outgroup sequences are not available. However, as PPi-PEPCK possesses insertion sequences forming appendage structures (lobes 1–4) that are absent in ATP-PEPCK and GTP-PEPCK, it is most probable that, at first, the ancestor of PPi-PEPCK diverged from the ancestor of three types of PEPCKs, and then the ancestor of ATP-PEPCK and GTP-PEPCK diverged (Fig. 5). Both ATP- and GTP-PEPCKs exist in various bacteria and archaea and follow a chimeric distribution. For example, most alpha-, gamma-, and epsilonproteobacteria and approximately half of all deltaproteobacteria have ATP-type PEPCKs, whereas nearly half of all betaproteobacteria and deltaproteobacteria possess GTP-type PEPCKs. Furthermore, in the case of eukaryotes, ATP-PEPCKs exist in yeasts and plants, whereas GTP-dependent PEPCKs are found in higher organisms, including animals and insects. Phylogenic analyses indicate that most archaeal and bacterial ATP- and GTP-PEPCKs sequences are separated by significant values (Fig. S8). Taken together, these results suggest that the separation of ATP-PEPCK and GTP-PEPCK occurred before the birth of the last universal common ancestor (LUCA). To sum up these discussions, the ancestor of PPi-PEPCK may also separate from the common ancestor of the three types of PEPCKs before the LUCA. The three types of PEPCKs may have evolved independently for a sufficiently long period to lose amino acid sequence similarity but still retain the active site, with the exception of the purine-binding region.
      Figure thumbnail gr5
      Figure 5Distribution of the three types of PEPCKs in extant organisms and the predicted evolutionary history.
      It remains uncertain whether the common ancestor of the three types of PEPCKs possessed PPi-PEPCK specific lobes 1–4 or whether the appendage structures were inserted after the ancestor of PPi-PEPCK diverged from the common ancestor of the three types of PEPCKs. If lobes 1–4 were added to ancestral PEPCK by lateral gene transfer, then sequences and/or structures that were the source of lobes 1–4 might remain in protein sequences or structures of extant organisms. However, BLASTP and PDB searches conducted using lobes 1–4 as queries found no sequences or functionally conserved structures like the core structure with statistically significant similarities. Discovery of enzymes that share the origin with PEPCKs is required to estimate the presence or absence of the appendage structures and substrate specificity of the common ancestor of PEPCKs.
      The evolutionary history of the three types of PEPCKs is clearly distinct from that of other kinases. In the case of PFK, the PPi type has high amino acid sequence similarity to ATP types, and PPi-PFK appears to have been derived from ATP-PFK in multiple events that have occurred relatively recently and involved substitution of one or two Gly residues in the active site with bulky ones (
      • Bapteste E.
      • Moreira D.
      • Philippe H.
      Rampant horizontal gene transfer and phospho-donor change in the evolution of the phosphofructokinase.
      ,
      • Chi A.
      • Kemp R.G.
      The primordial high energy compound: ATP or inorganic pyrophosphate?.
      ). In contrast, separation of PPi-PEPCK and the ATP- and GTP-dependent functional homologs occurred only once, most probably before the LUCA arose. Furthermore, a drastic change of the structure (insertion or deletion of multiple appendage structures) occurred when the ancestor of ATP- and GTP-PEPCK and PPi-PEPCK were separated. In conclusion, the present structure-based analyses of PPi-PEPCK have helped determine the evolutionary history of PEPCKs, which could not have been detected from the amino acid sequence alone.

      Materials and methods

      Plasmid construction

      AiPEPCK (WP_043560275.1) was PCR-amplified from genomic DNA (JGD12771) purchased from RIKEN BRC, which is participating in the National Bio-Resource Project of MEXT, Japan using the primers 5′-TCGAAGGTAGGCATAATGTCCGTAGTCGAACGC-3′ and 5′-ATTCGGATCCCTCGATCAGACGAACCTGGGCTG-3′ and was then cloned into pCold GST plasmids (Takara) cut with NdeI and XhoI using the In-Fusion HD cloning system (Takara).

      Overexpression and purification of recombinant PPi-PEPCKs

      E. coli BL21 Star (DE3) (Life Technologies) was used for expression of AiPEPCK. Host cells transformed with expression plasmids were inoculated into 400 ml of Luria–Bertani medium in a 1-liter conical flask containing 50 mg liter−1 ampicillin. After cultivating the cells aerobically at 37 °C until A600 reached ∼0.5, protein expression was induced by cooling the culture on ice for 30 min and adding 0.1 mm isopropyl 1-thio-β-d-galactopyranoside to the medium, followed by overnight cultivation at 15 °C. To obtain AiPEPCK labeled with selenomethionine, the host cells were cultivated in 400 ml M9 minimal medium supplemented with 20 mg liter−1 thiamine, biotin, adenosine, guanosine, cytidine, and thymidine and 50 mg liter−1 ampicillin at 37 °C. When the A600 of the culture reached ∼0.5, 100 mg liter−1 (final concentration) of Lys, Phe, and Thr; 50 mg liter−1 (final concentration) of Ile, Leu, and Val; and 60 mg liter−1 (final concentration) of selenomethionine were added to the medium, and the cells were further cultivated until A600 reached ∼0.7. Protein expression was then induced as described above.
      Harvested cells (∼7 g of wet cells from 2 liters of culture) were disrupted by sonication in GST buffer (20 mm Tris-HCl (pH 8.0), 300 mm NaCl, 1 mm DTT, and 3 ml g−1 of wet cells), and cell debris was removed by centrifugation. The supernatant was then mixed with 0.5 ml of GSH-Sepharose 4B (GE Healthcare) and incubated for 30 min at 4 °C. The mixture was then applied to an open column and washed with 30 bed volumes of GST buffer. The beads were suspended with 125 μl of GST buffer, and the N-terminal GST tag was digested by reaction with human rhinovirus 3C protease at 4 °C overnight. The cleaved protein was eluted with 2 bed volumes of GST buffer and then diluted with the same volume of 0.2 mm CoCl2 to give a NaCl concentration of 150 mm. The resulting solution was applied to a MonoQ HR 5/5 column (bed volume, 1 ml; GE Healthcare) equilibrated with 10 mm Tris-HCl (pH 8.0) containing150 mm NaCl and 0.1 mm CoCl2. Proteins were eluted by increasing the NaCl gradient from 150 to 650 mm over 20 column volumes at a flow rate of 1.0 ml min−1. The purified protein was concentrated to ∼10 mg ml−1 using a 4-ml Vivaspin concentrator (50-kDa cutoff, Vivascience) in 10 mm Tris-HCl (pH 8.0), 150 mm NaCl, and 0.1 mm CoCl2. Protein concentrations were measured using Bio-Rad DC protein assay dye and bovine γ-globulin as a standard.

      Gel filtration

      To determine the quaternary structure of AiPEPCK, gel filtration was performed using a Superdex 200 (10/300) column equilibrated with 20 mm Tris-HCl (pH 8.0) supplemented with 150 mm NaCl at a flow rate of 1 ml min−1. Gel filtration standard (Bio-Rad, catalog no. 1511901) was used as a standard.

      Crystallization, data collection, and preliminary X-ray analysis

      Crystallization experiments were performed using commercially available crystallization kits (Crystal Screen HT, Index HT (Hampton Research, Aliso Viejo, CA), and Wizard Screens I and II (Emerald BioSystems, Bainbridge Island, WA) at 293 K in 96-well VIOLAMO sitting-drop protein crystallization plates (AS ONE Co., Osaka, Japan) with a Gryphon protein crystallization system (Art Robbins Instruments, Sunnyvale, CA). A sitting drop was prepared by mixing 0.2 μl of protein solution and 0.2 μl of reservoir solution and equilibrated against 40 μl of reservoir solution. After optimizing the crystallization conditions, the crystals obtained using a reservoir solution consisting of 100 mm HEPES-NaOH (pH 7.5), 30% (w/v) PEG 400, and 200 mm MgCl2 were used for data collection.
      The selenomethionine-modified AiPEPCK crystals were soaked in crystallization solution supplemented with 25% (v/v) ethylene glycol as a cryoprotectant, picked up using Dual Thickness MicroMountsTM (MiTeGen, Ithaca, NY), and cooled in a liquid nitrogen stream. The X-ray diffraction data (1800 images) were collected using a PILATUS 6 m detector on beamline BL-17A at the Photon Factory (Tsukuba, Japan) with the following parameters: wavelength, 0.97887 Å (Se peak); oscillation angle, 0.2°; exposure time, 0.5 s; crystal-to-detector distance, 485.7 mm.
      The diffraction data were indexed, integrated, and scaled using XDS (
      • Kabsch W.
      XDS. Xds.
      ) and AIMLESS (
      • Evans P.R.
      • Murshudov G.N.
      How good are my data and what is the resolution?.
      ). The obtained crystal belonged to the space group P3121 with unit cell parameters of a = b = 160.4 Å and c = 200.2 Å. The initial model was solved by single-wavelength anomalous dispersion phasing using AutoSol of the PHENIX program suite (
      • Terwilliger T.C.
      • Adams P.D.
      • Read R.J.
      • McCoy A.J.
      • Moriarty N.W.
      • Grosse-Kunstleve R.W.
      • Afonine P.V.
      • Zwart P.H.
      • Hung L.-W.
      Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard.
      ). Iterative model building and refinement cycles were performed using COOT (
      • Emsley P.
      • Cowtan K.
      Coot: model-building tools for molecular graphics.
      ) and PHENIX.REFINE (
      • Adams P.D.
      • Grosse-Kunstleve R.W.
      • Hung L.-W.
      • Ioerger T.R.
      • McCoy A.J.
      • Moriarty N.W.
      • Read R.J.
      • Sacchettini J.C.
      • Sauter N.K.
      • Terwilliger T.C.
      PHENIX: building new software for automated crystallographic structure determination.
      ). The final model was refined by Refmac5 (
      • Murshudov G.N.
      • Skubák P.
      • Lebedev A.A.
      • Pannu N.S.
      • Steiner R.A.
      • Nicholls R.A.
      • Winn M.D.
      • Long F.
      • Vagin A.A.
      REFMAC5 for the refinement of macromolecular crystal structures.
      ) with twin refinement (twin fraction of 0.062) and local noncrystallographic symmetry restraint to an Rwork of 0.196 and Rfree of 0.241. Data collection and refinement statistics are summarized in Table 1. All structures were depicted using PyMOL viewer (Schrödinger, Tokyo, Japan).

      Construction of structure-based amino acid sequence alignment

      6K31 (A. israelii PPi-PEPCK), 2PXZ (E. coli ATP-PEPCK), and 3DT7 (rat cytosolic GTP-PEPCK) (
      • Sullivan S.M.
      • Holyoak T.
      Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection.
      ) were used for structure-based amino acid sequence alignment. Structures with complete sequences were reconstructed as follows because the PDB coordinate files lacked some amino acid residues. Complete sequences of each PDB structure were obtained from GenBank. Self-homology modeling with each complete sequence was performed using SWISS-MODEL (
      • Waterhouse A.
      • Bertoni M.
      • Bienert S.
      • Studer G.
      • Tauriello G.
      • Gumienny R.
      • Heer F.T.
      • de Beer T.A.P.
      • Rempfer C.
      • Bordoli L.
      • Lepore R.
      • Schwede T.
      SWISS-MODEL: homology modelling of protein structures and complexes.
      ). The first through seventeenth and first through eighth sequences of AiPEPCK and ATP-PEPCK, respectively, were added manually because the program was unable to determine the topology.
      The structure-based alignment of the reconstructed and full-length PEPCKs was performed using the MultiSeq plugin (
      • Roberts E.
      • Eargle J.
      • Wright D.
      • Luthey-Schulten Z.
      MultiSeq: unifying sequence and structure data for evolutionary analysis.
      ) in the VMD program (
      • Humphrey W.
      • Dalke A.
      • Schulten K.
      VMD: visual molecular dynamics.
      ) and visualized with ESPript 3.0 (
      • Robert X.
      • Gouet P.
      Deciphering key features in protein structures with the new ENDscript server.
      ). Secondary structures were predicted using PyMOL.

      Author contributions

      Y. C. conceptualization; Y. C. data curation; Y. C., T. M., and Y. S. formal analysis; Y. C. investigation; Y. C. methodology; Y. C. and T. M. writing-original draft; Y. C., K. T., and M. T. project administration; T. M. validation; T. M. visualization; K. T., M. T., and T. N. writing-review and editing.

      Acknowledgments

      We thank Mihoko Imada (National Institute of Infectious Diseases) and Keiko Usui (Japan Agency for Marine-Earth Science and Technology; JAMSTEC) for plasmid construction. The synchrotron radiation experiments were performed on beamline BL-17A at the Photon Factory with approval from the High Energy Accelerator Research Organization (proposal 2016G648 ).

      References

        • Ma B.
        • Meredith C.
        • Schaefer III, H.F.
        Pyrophosphate structures and reactions: evaluation of electrostatic effects on the pyrophosphates with and without alkali cations.
        J. Phys. Chem. 1994; 98: 8216-8223
        • Baltscheffsky M.
        • Baltscheffsky H.
        Inorganic pyrophosphate and inorganic pyrophosphatases.
        Molecular Mechanisms in Bioenergetics. 1992; 23: 2331-2348
        • Kornberg A.
        Inorganic polyphosphate: toward making a forgotten polymer unforgettable.
        J. Bacteriol. 1995; 177 (7836277): 491-496
        • Kulaev I.S.
        • Vagabov V.M.
        Polyphosphate metabolism in micro-organisms.
        Adv. Microb. Physiol. 1983; 24 (6320606): 83-171
        • Reeves R.
        How useful is energy in inorganic pyrophosphate.
        Trends Biochem. Sci. 1976; 1: 53-55
        • Mertens E.
        Pyrophosphate-dependent phosphofructokinase, an anaerobic glycolytic enzyme?.
        FEBS Lett. 1991; 285 (1648508): 1-5
        • Dang T.
        • Ingram-Smith C.
        Investigation of pyrophosphate versus ATP substrate selection in the Entamoeba histolytica acetate kinase.
        Sci. Rep. 2017; 7 (28724909): 5912
        • Thauer R.K.
        • Jungermann K.
        • Decker K.
        Energy conservation in chemotrophic anaerobic bacteria.
        Bacteriol. Rev. 1977; 41 (860983): 100-180
        • Coombs G.H.
        • Müller M.
        Energy metabolism in anaerobic protozoa.
        Biochemistry and Molecular Biology of Parasites. 1995; : 33-47
        • Nagata R.
        • Fujihashi M.
        • Sato T.
        • Atomi H.
        • Miki K.
        Identification of a pyrophosphate-dependent kinase and its donor selectivity determinants.
        Nat. Commun. 2018; 9 (29720581): 1765
        • Liu C.-L.
        • Hart N.
        • Peck Jr., H.D.
        Inorganic pyrophosphate: energy source for sulfate-reducing bacteria of the genus Desulfotomaculum.
        Science. 1982; 217 (17791517): 363-364
        • Carlisle S.M.
        • Blakeley S.D.
        • Hemmingsen S.M.
        • Trevanion S.J.
        • Hiyoshi T.
        • Kruger N.J.
        • Dennis D.T.
        Pyrophosphate-dependent phosphofructokinase. Conservation of protein sequence between the α- and β-subunits and with the ATP-dependent phosphofructokinase.
        J. Biol. Chem. 1990; 265 (2170409): 18366-18371
        • Bapteste E.
        • Moreira D.
        • Philippe H.
        Rampant horizontal gene transfer and phospho-donor change in the evolution of the phosphofructokinase.
        Gene. 2003; 318 (14585511): 185-191
        • Chi A.
        • Kemp R.G.
        The primordial high energy compound: ATP or inorganic pyrophosphate?.
        J. Biol. Chem. 2000; 275 (11001940): 35677-35679
        • Alves A.M.
        • Meijer W.G.
        • Vrijbloed J.W.
        • Dijkhuizen L.
        Characterization and phylogeny of the pfp gene of Amycolatopsis methanolica encoding PPi-dependent phosphofructokinase.
        J. Bacteriol. 1996; 178 (8550409): 149-155
        • Müller M.
        • Lee J.A.
        • Gordon P.
        • Gaasterland T.
        • Sensen C.W.
        Presence of prokaryotic and eukaryotic species in all subgroups of the PPi-dependent group II phosphofructokinase protein family.
        J. Bacteriol. 2001; 183 (11673446): 6714-6716
        • Reeves R.E.
        • Guthrie J.D.
        Acetate kinase (pyrophosphate): a fourth pyrophosphate-dependent kinase from Entamoeba histolytica.
        Biochem. Biophys. Res. Commun. 1975; 66 (172079): 1389-1395
        • Ingram-Smith C.
        • Martin S.R.
        • Smith K.S.
        Acetate kinase: not just a bacterial enzyme.
        Trends Microbiol. 2006; 14 (16678422): 249-253
        • Fowler M.L.
        • Ingram-Smith C.
        • Smith K.S.
        Novel pyrophosphate-forming acetate kinase from the protist Entamoeba histolytica.
        Eukaryot. Cell. 2012; 11 (22903977): 1249-1256
        • Matte A.
        • Tari L.W.
        • Goldie H.
        • Delbaere L.T.
        Structure and mechanism of phosphoenolpyruvate carboxykinase.
        J. Biol. Chem. 1997; 272 (9139042): 8105-8108
        • Matte A.
        • Goldie H.
        • Sweet R.M.
        • Delbaere L.T.
        Crystal structure of Escherichia coli phosphoenolpyruvate carboxykinase: a new structural family with the P-loop nucleoside triphosphate hydrolase fold.
        J. Mol. Biol. 1996; 256 (8609605): 126-143
        • Dunten P.
        • Belunis C.
        • Crowther R.
        • Hollfelder K.
        • Kammlott U.
        • Levin W.
        • Michel H.
        • Ramsey G.B.
        • Swain A.
        • Weber D.
        • Wertheimer S.J.
        Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site.
        J. Mol. Biol. 2002; 316 (11851336): 257-264
        • Siu P.M.
        • Wood H.G.
        • Stjernholm R.L.
        Fixation of CO2 by phosphoenolpyruvic carboxytransphosphorylase.
        J. Biol. Chem. 1961; 236: PC21-PC22
        • Siu P.M.
        • Wood H.G.
        Phosphoenolpyruvic carboxytransphosphorylase, a CO2 fixation enzyme from propionic acid bacteria.
        J. Biol. Chem. 1962; 237 (13977785): 3044-3051
        • Lochmüller H.
        • Wood H.G.
        • Davis J.J.
        Phosphoenolpyruvate carboxytransphosphorylase II: crystallization and properties.
        J. Biol. Chem. 1966; 241 (4288896): 5678-5691
        • Reeves R.E.
        Phosphopyruvate carboxylase from Entamoeba histolytica.
        Biochim. Biophys. Acta. 1970; 220 (4395132): 346-349
        • Haberland M.E.
        • Willard J.M.
        • Wood H.G.
        Phosphoenolpyruvate carboxytransphosphorylase: VI: catalytic and physical structures.
        Biochemistry. 1972; 11 (4333941): 712-722
        • Chiba Y.
        • Kamikawa R.
        • Nakada-Tsukui K.
        • Saito-Nakano Y.
        • Nozaki T.
        Discovery of PPi-type phosphoenolpyruvate carboxykinase genes in eukaryotes and bacteria.
        J. Biol. Chem. 2015; 290 (26269598): 23960-23970
        • Krissinel E.
        • Henrick K.
        Inference of macromolecular assemblies from crystalline state.
        J. Mol. Biol. 2007; 372 (17681537): 774-797
        • Holm L.
        • Laakso L.M.
        Dali server update.
        Nucleic Acids Res. 2016; 44 (27131377): W351-W355
        • Tari L.W.
        • Matte A.
        • Goldie H.
        • Delbaere L.T.
        Mg2+–Mn2+ clusters in enzyme-catalyzed phosphoryl-transfer reactions.
        Nat. Struct. Mol. Biol. 1997; 4 (9406547): 990-994
        • Mahajan A.
        • Yuan C.
        • Lee H.
        • Chen E.S.
        • Wu P.-Y.
        • Tsai M.-D.
        Structure and function of the phosphothreonine-specific FHA domain.
        Sci. Signal. 2008; 1 (19109241): re12-re112
        • Kosinski J.
        • Mosalaganti S.
        • von Appen A.
        • Teimer R.
        • DiGuilio A.L.
        • Wan W.
        • Bui K.H.
        • Hagen W.J.
        • Briggs J.A.
        • Glavy J.S.
        • Hurt E.
        • Beck M.
        Molecular architecture of the inner ring scaffold of the human nuclear pore complex.
        Science. 2016; 352 (27081072): 363-365
        • Park Y.B.
        • Hohl M.
        • Padjasek M.
        • Jeong E.
        • Jin K.S.
        • Krel A.
        • Petrini J.H.
        • Cho Y.
        Eukaryotic Rad50 functions as a rod-shaped dimer.
        Nat. Struct. Mol. Biol. 2017; 24 (28134932): 248-257
        • Iwabuchi N.
        • Maejima K.
        • Kitazawa Y.
        • Miyatake H.
        • Nishikawa M.
        • Tokuda R.
        • Koinuma H.
        • Miyazaki A.
        • Nijo T.
        • Oshima K.
        • Yamaji Y.
        • Namba S.
        Crystal structure of phyllogen, a phyllody-inducing effector protein of phytoplasma.
        Biochem. Biophys. Res. Commun. 2019; 513 (31010685): 952-957
        • Yue P.
        • Zhang Y.
        • Mei K.
        • Wang S.
        • Lesigang J.
        • Zhu Y.
        • Dong G.
        • Guo W.
        Sec3 promotes the initial binary t-SNARE complex assembly and membrane fusion.
        Nat. Commun. 2017; 8 (28112172): 14236
        • Biou V.
        • Yaremchuk A.
        • Tukalo M.
        • Cusack S.
        The 2.9 Å crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNASer.
        Science. 1994; 263 (8128220): 1404-1410
        • Tari L.W.
        • Matte A.
        • Pugazhenthi U.
        • Goldie H.
        • Delbaere L.T.
        Snapshot of an enzyme reaction intermediate in the structure of the ATP–Mg2+–oxalate ternary complex of Escherichia coli PEP carboxykinase.
        Nat. Struct. Mol. Biol. 1996; 3 (8599762): 355-363
        • Sullivan S.M.
        • Holyoak T.
        Structures of rat cytosolic PEPCK: insight into the mechanism of phosphorylation and decarboxylation of oxaloacetic acid.
        Biochemistry. 2007; 46 (17685635): 10078-10088
        • Carlson G.M.
        • Holyoak T.
        Structural insights into the mechanism of phosphoenolpyruvate carboxykinase catalysis.
        J. Biol. Chem. 2009; 284 (19638345): 27037-27041
        • Delbaere L.T.
        • Sudom A.M.
        • Prasad L.
        • Leduc Y.
        • Goldie H.
        Structure/function studies of phosphoryl transfer by phosphoenolpyruvate carboxykinase.
        Biochim. Biophys. Acta. 2004; 1697 (15023367): 271-278
        • Fukuda W.
        • Fukui T.
        • Atomi H.
        • Imanaka T.
        First characterization of an archaeal GTP-dependent phosphoenolpyruvate carboxykinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
        J. Bacteriol. 2004; 186 (15231795): 4620-4627
        • Holyoak T.
        • Sullivan S.M.
        • Nowak T.
        Structural insights into the mechanism of PEPCK catalysis.
        Biochemistry. 2006; 45 (16819824): 8254-8263
        • Cotelesage J.J.
        • Prasad L.
        • Zeikus J.G.
        • Laivenieks M.
        • Delbaere L.T.
        Crystal structure of Anaerobiospirillum succiniciproducens PEP carboxykinase reveals an important active site loop.
        Int. J. Biochem. Cell Biol. 2005; 37 (15890557): 1829-1837
        • Trapani S.
        • Linss J.
        • Goldenberg S.
        • Fischer H.
        • Craievich A.F.
        • Oliva G.
        Crystal structure of the dimeric phosphoenolpyruvate carboxykinase (PEPCK) from Trypanosoma cruzi at 2 Å resolution.
        J. Mol. Biol. 2001; 313 (11700062): 1059-1072
        • Jurado L.A.
        • Machín I.
        • Urbina J.A.
        Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent): transition metal ion requirement for activity and sulfhydryl group reactivity.
        Biochim. Biophys. Acta. 1996; 1292 (8547343): 188-196
        • Nakatsu T.
        • Kato H.
        • Oda J.
        Crystal structure of asparagine synthetase reveals a close evolutionary relationship to class II aminoacyl-tRNA synthetase.
        Nat. Struct. Biol. 1998; 5 (9437423): 15-19
        • Perutz M.F.
        • Kendrew J.C.
        • Watson H.C.
        Structure and function of haemoglobin: II: some relations between polypeptide chain configuration and amino acid sequence.
        J. Mol. Biol. 1965; 13: 669-678
        • Babbitt P.C.
        • Gerlt J.A.
        Understanding enzyme superfamilies chemistry as the fundamental determinant in the evolution of new catalytic activities.
        J. Biol. Chem. 1997; 272 (9388188): 30591-30594
        • Hanefeld U.
        • Gardossi L.
        • Magner E.
        Understanding enzyme immobilisation.
        Chem. Soc. Rev. 2009; 38 (19169460): 453-468
        • Kabsch W.
        XDS. Xds.
        Acta Crystallogr. D Biol. Crystallogr. 2010; 66 (20124692): 125-132
        • Evans P.R.
        • Murshudov G.N.
        How good are my data and what is the resolution?.
        Acta Crystallogr. D Biol. Crystallogr. 2013; 69 (23793146): 1204-1214
        • Terwilliger T.C.
        • Adams P.D.
        • Read R.J.
        • McCoy A.J.
        • Moriarty N.W.
        • Grosse-Kunstleve R.W.
        • Afonine P.V.
        • Zwart P.H.
        • Hung L.-W.
        Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard.
        Acta Crystallogr. D Biol. Crystallogr. 2009; 65 (19465773): 582-601
        • Emsley P.
        • Cowtan K.
        Coot: model-building tools for molecular graphics.
        Acta Crystallogr. D Biol. Crystallogr. 2004; 60 (15572765): 2126-2132
        • Adams P.D.
        • Grosse-Kunstleve R.W.
        • Hung L.-W.
        • Ioerger T.R.
        • McCoy A.J.
        • Moriarty N.W.
        • Read R.J.
        • Sacchettini J.C.
        • Sauter N.K.
        • Terwilliger T.C.
        PHENIX: building new software for automated crystallographic structure determination.
        Acta Crystallogr. D Biol. Crystallogr. 2002; 58 (12393927): 1948-1954
        • Murshudov G.N.
        • Skubák P.
        • Lebedev A.A.
        • Pannu N.S.
        • Steiner R.A.
        • Nicholls R.A.
        • Winn M.D.
        • Long F.
        • Vagin A.A.
        REFMAC5 for the refinement of macromolecular crystal structures.
        Acta Crystallogr. D Biol. Crystallogr. 2011; 67 (21460454): 355-367
        • Sullivan S.M.
        • Holyoak T.
        Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18772387): 13829-13834
        • Waterhouse A.
        • Bertoni M.
        • Bienert S.
        • Studer G.
        • Tauriello G.
        • Gumienny R.
        • Heer F.T.
        • de Beer T.A.P.
        • Rempfer C.
        • Bordoli L.
        • Lepore R.
        • Schwede T.
        SWISS-MODEL: homology modelling of protein structures and complexes.
        Nucleic Acids Res. 2018; 46 (29788355): W296-W303
        • Roberts E.
        • Eargle J.
        • Wright D.
        • Luthey-Schulten Z.
        MultiSeq: unifying sequence and structure data for evolutionary analysis.
        BMC Bioinformatics. 2006; 7 (16914055): 382
        • Humphrey W.
        • Dalke A.
        • Schulten K.
        VMD: visual molecular dynamics.
        J. Mol. Graphics. 1996; 14 (27, 28, 8744570): 33-38
        • Robert X.
        • Gouet P.
        Deciphering key features in protein structures with the new ENDscript server.
        Nucleic Acids Res. 2014; 42 (24753421): W320-W324