Structural requirements for N-trimethylation of lysine 115 of calmodulin

Calmodulin is trimethylated at lysine 115 by a highly specific methyltransferase that utilizes S-adenosylmethionine as a co-substrate. Lysine 115 is found within a highly conserved six-amino acid loop (LGEKLT) that forms a 90 degrees turn between EF-hand III and EF-hand IV in the carboxyl-terminal lobe. In the present work a mutagenesis approach was used to investigate the structural features of the carboxyl-terminal lobe that lead to the specificity of calmodulin methylation. Three structural regions within the carboxyl-terminal lobe appear to be involved in methyltransferase recognition: the highly conserved six-amino acid loop-turn region that contains lysine 115 as well as the adjacent alpha-helices (helix 6 and helix 7) from EF-hands III and IV. Site-directed mutagenesis of residues in the loop show that three residues, glycine 113, glutamate 114, and leucine 116 are essential for methylation. In addition, subdomain (individual helix or Ca(2+) binding loop) exchange mutants show that the substitutions of either helix 6 (EF-hand III) with helix 2 (EF-hand I) or helix 7 (EF-hand IV) with helix 3 (EF-hand II) compromises methylation. Charge-to-alanine mutations in helix 7 show that substitution of conserved charged residues at positions 118, 120, 122, 126, and 127 reduced lysine 115 methylation rates, suggesting possible electrostatic interactions between this helix and the methyltransferase. Single substitutions in helix 6 did not affect calmodulin methylation, suggesting this region may play a more indirect role in stabilizing the conformation of the methyltransferase recognition sequence.     

Influence of pH on NMR structure and stability of the human prion protein globular domain

The NMR structure of the globular domain of the human prion protein (hPrP) with residues 121-230 at pH 7.0 shows the same global fold as the previously published structure determined at pH 4.5. It contains three alpha-helices, comprising residues 144-156, 174-194, and 200-228, and a short anti-parallel beta-sheet, comprising residues 128-131 and 161-164. There are slight, strictly localized, conformational changes at neutral pH when compared with acidic solution conditions: helix alpha1 is elongated at the C-terminal end with residues 153-156 forming a 310-helix, and the population of helical structure in the C-terminal two turns of helix alpha 2 is increased. The protonation of His155 and His187 presumably contributes to these structural changes. Thermal unfolding monitored by far UV CD indicates that hPrP-(121-230) is significantly more stable at neutral pH. Measurements of amide proton protection factors map local differences in protein stability within residues 154-157 at the C-terminal end of helix alpha 1 and residues 161-164 of beta-strand 2. These two segments appear to form a separate domain that at acidic pH has a larger tendency to unfold than the overall protein structure. This domain could provide a "starting point" for pH-induced unfolding and thus may be implicated in endosomic PrPC to PrPSc conformational transition resulting in transmissible spongiform encephalopathies.     

NMR characterization of three-disulfide variants of lysozyme, C64A/C80A, C76A/C94A, and C30A/C115A--a marginally stable state in folded proteins

Our earlier NMR study showed that a two-disulfide variant of hen lysozyme containing intra-alpha-domain disulfide bridges, C6-C127 and C30-C115, is partially folded, with the alpha domain tightly folded to the nativelike conformation and the beta domain flexible or unfolded. With a view that the formation of a third disulfide bridge is a key for the accomplishment of the overall chain fold, three-dimensional structures of three-disulfide variants of hen lysozyme lacking one disulfide bridge (C64A/C80A, C76A/C94A, and C30A/C115A) were studied in detail using NMR spectroscopy. Amide hydrogen exchange rates were measured to estimate the degree of conformational fluctuation in a residue-specific manner. The structure of C76A/C94A was found to be quite similar to that of the wild type, except for the peptide segment of residues 74-78. The structure of C64A/C80A was considerably disordered in the entire region of the loop (residues 62-79). Further, it was found that a network of hydrogen bonds within the beta sheet and the 3(10) helix in the beta domain were disrupted and fluctuating. In C30A/C115A, the D helix was unstructured and the interface of the B helix with the D helix was significantly perturbed. However, the structural disorder generated in the hydrophobic core of the alpha domain was prevented by the C helix from propagating toward the beta domain. A marginally stable state in folded proteins is discussed based on the structures remaining in each three-disulfide variant.     

Native-state conformational dynamics of GART: a regulatory pH-dependent coil-helix transition examined by electrostatic calculations

Glycinamide ribonucleotide transformylase (GART) undergoes a pH-dependent coil-helix transition with pK(a) approximately 7. An alpha-helix is formed at high pH spanning 8 residues of a 21-residue-long loop, comprising the segment Thr120-His121-Arg122-Gln123-Ala124-Leu125-Glu126-Asn127. To understand the electrostatic nature of this loop-helix, called the activation loop-helix, which leads to the formation and stability of the alpha-helix, pK(a) values of all ionizable residues of GART have been calculated, using Poisson-Boltzmann electrostatic calculations and crystallographic data. Crystallographic structures of high and low pH E70A GART have been used in our analysis. Low pK(a) values of 5.3, 5.3, 3.9, 1.7, and 4.7 have been calculated for five functionally important histidines, His108, His119, His121, His132, and His137, respectively, using the high pH E70A GART structure. Ten theoretical single and double mutants of the high pH E70A structure have been constructed to identify pairwise interactions of ionizable residues, which have aided in elucidating the multiplicity of electrostatic interactions of the activation loop-helix, and the impact of the activation helix on the catalytic site. Based on our pK(a) calculations and structural data, we propose that: (1) His121 forms a molecular switch for the coil-helix transition of the activation helix, depending on its protonation state; (2) a strong electrostatic interaction between His132 and His121 is observed, which can be of stabilizing or destabilizing nature for the activation helix, depending on the relative orientation and protonation states of the rings of His121 and His132; (3) electrostatic interactions involving His119 and Arg122 play a role in the stability of the activation helix; and (4) the activation helix contains the helix-promoting sequence Arg122-Gln123-Ala124-Leu125-Glu126, but its alignment relative to the N and C termini of the helix is not optimal, and is possibly of a destabilizing nature. Finally, we provide electrostatic evidence that the formation and closure of the activation helix create a hydrophobic environment for catalytic-site residue His108, to facilitate catalysis.     

The solution structure of the recombinant hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803 in its hemichrome state

The product of the cyanobacterium Synechocystis sp. PCC 6803 gene slr2097 is a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Recombinant, ferric heme-reconstituted Synechocystis sp. PCC 6803 hemoglobin displays bis-histidine coordination of the iron ion. In addition, this protein is capable of covalently attaching a reactive histidine to the heme 2-vinyl group. The structure of the protein in the low-spin ferric state with intact vinyl substituents was solved by NMR methods. It was found that the structure differs from that of known truncated hemoglobins primarily in the orientation of the E helix, which carries His46 (E10) as the distal ligand to the iron; the length and orientation of the F helix, which carries His70 (F8) as the proximal ligand to the iron; and the H-helix, which carries His117 (H16), the reactive histidine. Regions of enhanced flexibility include the short A helix, the loop connecting the E and F helices, and the last seven residues at the carboxy end. The structural data allowed for the rationalization of physical properties of the cyanobacterial protein, such as fast on-rate for small ligand binding, unstable apoprotein fold, and cross-linking ability. Comparison to the truncated hemoglobin from the green alga Chlamydomonas eugametos also suggested how the endogenous hexacoordination affected the structure.     

A comparison of the predicted and X-ray structures of angiogenin. Implications for further studies of model building of homologous proteins

The three-dimensional structure of human angiogenin has been determined by X-ray crystallography and is compared here with an earlier model which predicted its structure, based on the homology of angiogenin with bovine pancreatic ribonuclease A. Comparison of the predicted model and crystal structure shows that the active-site histidine residues and the core of the angiogenin molecule, including most of the-strands and-helices, were predicted reasonably well. However, the structure of the surface loop regions and residues near the truncated C-terminus differs significantly. The C-terminal segment includes the active-site residues Asp-116, Gln-117, and Ser-118; Gln-117 in particular has been shown to be important in affecting the ribonucleolytic activity of angiogenin. Also, the orientation of one helix in the model differed from the orientation observed experimentally by about 20°, resulting in a large displacement of this chain segment. The difficulty encountered in predicting the surface loop regions has led to a new algorithm [Palmer and Scheraga (1991),J. Comput. Chem.,12, 505–526; (1992),J. Comput. Chem.,13, 329–350] for predicting the conformations of surface loops.     

Human bile salt-stimulated lipase has a high frequency of size variation due to a hypervariable region in exon 11.

The apparent molecular mass of human milk bile salt-stimulated lipase (BSSL) varies between mothers. The molecular basis for this is unknown, but indirect evidence has suggested the differences to reside in a region of repeats located in the C-terminal part of the protein. We here report that a polymorphism within exon 11 of the BSSL gene is the explanation for the molecular variants of BSSL found in milk. By Southern blot hybridization we analyzed the BSSL gene from mothers known to have BSSL of different molecular masses in their milk. A polymorphism was found within exon 11, previously shown to consist of 16 near identical repeats of 33 bp each. We detected deletions or, in one case, an insertion corresponding to the variation in molecular mass of the BSSL protein found in milk from the respective woman. Furthermore, we found that 56%, out of 295 individuals studied, carry deletions or insertions within exon 11 in one or both alleles of the BSSL gene. Hence, this is a hypervariable region and the current understanding that exon 11 in the human BSSL gene encodes 16 repeats is an oversimplification and needs to be revisited. Natural variation in the molecular mass of BSSL may have clinical implications.     

Conformational flexibility in T4 endonuclease VII revealed by crystallography: implications for substrate binding and cleavage

The structure of the N62D mutant of the junction-resolving endonuclease VII (EndoVII) from phage T4 has been refined at 1.3 A, and a second wild-type crystal form solved and refined at 2.8 A resolution. Comparison of the mutant with the wild-type protein structure in two different crystal environments reveals considerable conformational flexibility at the dimer level affecting the substrate-binding cleft, the dimerization interface and the orientation of the C-terminal domains. The opening of the DNA-binding cleft, the orientation of the C-terminal domains relative to the central dimerization domain as well as the relative positioning of helices in the dimerization interface appear to be sensitive to the crystal packing environment. The highly unexpected rearrangement within the extended hydrophobic interface does change the contact surface area but keeps the number of hydrophobic contacts about the same and will therefore not require significant energy input. The conformational flexibility most likely is of functional significance for the broad substrate specificity of EndoVII. Binding of sulphate ions in the mutant structure and their positions relative to the active-site metal ions and residues known to be essential for catalysis allows us to propose a possible catalytic mechanism. A comparison with the active-site geometries of other magnesium-dependent nucleases, among them the homing endonuclease I-PpoI and Serratia endonuclease, shows common features, suggesting related catalytic mechanisms.     

Modeling the active-site structure of the cbb3-type oxidase from Rhodobacter sphaeroides

The active site of the heme-copper oxidases comprises a redox-active high-spin heme and a tris-histidine copper center Cu B. Two amino acids in the close vicinity of the metals, a tyrosine and a tryptophan from helix 6, have been shown to be absolutely required for the catalytic function and should be considered part of the active site. Additionally, amino acid residues from interhelical loops strongly modify the activity. In a separate subfamily of heme-copper oxidases, the cbb 3-type oxidases, the metal centers are identical, the tyrosine is found in helix 7, but nothing is known of the corresponding tryptophan or of the involvement of the loop residues. We have observed a conserved aromatic cluster in the known oxidase structures, including the essential tryptophan and loop residues, and refined our earlier model of the cbb 3-type oxidase from Rhodobacter sphaeroides to test the feasibility of a similar structure. In the refined model, the interactions around the Delta-propionate of the high-spin heme resemble closely those seen in crystal structures of other terminal oxidases. Two alternative models (G- and C-models) that differ for the positioning of conserved tryptophans in helix 6, are presented. Molecular dynamics simulations on the catalytic subunit of the cbb 3-type oxidase model result in a conformational change of the active-site tyrosine, which may be related to different ligand-binding properties of the cbb 3-type oxidases. The relationship between sequence and functional data for defining the subfamily is discussed.     

Dynamic features of cAMP-dependent protein kinase revealed by apoenzyme crystal structure

To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other protein kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.     

Functional and structural changes due to a serine to alanine mutation in the active-site flap of enolase

Crystallographic and kinetic methods have been used to characterize a site-specific variant of yeast enolase in which Ser 39 in the active-site flap has been changed to Ala. In the wild-type enzyme, the carbonyl and hydroxyl groups of Ser 39 chelate the second equivalent of divalent metal ion, effectively anchoring the flap over the fully liganded active site. With Mg(2+) as the activating cation, S39A enolase has <0.01% of wild-type activity as reported previously [J.M. Brewer, C.V. Glover, M.J. Holland, L. Lebioda, Biochim. Biophys. Acta 1383 (2) (1998) 351-355]. Measurements of (2)H kinetic isotope effects indicate that the proton abstraction from 2-phosphoglycerate (2-PGA) is significantly rate determining. Analysis of the isotope effects provides information on the relative rates of formation and breakdown of the enolate intermediate. Moreover, assays with different species of divalent metal ions reveal that with S39A enolase (unlike the case of wild-type enolase), more electrophilic metal ions promote higher activities. The kinetic results with the S39A variant support the notions that a rate-limiting product release lowers the activity of wild-type enolase with more electrophilic metal ions and that the metal ions are used to acidify the C2-proton of 2-PGA. The S39A enolase was co-crystallized with Mg(2+) and the inhibitor phosphonoacetohydroxamate (PhAH). The structure was solved and refined at a resolution of 2.1 A. The structure confirms the conjecture that the active-site flap is opened in the mutant protein. PhAH chelates to both Mg ions as in the corresponding structure of the wild-type complex. Positions of the side chains of catalytic groups, Lys 345 and Glu 211, and of "auxiliary" residues Glu 168 and Lys 396 are virtually unchanged relative to the complex with the wild-type protein. His 159, which hydrogen bonds to the phosphonate oxygens in the wild-type complex, is 5.7 A from the closest phosphonate oxygen, and the loop (154-166) containing His 159 is shifted away from the active center. A peripheral loop, Glu 251-Gly 275, also moves to open access to the active site.     

Structure of a biologically active fragment of human serum apolipoprotein C-II in the presence of sodium dodecyl sulfate and dodecylphosphocholine

We have studied the three-dimensional structure of a biologically active peptide of apolipoprotein C-II (apoC-II) in the presence of lipid mimetics by CD and NMR spectroscopy. This peptide, corresponding to residues 44-79 of apoC-II, has been shown to reverse the symptoms of genetic apoC-II deficiency in a human subject. A comparison of alpha-proton secondary shifts and CD spectroscopic data indicates that the structure of apoC-II(44-79) is similar in the presence of dodecylphosphocholine and sodium dodecyl sulfate. The three-dimensional structure of apoC-II(44-79) in the presence of sodium dodecyl sulfate, determined by relaxation matrix calculations, contains two amphipathic helical domains formed by residues 50-58 and 67-75, separated by a non-helical linker centered at Tyr63. The C-terminal helix is terminated by a loop formed by residues 76-79. The C-terminal helix is better defined and has a larger hydrophobic face than the N-terminal helix, which leads us to propose that the C-terminal helix together with the non-helical Ile66 constitute the primary lipid binding domain of apoC-II(44-79). Based on our structure we suggest a new mechanism of lipoprotein lipase activation in which both helices of apoC-II(44-79) remain lipid bound, while the seven-residue interhelical linker extends away from the lipid surface in order to project Tyr63 into the apoC-II binding site of lipoprotein lipase.     

Identification of four GyrA residues involved in the DNA breakage-reunion reaction of DNA gyrase

DNA supercoiling by DNA gyrase involves the cleavage of a DNA helix, the passage of another helix through the break, and the religation of the first helix. The cleavage-religation reaction involves the formation of a 5'-phosphotyrosine intermediate with the GyrA subunit of the gyrase (A(2)B(2)) complex. We report the characterization of mutations near the active-site tyrosine residue in GyrA predicted to affect the cleavage-religation reaction of gyrase. We find that mutations at Arg32, Arg47, His78 and His80 inhibit DNA supercoiling and other reactions of gyrase. These effects are caused by the involvement of these residues in the DNA cleavage reaction; religation is largely unaffected by these mutations. We show that these residues cooperate with the active-site tyrosine residue on the opposite subunit of the GyrA dimer during the cleavage-religation reaction.     

Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution.

Ubiquitin C-terminal hydrolases catalyze the removal of adducts from the C-terminus of ubiquitin. We have determined the crystal structure of the recombinant human Ubiquitin C-terminal Hydrolase (UCH-L3) by X-ray crystallography at 1.8 A resolution. The structure is comprised of a central antiparallel beta-sheet flanked on both sides by alpha-helices. The beta-sheet and one of the helices resemble the well-known papain-like cysteine proteases, with the greatest similarity to cathepsin B. This similarity includes the UCH-L3 active site catalytic triad of Cys95, His169 and Asp184, and the oxyanion hole residue Gln89. Papain and UCH-L3 differ, however, in strand and helix connectivity, which in the UCH-L3 structure includes a disordered 20 residue loop (residues 147-166) that is positioned over the active site and may function in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain-like enzymes, we propose a model describing the binding of ubiquitin to UCH-L3. The UCH-L3 active site cleft appears to be masked in the unliganded structure by two different segments of the enzyme (residues 9-12 and 90-94), thus implying a conformational change upon substrate binding and suggesting a mechanism to limit non-specific hydrolysis.     

Consolidation of the thioredoxin fold by peptide recognition: interaction between E. coli thioredoxin fragments 1-93 and 94-108

Escherichia coli thioredoxin (TRX) catalyzes redox reactions via the reversible oxidation of the conserved active center WCGPC. TRX is a monomeric alpha/beta protein with a fold characterized by a central beta-sheet surrounded by alpha-helical elements. The interaction of the C-terminal alpha-helix (helix 5) of TRX against the remainder of the protein involves the close packing of hydrophobic surfaces, opening the possibility of studying a fine-tuned molecular recognition phenomenon. To evaluate the relevance of this interaction on the folding mechanism of TRX, we characterize TRX1-93, a truncated variant of TRX devoid of the last stretch of 15 amino acid residues that includes helix 5. TRX1-93 may possibly represent a molecular form where the folding process becomes interrupted, giving rise to a structure exhibiting the features of a molten globule state. This was assessed by circular dichroism, intrinsic fluorescence, binding of the probe ANS, size-exclusion chromatography, limited proteolysis, and calorimetry. Remarkably, fragment TRX1-93 interacts with peptide TRX94-108 (KD approximately 2-12 microM), bringing forth the restoration of native-like signatures and enzymic function. This represents a molecular event of reciprocal structure selection where both partners gain order, thus leading to long-range consequences on conformation. In this context, the binding of the C-terminal helix could signify a late event in the consolidation of the overall TRX fold.     

Solution structure of the catalytic domain of gammadelta resolvase. Implications for the mechanism of catalysis

The site-specific DNA recombinase, gammadelta resolvase, from Escherichia coli catalyzes recombination of res site-containing plasmid DNA to two catenated circular DNA products. The catalytic domain (residues 1-105), lacking a C-terminal dimerization interface, has been constructed and the NMR solution structure of the monomer determined. The RMSD of the NMR conformers for residues 2-92 excluding residues 37-45 and 64-73 is 0.41 A for backbone atoms and 0.88 A for all heavy atoms. The NMR solution structure of the monomeric catalytic domain (residues 1-105) was found to be formed by a four-stranded parallel beta-sheet surrounded by three helices. The catalytic domain (residues 1-105), deficient in the C-terminal dimerization domain, was monomeric at high salt concentration, but displayed unexpected dimerization at lower ionic strength. The unique solution dimerization interface at low ionic strength was mapped by NMR. With respect to previous crystal structures of the dimeric catalytic domain (residues 1-140), differences in the average conformation of active-site residues were found at loop 1 containing the catalytic S10 nucleophile, the beta1 strand containing R8, and at loop 3 containing D67, R68 and R71, which are required for catalysis. The active-site loops display high-frequency and conformational backbone dynamics and are less well defined than the secondary structures. In the solution structure, the D67 side-chain is proximal to the S10 side-chain making the D67 carboxylate group a candidate for activation of S10 through general base catalysis. Four conserved Arg residues can function in the activation of the phosphodiester for nucleophilic attack by the S10 hydroxyl group. A mechanism for covalent catalysis by this class of recombinases is proposed that may be related to dimer interface dissociation.     

Crystal structures of Streptococcus pyogenes Dpr reveal a dodecameric iron-binding protein with a ferroxidase site

DNA-binding protein from starved cells (Dps)-like proteins are key factors involved in oxidative stress protection in bacteria. They bind and oxidize iron, thus preventing the formation of harmful reactive oxygen species that can damage biomolecules, particularly DNA. Dps-like proteins are composed of 12 identical subunits assembled in a spherical structure with a hollow central cavity. The iron oxidation occurs at 12 intersubunit sites located at dimer interfaces. Streptococcus pyogenes Dps-like peroxide resistance protein (Dpr) has been previously found to protect the catalase-lacking S. pyogenes bacterium from oxidative stress. We have determined the crystal structure of S. pyogenes Dpr, the second Dpr structure from a streptococcal bacterium, in iron-free and iron-bound forms at 2.0- and 1.93-Å resolution, respectively. The iron binds to well-conserved sites at dimer interfaces and is coordinated directly to Asp77 and Glu81 from one monomer, His50 from a twofold symmetry-related monomer, a glycerol molecule, and a water molecule. Upon iron binding, Asp77 and Glu81 change conformation. Site-directed mutagenesis of active-site residues His50, His62, Asp66, Asp77, and Glu81 to Ala revealed a dramatic decrease in iron incorporation. A short helix at the N-terminal was found in a different position compared with other Dps-like proteins. Two types of pores were identified in the dodecamer. Although the N-terminal pore was found to be similar to that of other Dps-like proteins, the C-terminal pore was found to be blocked by bulky Tyr residues instead of small residues present in other Dps-like proteins.     

A Model of the Quaternary Structure of Enolases, Based on Structural and Evolutionary Analysis of the Octameric Enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pH_o) for activity is 8.1–8.2, and the K _m for 2-PGA is approximately 0.67 mM. Using the dimeric Cα structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86–96) and the loop between helix L and strand 1 (HL–S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL–S1 loop is truncated by 4–6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL–S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.