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Iron is an essential element for various lifeforms but is largely insoluble due to the oxygenation of Earth’s atmosphere and oceans during the Proterozoic era. Metazoans evolved iron transport glycoproteins, like transferrin (Tf) and lactoferrin (Lf), to keep iron in a non-toxic, usable form, while maintaining a low free iron concentration in the body that is unable to sustain bacterial growth. To survive on the mucosal surfaces of the human respiratory tract where it exclusively resides, the Gram-negative bacterial pathogen Moraxella catarrhalis utilizes surface receptors for acquiring iron directly from human Tf and Lf. The receptors are comprised of a surface lipoprotein to capture iron-loaded Tf or Lf and deliver it to a TonB-dependent transporter (TBDT) for removal of iron and transport across the outer membrane. The subsequent transport of iron into the cell is normally mediated by a periplasmic iron-binding protein and inner membrane transport complex, which has yet to be determined for Moraxella catarrhalis. We identified two potential periplasm to cytoplasm transport systems and performed structural and functional studies with the periplasmic binding proteins (FbpA and AfeA) to evaluate their role. Growth studies with strains deleted in the fbpA or afeA gene demonstrated that FbpA, but not AfeA, was required for growth on human Tf or Lf. The crystal structure of FbpA with bound iron in the open conformation was obtained, identifying three tyrosine ligands that were required for growth on Tf or Lf. Computational modeling of the YfeA homologue, AfeA, revealed conserved residues involved in metal binding.

Succinyl-CoA synthetase (SCS) catalyzes a three-step reaction in the citric acid cycle with succinyl-phosphate proposed as a catalytic intermediate. However, there are no structural data to show the binding of succinyl-phosphate to SCS. Recently, the catalytic mechanism underlying acetyl-CoA production by ATP-citrate lyase (ACLY) has been debated. The enzyme belongs to the family of acyl-CoA synthetases (nucleoside diphosphate-forming) for which SCS is the prototype. It was postulated that the amino-terminal portion catalyzes the full reaction and the carboxy-terminal portion plays only an allosteric role. This interpretation was based on the partial loss of the catalytic activity of ACLY when Glu599 was mutated to Gln or Ala, and on the interpretation that the phospho-citryl-CoA intermediate was trapped in the 2.85 Å resolution structure from cryogenic electron microscopy (cryo-EM). To better resolve the structure of the intermediate bound to the E599Q mutant, the equivalent mutation, E105αQ, was made in human GTP-specific SCS. The structure of the E105αQ mutant shows succinyl-phosphate bound to the enzyme at 1.58 Å resolution when the mutant, after phosphorylation in solution by Mg2+-ATP, was crystallized in the presence of magnesium ions, succinate and desulfo-CoA. The E105αQ mutant is still active but has a specific activity that is 120-fold less than that of the wild-type enzyme, with apparent Michaelis constants for succinate and CoA that are 50-fold and 11-fold higher, respectively. Based on this high-resolution structure, the cryo-EM maps of the E599Q ACLY complex reported previously should have revealed the binding of citryl-phosphate and CoA and not phospho-citryl-CoA.

Succinyl-CoA synthetase (SCS) catalyzes a reversible reaction that is the only substrate-level phosphorylation in the citric acid cycle. One of the essential steps for the transfer of the phosphoryl group involves the movement of the phosphohistidine loop between active site I, where CoA, succinate and phosphate bind, and active site II, where the nucleotide binds. Here, the first crystal structure of SCS revealing the conformation of the phosphohistidine loop in site II of the porcine GTP-specific enzyme is presented. The phosphoryl transfer bridges a distance of 29 Å between the binding sites for phosphohistidine in site I and site II, so these crystal structures support the proposed mechanism of catalysis by SCS. In addition, a second succinate-binding site was discovered at the interface between the α- and β-subunits of SCS, and another magnesium ion was found that interacts with the side chains of Glu141β and Glu204β via water-mediated interactions. These glutamate residues interact with the active-site histidine residue when it is bound in site II.

Succinyl-CoA synthetase (SCS) catalyzes the only substrate-level phosphorylation step in the tricarboxylic acid cycle. Human GTP-specific SCS (GTPSCS), an αβ-heterodimer, was produced inEscherichia coli. The purified protein crystallized from a solution containing tartrate, CoA and magnesium chloride, and a crystal diffracted to 1.52 Å resolution. Tartryl-CoA was discovered to be bound to GTPSCS. The CoA portion lies in the amino-terminal domain of the α-subunit and the tartryl end extends towards the catalytic histidine residue. The terminal carboxylate binds to the phosphate-binding site of GTPSCS.

Hydroxycitrate from the fruit ofGarcinia cambogia[i.e.(2S,3S)-2-hydroxycitrate] is the best-known inhibitor of ATP-citrate lyase. Well diffracting crystals showing how the inhibitor binds to human ATP-citrate lyase were grown by modifying the protein. The protein was modified by introducing cleavage sites forTobacco etch virusprotease on either side of a disordered linker. The protein crystallized consisted of residues 2–425-ENLYFQ and S-488–810 of human ATP-citrate lyase. (2S,3S)-2-Hydroxycitrate binds in the same orientation as citrate, but the citrate-binding domain (residues 248–421) adopts a different orientation with respect to the rest of the protein (residues 4–247, 490–746 and 748–809) from that previously seen. For the first time, electron density was evident for the loop that contains His760, which is phosphorylated as part of the catalytic mechanism. The pro-Scarboxylate of (2S,3S)-2-hydroxycitrate is available to accept a phosphoryl group from His760. However, when co-crystals were grown with ATP and magnesium ions as well as either the inhibitor or citrate, Mg2+-ADP was bound and His760 was phosphorylated. The phosphoryl group was not transferred to the organic acid. This led to the interpretation that the active site is trapped in an open conformation. The strategy of designing cleavage sites to remove disordered residues could be useful in determining the crystal structures of other proteins.

ATP‐citrate lyase (ACLY) catalyzes production of acetyl‐CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)‐terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C‐terminal portion of ACLY, full‐length C. limicola ACLY was cleaved, first non‐specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C‐terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes., Natural Sciences and Engineering Research Council of Canada, Grant/Award Number: Discovery Grant; Spanish Ministry of Science and Innovations, Grant/Award Numbers: BES‐2017‐080368, BIO2015‐64216‐P, MDM2014‐0435; Argonne National Laboratory, Grant/Award Number: DE‐AC02‐06CH11357; Canadian Institutes of Health Research; National Research Council Canada; Western Economic Diversification Canada; Government of Saskatchewan; University of Saskatchewan; Natural Sciences and Engineering Research Council of Canada (NSERC); Canada Foundation for Innovation; Queen Elizabeth II Scholarship; Canada Graduate Scholarship—Master's Program; NSERC

Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of `gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg2+-ADP was determined, as well as the structures of four proteins with single mutations, K46βE, K114βD, V113βL and L227βF, and one with two mutations, K46βE/K114βD. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20β, Gly111β and Val113β. The O atom of the side chain of Gln20β interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111β. It is the different conformations of the backbone at Gln20β, of the side chain of Gln20β and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227βF mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46βE/K114βD double mutant ofBlastocystis hominisSCS binds GTP better than ATP according to kinetic assays, only the complex with Mg2+-ADP was obtained.

Author(s): James, Michael NG; Fraser, Marie E; Khan, Amir R; Parrish, Jonathan C; Smith, Whitney W; Bartlett, Paul A

Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Å resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate.

Substrate initially binds to β-galactosidase (Escherichia coli) at a 'shallow' site. It then moves ∼3Å to a 'deep' site and the transition state forms. Asn460 interacts in both sites, forming a water bridge interaction with the O3 hydroxyl of the galactosyl moiety in the shallow site and a direct H-bond with the O2 hydroxyl of the transition state in the deep site. Structural and kinetic studies were done with β-galactosidases with substitutions for Asn460. The substituted enzymes have enhanced substrate affinity in the shallow site indicating lower E·substrate complex energy levels. They have poor transition state stabilization in the deep site that is manifested by increased energy levels of the E·transition state complexes. These changes in stability result in increased activation energies and lower k(cat) values. Substrate affinity to N460D-β-galactosidase was enhanced through greater binding enthalpy (stronger H-bonds through the bridging water) while better affinity to N460T-β-galactosidase occurred because of greater binding entropy. The transition states are less stable with N460S- and N460T-β-galactosidase because of the weakening or loss of the important bond to the O2 hydroxyl of the transition state. For N460D-β-galactosidase, the transition state is less stable due to an increased entropy penalty.

Catalysis by succinyl-CoA:3-oxoacid CoA transferase proceeds through a thioester intermediate in which CoA is covalently linked to the enzyme. To determine the conformation of the thioester intermediate, crystals of the pig enzyme were grown in the presence of the substrate acetoacetyl-CoA. X-ray diffraction data show the enzyme in both the free form and covalently bound to CoA via Glu305. In the complex, the protein adopts a conformation in which residues 267-275, 280-287, 357-373, and 398-477 have shifted toward Glu305, closing the enzyme around the thioester. Enzymes provide catalysis by stabilizing the transition state relative to complexes with substrates or products. In this case, the conformational change allows the enzyme to interact with parts of CoA distant from the reactive thiol while the thiol is covalently linked to the enzyme. The enzyme forms stabilizing interactions with both the nucleotide and pantoic acid portions of CoA, while the interactions with the amide groups of the pantetheine portion are poor. The results shed light on how the enzyme uses the binding energy for groups remote from the active center of CoA to destabilize atoms closer to the active center, leading to acceleration of the reaction by the enzyme.

Succinyl-CoA synthetase (SCS) is an enzyme of the citric acid cycle and is thus found in most species. To date, there are no structures available of SCS from a thermophilic organism. To investigate how the enzyme adapts to higher temperatures, SCS from Thermus aquaticus was cloned, overexpressed, purified and crystallized. Attempts to crystallize the enzyme were thwarted by proteolysis of the beta-subunit and preferential crystallization of the truncated form. Crystals of full-length SCS were grown after the purification protocol was modified to include frequent additions of protease inhibitors. The resulting crystals, which diffract to 2.35 A resolution, are of the protein in complex with Mn2+-GDP.

It is shown here that Escherichia coli beta-galactosidase has a second Mg2+ binding site that is important for activity. Binding of Mg2+ to the second site caused the k(cat) (with oNPG as the substrate) to increase about 100 s(-1); the Km was not affected. The Kd for binding the second Mg2+ is about 10(-4)M. Since the concentration of free Mg2+ in E. coli is about 1-2 mM, the second site is physiologically significant. Non-polar substitutions (Ala or Leu) for Glu-797, a residue in an active site loop, eliminated the k(cat) increase. This indicates that the second Mg2+ site is near to Glu-797. The Ki values of transition state analogs were decreased by small but statistically significant amounts when the second Mg2+ site was occupied and Arrhenius plots showed that less entropic activation energy is required when the second site is occupied. These inhibitor and temperature results suggest that binding of the second Mg2+ helps to order the active site for stabilization of the transition state.

Pig GTP-specific succinyl-CoA synthetase is an αβ-heterodimer. The crystal structure of the complex with the substrate CoA was determined at 2.1 Å resolution. The structure shows CoA bound to the amino-terminal domain of the α-subunit, with the free thiol extending from the adenine portion into the site where the catalytic histidine residue resides.

Several serotypes of Escherichia coli produce protein toxins closely related to Shiga toxin (Stx) from Shigella dysenteriae serotype 1. These Stx-producing E. coli cause outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in humans, with the latter being more likely if the E. coli produce Stx2 than if they only produce Stx1. To investigate the differences among the Stxs, which are all AB(5) toxins, the crystal structure of Stx2 from E. coli O157:H7 was determined at 1.8-A resolution and compared with the known structure of Stx. Our major finding was that, in contrast to Stx, the active site of the A-subunit of Stx2 is accessible in the holotoxin, and a molecule of formic acid and a water molecule mimic the binding of the adenine base of the substrate. Further, the A-subunit adopts a different orientation with respect to the B-subunits in Stx2 than in Stx, due to interactions between the carboxyl termini of the B-subunits and neighboring regions of the A-subunit. Of the three types of receptor-binding sites in the B-pentamer, one has a different conformation in Stx2 than in Stx, and the carboxyl terminus of the A-subunit binds at another. Any of these structural differences might result in different mechanisms of action of the two toxins and the development of hemolytic uremic syndrome upon exposure to Stx2.

Ketoacidosis affects patients who are deficient in the enzyme activity of succinyl-CoA:3-ketoacid CoA transferase (SCOT), since SCOT catalyses the activation of acetoacetate in the metabolism of ketone bodies. Thus far, structure/function analysis of the mammalian enzyme has been predicted based on the three-dimensional structure of a CoA transferase determined from an anaerobic bacterium that utilizes its enzyme for glutamate fermentation. To better interpret clinical data, we have determined the structure of a mammalian CoA transferase from pig heart by X-ray crystallography to 2.5 A resolution. Instrumental to the structure determination were selenomethionine substitution and the use of argon during purification and crystallization. Although pig heart SCOT adopts an alpha/beta protein fold, resembling the overall fold of the bacterial CoA transferase, several loops near the active site of pig heart SCOT follow different paths than the corresponding loops in the bacterial enzyme, accounting for differences in substrate specificities. Two missense mutations found associated with SCOT of ketoacidosis patients were mapped to a location in the structure that might disrupt the stabilization of the amino-terminal strand and thereby interfere with the proper folding of the protein into a functional enzyme.

Pig heart CoA transferase (EC 2.8.3.5) has been shown previously to adopt a homodimeric structure, in which each subunit has a molecular weight of 52 197 and consists of N- and C-domains linked by a hydrophilic linker or "hinge". Here we identify and characterize a second oligomeric form constituent in purified enzyme preparations, albeit at low concentrations. Both species catalyze the transfer of CoA with similar values for k(cat) and K(M). This second form sediments more rapidly than the homodimer under the conditions of conventional sedimentation velocity and active enzyme centrifugation. Apparent molecular weight values determined by sedimentation equilibrium and gel filtration chromatography are 4-fold greater than the subunit molecular weight, confirming that this form is a homotetramer. The subunits of both oligomeric forms are indistinguishable with respect to molecular mass, far-UV CD, intrinsic tryptophan fluorescence, and equilibrium unfolding. Dissociation of the homotetramer to the homodimer occurs very slowly in benign solutions containing high salt concentrations (0.25-2.0 M KCl). The homotetramer is fully converted to homodimer during refolding from denaturant at low protein concentrations. Disruption of the hydrophilic linker between the N- and C-domains by mutagenesis or mild proteolysis causes a decrease in the relative amount of the larger conformer. The homotetramer is stabilized by interactions involving the helical hinge region, and a substantial kinetic barrier hinders interconversion of the two oligomeric species under nondenaturing conditions.

Succinyl-CoA synthetase (SCS) carries out the substrate-level phosphorylation of GDP or ADP in the citric acid cycle. A molecular model of the enzyme from Escherichia coli, crystallized in the presence of CoA, has been refined against data collected to 2.3 A resolution. The crystals are of space group P4322, having unit cell dimensions a = b = 98.68 A, c = 403.76 A and the data set includes the data measured from 23 crystals. E. coli SCS is an (αβ)2-tetramer; there are two copies of each subunit in the asymmetric unit of the crystals. The crystal packing leaves two choices for which pair of αβ-dimers form the physiologically relevant tetramer. The copies of the αβ-dimer are similar, each having one active site where the phosphorylated histidine residue and the thiol group of CoA are found. CoA is bound in an extended conformation to the nucleotide-binding motif in the N-terminal domain of the α-subunit. The phosphoryl group of the phosphorylated histidine residue is positioned at the amino termini of two α-helices, one from the C-terminal domain of the α-subunit and the other from the C-terminal domain of the β-subunit. These two domains have similar topologies, despite only 14% sequence identity. By analogy to other nucleotide-binding proteins, the binding site for the nucleotide may reside in the N-terminal domain of the β-subunit. If this is so, the catalytic histidine residue would have to move about 35 A to react with the nucleotide.

Succinyl-CoA synthetase (SCS) catalyzes the substrate-level phosphorylation step of the citric acid cycle. The enzyme from Escherichia coli is an (αβ)2-heterotetramer with two active sites, one in each αβ-dimer. To determine whether the two active sites could function independently, mutations were made to split the tetramer into αβ-dimers. Because two choices for the tetramer (I and II) were possible from the X-ray crystallographic analyses, mutations were made at two different interfaces. All mutations based on tetramer I resulted in an intact tetramer. Of the two mutants based on tetramer II, one was insoluble and the other, where M156β, Y158β, R161β and E162β were changed to D, D, E and R, respectively, was a dimer. This quaternary structure was confirmed by fast protein liquid chromatography, blue native PAGE and ultracentrifugation. The DDER mutant has kinetic parameters similar to the tetrameric E. coli enzyme. Like the tetrameric enzyme, it shows ATP-facilitated dethiophosphorylation, proving that this property is a single-site effect. The ATP-facilitated dethiophosphorylation is inhibited by phosphate. It is concluded that dimerization of αβ-dimers is not a prerequisite for catalytic competency nor for alternating sites cooperativity in the tetramer. The rationale behind the dimer-of-dimers in E. coli SCS is still not known, but increased solubility, increased stability and in vivo interactions of the tetramer with other proteins are still possibilities.

Macrocyclic inhibitor 1 {methyl [cyclo-7[(2R)-((N-valyl) amino)-2-(hydroxyl-(1S)-1-methyoxycarbonyl-2-phenylethoxy)phosphinyloxyethyl]-1-naphthaleneacetamide] sodium salt} was designed according to the conformation of the acyclic analogue Iva-l-Val-l-Val-l-LeuP-(O)Phe-OMe [LeuP = the phosphinic acid and analogue of l-leucine; (O)Phe = l-3-phenyllactic acid; OMe = methyl ester] (4) bound to penicillopepsin, by linking the P1 and P3 side chains of the penicillopepsin inhibitor. This compound and its two acyclic derivatives, {methyl (2S)-[1-(((N-Formyl)-l-valyl)amino-2-(2-naphthyl)ethyl)hydroxyphosphinyloxy]-3-phenylpropanoate, sodium salt} (2) and {methyl (2S)-[1-(((N-(1-naphthaleneacetyl))-l-valyl)aminomethyl)hydroxyphosphinyloxy]-3-phenylpropanoate, sodium salt} (3), have been synthesized and evaluated as inhibitors of penicillopepsin. Their binding affinity to the enzyme was found to be inversely related to the predicted degree of conformational flexibility across the series: 3 (Ki = 110 μM), 2 (Ki = 7....

Mutations in the erythroid-specific aminolevulinic acid synthase gene (ALAS2) cause X-linked sideroblastic anemia (XLSA) by reducing mitochondrial enzymatic activity. Surprisingly, a patient with the classic XLSA phenotype had a novel exon 11 mutation encoding a recombinant enzyme (p.Met567Val) with normal activity, kinetics, and stability. Similarly, both an expressed adjacent XLSA mutation, p.Ser568Gly, and a mutation (p.Phe557Ter) lacking the 31 carboxyl-terminal residues also had normal or enhanced activity, kinetics, and stability. Because ALAS2 binds to the β subunit of succinyl-CoA synthetase (SUCLA2), the mutant proteins were tested for their ability to bind to this protein. Wild type ALAS2 bound strongly to a SUCLA2 affinity column, but the adjacent XLSA mutant enzymes and the truncated mutant did not bind. In contrast, vitamin B6-responsive XLSA mutations p.Arg452Cys and p.Arg452His, with normal in vitro enzyme activity and stability, did not interfere with binding to SUCLA2 but instead had loss of positive cooperativity for succinyl-CoA binding, an increased K(m) for succinyl-CoA, and reduced vitamin B6 affinity. Consistent with the association of SUCLA2 binding with in vivo ALAS2 activity, the p.Met567GlufsX2 mutant protein that causes X-linked protoporphyria bound strongly to SUCLA2, highlighting the probable role of an ALAS2-succinyl-CoA synthetase complex in the regulation of erythroid heme biosynthesis.

Succinyl-CoA synthetase (SCS) from Thermus aquaticus was characterized biochemically via measurements of the activity of the enzyme and determination of its quaternary structure as well as its stability and refolding properties. The enzyme is most active between pH 8.0 and 8.4 and its activity increases with temperature to about 339 K. Gel-filtration chromatography and sedimentation equilibrium under native conditions demonstrated that the enzyme is a heterotetramer of two α-subunits and two β-subunits. The activity assays showed that the enzyme uses either ADP/ATP or GDP/GTP, but prefers GDP/GTP. This contrasts with Escherichia coli SCS, which uses GDP/GTP but prefers ADP/ATP. To understand the nucleotide preference, T. aquaticus SCS was crystallized in the presence of GDP, leading to the determination of the structure in complex with GDP-Mn(2+). A water molecule and Pro20β in T. aquaticus take the place of Gln20β in pig GTP-specific SCS, interacting well with the guanine base and other residues of the nucleotide-binding site. This leads to the preference for GDP/GTP, but does not hinder the binding of ADP/ATP.

Tetraloop-receptor interactions are prevalent structural units in RNAs, and include the GAAA/11-nt and GNRA-minor groove interactions. In this study, we have compiled a set of 78 nonredundant loop-helix interactions from X-ray crystal structures, and examined them for the extent of their sequence and structural variation. Of the 78 interactions in the set, only four were classical GAAA/11-nt motifs, while over half (48) were GNRA-minor groove interactions. The GNRA-minor groove interactions were not a homogeneous set, but were divided into five subclasses. The most predominant subclass is characterized by two triple base pair interactions in the minor groove, flanked by two ribose zipper contacts. This geometry may be considered the “standard” GNRA-minor groove interaction, while the other four subclasses are alternative ways to form interfaces between a minor groove and tetraloop. The remaining 26 structures in the set of 78 have loops interacting with mostly idiosyncratic receptors. Among the entire set, a number of sequence-structure correlations can be identified, which may be used as initial hypotheses in predicting three-dimensional structures from primary sequences. Conversely, other sequence patterns are not predictive; for example, GAAA loop sequences and GG/CC receptors bind to each other with three distinct geometries. Finally, we observe an example of structural evolution in group II introns, in which loop-receptor motifs are substituted for each other while maintaining the larger three-dimensional geometry. Overall, the study gives a more complete view of RNA loop-helix interactions that exist in nature.

A loop (residues 794–803) at the active site of β-galactosidase (Escherichia coli) opens and closes during catalysis. The α and β carbons of Ser-796 form a hydrophobic connection to Phe-601 when the loop is closed while a connection via two H-bonds with the Ser hydroxyl occurs with the loop open. β-Galactosidases with substitutions for Ser-796 were investigated. Replacement by Ala strongly stabilizes the closed conformation because of greater hydrophobicity and loss of H-bonding ability while replacement with Thr stabilizes the open form through hydrophobic interactions with its methyl group. Upon substitution with Asp much of the defined loop structure is lost. The different open-closed equilibria cause differences in the stabilities of the enzyme · substrate and enzyme · transition state complexes and of the covalent intermediate that affect the activation thermodynamics. With Ala, large changes of both the galactosylation (k2) and degalactosylation (k3) rates occur. With Thr and Asp, the k2 and k3 were not changed as much but large ΔH‡ and TΔS‡ changes showed that the substitutions caused mechanistic changes. Overall, the hydrophobic and H-bonding properties of Ser-796 result in interactions strong enough to stabilize the open or closed conformations of the loop but weak enough to allow loop movement during the reaction.

Human ATP-citrate lyase (EC 2.3.3.8) is the cytoplasmic enzyme that catalyzes the production of acetyl-CoA from citrate, CoA and ATP. The amino-terminal portion of the enzyme, containing residues 1-817, was crystallized in the presence of tartrate, ATP and magnesium ions. The crystals diffracted to 2.3 A resolution. The structure shows ADP-Mg(2+) bound to the domain that possesses the ATP-grasp fold. The structure demonstrates that this crystal form could be used to investigate the structures of complexes with inhibitors of ATP-citrate lyase that bind at either the citrate- or ATP-binding site.

ATP-citrate lyase (ACLY) catalyzes the conversion of citrate and CoA into acetyl-CoA and oxaloacetate, coupled with the hydrolysis of ATP. In humans, ACLY is the cytoplasmic enzyme linking energy metabolism from carbohydrates to the production of fatty acids. In situ proteolysis of full-length human ACLY gave crystals of a truncated form, revealing the conformations of residues 2-425, 487-750, and 767-820 of the 1101-amino acid protein. Residues 2-425 form three domains homologous to the beta-subunit of succinyl-CoA synthetase (SCS), while residues 487-820 form two domains homologous to the alpha-subunit of SCS. The crystals were grown in the presence of tartrate or the substrate, citrate, and the structure revealed the citrate-binding site. A loop formed by residues 343-348 interacts via specific hydrogen bonds with the hydroxyl and carboxyl groups on the prochiral center of citrate. Arg-379 forms a salt bridge with the pro-R carboxylate of citrate. The pro-S carboxylate is free to react, providing insight into the stereospecificity of ACLY. Because this is the first structure of any member of the acyl-CoA synthetase (NDP-forming) superfamily in complex with its organic acid substrate, locating the citrate-binding site is significant for understanding the catalytic mechanism of each member, including the prototype SCS. Comparison of the CoA-binding site of SCSs with the similar structure in ACLY showed that ACLY possesses a different CoA-binding site. Comparisons of the nucleotide-binding site of SCSs with the similar structure in ACLY indicates that this is the ATP-binding site of ACLY.

The molecular structures of three phosphorus-based peptide inhibitors of aspartyl proteinases complexed with penicillopepsin [1, Iva-L-Val-L-Val-StaPOEt [Iva = isovaleryl, StaP = the phosphinic acid analogue of statine [(S)-4-amino-(S)-3-hydroxy-6-methylheptanoic acid] (IvaVVStaPOEt)]; 2, Iva-L-Val-L-Val-L-LeuP-(O)Phe-OMe [LeuP = the phosphinic acid analogue of L-leucine; (O)Phe = L-3-phenyllactic acid; OMe = methyl ester] [Iva VVLP(O)FOMe]; and 3, Cbz-L-Ala-L-Ala-L-LeuP-(O)-Phe-OMe (Cbz = benzyloxycarbonyl) [CbzAALP(O)FOMe]] have been determined by X-ray crystallography and refined to crystallographic agreement factors, R ( = sigma parallel to F0 magnitude of - Fc parallel to/sigma magnitude of F0), of 0.132, 0.131, and 0.134, respectively. These inhibitors were designed to be structural mimics of the tetrahederal transition-state intermediate encountered during aspartic proteinase catalysis. They are potent inhibitors of penicillopepsin with Ki values of 1, 22 nM; 2, 2.8 nM; and 3, 1600 nM, respectively [Bartlett, P. A., Hanson, J. E., & Giannousis, P. P. (1990) J. Org. Chem. 55, 6268-6274]. All three of these phosphorus-based inhibitors bind virtually identically in the active site of penicillopepsin in a manner that closely approximates that expected for the transition state [James, M. N. G., Sielecki, A.R., Hayakawa, K., & Gelb, M. H. (1992) Biochemistry 31, 3872-3886]. The pro-S oxygen atom of the two phosphonate inhibitors and of the phosphinate group of the StaP inhibitor make very short contact distances (approximately 2.4 A) to the carboxyl oxygen atom, O delta 1, of Asp33 on penicillopepsin. We have interpreted this distance and the stereochemical environment of the carboxyl and phosphonate groups in terms of a hydrogen bond that most probably has a symmetric single-well potential energy function. The pro-R oxygen atom is the recipient of a hydrogen bond from the carboxyl group of Asp213. Thus, we are able to assign a neutral status to Asp213 and a partially negatively charged status to Asp33 with reasonable confidence. Similar very short hydrogen bonds involving the active site glutamic acid residues of thermolysin and carboxypeptidase A and the pro-R oxygen of bound phosphonate inhibitors have been reported [Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H., & Matthews, B. W. (1987) Biochemistry 26, 8542-8553; Kim, H., & Lipscomb, W. N. (1991) Biochemistry 30, 8171-8180].(ABSTRACT TRUNCATED AT 400 WORDS)

Recent X-ray crystal structures and solution NMR spectroscopy data for calcium- and integrin-binding protein 1 (CIB1) have all revealed a common EF-hand domain structure for the protein. However, the orientation of the two protein domains, the oligomerization state, and the conformations of the N- and C-terminal extensions differ among the structures. In this study, we examine whether the binding of glutathione or auxiliary Ca(2+) ions as observed in the crystal structures, occur in solution, and whether these interactions can influence the structure or dimerization of CIB1. In addition, we test the potential phosphatase activity of CIB1, which was hypothesized based on the glutathione binding site geometry observed in one of the crystal structures of the protein. Biophysical and biochemical experiments failed to detect glutathione binding, protein dimerization, or phosphatase activity for CIB1 under several solution conditions. However, our data identify low affinity (K(d), 10(-2)M) Ca(2+) binding events that influence the structures of the N- and C-terminal extensions of CIB1 under high (300 mM) Ca(2+) crystallization conditions. In addition to providing a rationale for differences amongst the various solution and crystal structures of CIB1, our results show that the impact of low affinity Ca(2+) binding events should be considered when analyzing and interpreting protein crystallographic structures determined in the presence of very high Ca(2+) concentrations.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) transfers CoA from succinyl-CoA to acetoacetate via a thioester intermediate with its active site glutamate residue, Glu 305. When CoA is linked to the enzyme, a cysteine residue can now be rapidly modified by 5,5'-dithiobis(2-nitrobenzoic acid), reflecting a conformational change of SCOT upon formation of the thioester. Since either Cys 28 or Cys 196 could be the target, each was mutated to Ser to distinguish between them. Like wild-type SCOT, the C196S mutant protein was modified rapidly in the presence of acyl-CoA substrates. In contrast, the C28S mutant protein was modified much more slowly under identical conditions, indicating that Cys 28 is the residue exposed on binding CoA. The specific activity of the C28S mutant protein was unexpectedly lower than that of wild-type SCOT. X-ray crystallography revealed that Ser adopts a different conformation than the native Cys. A chloride ion is bound to one of four active sites in the crystal structure of the C28S mutant protein, mimicking substrate, interacting with Lys 329, Asn 51, and Asn 52. On the basis of these results and the studies of the structurally similar CoA transferase from Escherichia coli, YdiF, bound to CoA, the conformational change in SCOT was deduced to be a domain rotation of 17 degrees coupled with movement of two loops: residues 321-329 that bury Cys 28 and interact with succinate or acetoacetate and residues 374-386 that interact with CoA. Modeling this conformational change has led to the proposal of a new mechanism for catalysis by SCOT.

Succinyl-CoA synthetase has a highly conserved cysteine residue, Cys123alpha in the Escherichia coli enzyme, that is located near the CoA-binding site and the active-site histidine residue. To test whether the succinyl moiety of succinyl-CoA is transferred to the thiol of Cys123alpha as part of the catalytic mechanism, this residue was mutated to alanine, serine, threonine and valine. Each mutant protein was catalytically active, although less active than the wild type. This proved that the specific formation of a thioester bond with Cys123alpha is not part of the catalytic mechanism. To understand why the mutations affected catalysis, the crystal structures of the four mutant proteins were determined. The alanine mutant showed no structural changes yet had reduced activity, suggesting that the size of the cysteine is important for optimal activity. These results explain why this cysteine residue is conserved in the sequences of succinyl-CoA synthetases from different sources.

Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active as an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to approximately 1.8 angstroms and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA.

Two isoforms of succinyl-CoA synthetase exist in mammals, one specific for ATP and the other for GTP. The GTP-specific form of pig succinyl-CoA synthetase has been crystallized in the presence of GTP and the structure determined to 2.1 A resolution. GTP is bound in the ATP-grasp domain, where interactions of the guanine base with a glutamine residue (Gln-20beta) and with backbone atoms provide the specificity. The gamma-phosphate interacts with the side chain of an arginine residue (Arg-54beta) and with backbone amide nitrogen atoms, leading to tight interactions between the gamma-phosphate and the protein. This contrasts with the structures of ATP bound to other members of the family of ATP-grasp proteins where the gamma-phosphate is exposed, free to react with the other substrate. To test if GDP would interact with GTP-specific succinyl-CoA synthetase in the same way that ADP interacts with other members of the family of ATP-grasp proteins, the structure of GDP bound to GTP-specific succinyl-CoA synthetase was also determined. A comparison of the conformations of GTP and GDP shows that the bases adopt the same position but that changes in conformation of the ribose moieties and the alpha- and beta-phosphates allow the gamma-phosphate to interact with the arginine residue and amide nitrogen atoms in GTP, while the beta-phosphate interacts with these residues in GDP. The complex of GTP with succinyl-CoA synthetase shows that the enzyme is able to protect GTP from hydrolysis when the active-site histidine residue is not in position to be phosphorylated.

Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 A resolution, respectively. YdiF is organized into tetramers, with each monomer having an open {alpha}/{beta} structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent {gamma}-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases

Succinyl-CoA:3-ketoacid CoA transferase (SCOT; EC 2.8.3.5) activates the acetoacetate in ketone bodies by transferring the CoA group from succinyl-CoA to acetoacetate to produce acetoacetyl-CoA and succinate. In the reaction, a glutamate residue at the active site of the enzyme forms a thioester bond with CoA and in this form the enzyme is subject to autolytic fragmentation. The crystal structure of pig heart SCOT has been solved and refined to 1.7 A resolution in a new crystal form. The structure shows the active-site glutamate residue in a conformation poised for autolytic fragmentation, with its side chain accepting one hydrogen bond from Asn281 and another from its own amide N atom. However, the conformation of this glutamate side chain would have to change for the residues that are conserved in the CoA transferases (Gln99, Gly386 and Ala387) to participate in stabilizing the tetrahedral transition states of the catalytic mechanism. The structures of a deletion mutant in two different crystal forms were also solved.

Succinyl-CoA synthetase catalyzes the reversible reaction succinyl-CoA + NDP + P(i)--succinate + CoA + NTP (N denoting adenosine or guanosine). The enzyme consists of two different subunits, designated alpha and beta. During the reaction, a histidine residue of the alpha-subunit is transiently phosphorylated. This histidine residue interacts with Glu 208 alpha at site I in the structures of phosphorylated and dephosphorylated Escherichia coli SCS. We postulated that Glu 197 beta, a residue in the nucleotide-binding domain, would provide similar stabilization of the histidine residue during the actual phosphorylation/dephosphorylation by nucleotide at site II. In this work, these two glutamate residues have been mutated individually to aspartate or glutamine. Glu 197 beta has been additionally mutated to alanine. The mutant proteins were tested for their ability to be phosphorylated in the forward or reverse direction. The aspartate mutant proteins can be phosphorylated in either direction, while the E208 alpha Q mutant protein can only be phosphorylated by NTP, and the E197 beta Q mutant protein can only be phosphorylated by succinyl-CoA and P(i). These results demonstrate that the length of the side chain at these positions is not critical, but that the charge is. Most significantly, the E197 beta A mutant protein could not be phosphorylated in either direction. Its crystal structure shows large differences from the wild-type enzyme in the conformation of two residues of the alpha-subunit, Cys 123 alpha-Pro 124 alpha. We postulate that in this conformation, the protein cannot productively bind succinyl-CoA for phosphorylation via succinyl-CoA and P(i).

The protein toxin produced by Shigella dysenteriae consists of one enzymatically active A subunit of 293 amino acid residues and five B subunits of 69 amino acid residues that are involved with cell attachment. The holotoxin has been purified by blue Sepharose and chromatofocusing column chromatography. Two crystal forms of purified holotoxin have been grown by vapor diffusion. One grows as fine needles, hexagonal in cross-section, which do not diffract well enough to characterize crystallographically. The second grows as thin plates that diffract to at least 3 A resolution. Their space group is P2(1)2(1)2(1) with unit cell dimensions of a = 132.0 A, b = 146.0 A and c = 82.5 A. The asymmetric unit of the crystals is likely to contain two AB5 units.

An enzyme in the human body that regulates lipogenesis and cholesterolgenesis is ATP-citrate lyase (ACLY) [1]. ACLY synthesizes acetyl-CoA and oxaloacetate from citrate, CoA and ATP, with citryl-CoA as an intermediate [1]. The product acetyl-CoA is involved in cell growth as well as embryonic and brain development [1]. Studies in mice suggest that ACLY is important during brain development as homozygous Acly knockout mouse embryos died early in development [2]. Knowledge of the reaction mechanism is limited and will be of use in understanding the energy flow in cells. Our current insight into the mechanism by which citryl-CoA is cleaved to form acetyl-CoA and oxaloacetate is based on sequence similarity of ACLY to citrate synthase (CS). Both enzymes have histidine and aspartic acid residues at similar positions in their sequences. We hypothesize that citryl-CoA binds at this site in ACLY and is cleaved into acetyl-CoA and oxaloacetate using these residues. To test this hypothesis, we have mutated these residues to alanine in both human ACLY and ACLY from the bacterium, Chlorobium limicola. Enzymatic activities of the mutant proteins were tested using a coupled-enzyme assay with malate dehydrogenase. The inactive mutants are being used in crystallization trials with substrates, since complexes of the intermediate citryl-CoA can be trapped on the protein. To date, the crystal structure of full-length ACLY has not been published. The structure of only the amino-terminal two-thirds of the human enzyme has been determined [3]; however, the part of the protein that is similar to CS is the carboxy-terminal portion. This work identifies the catalytic residues of ACLY and complements the previous structure determination, increasing our currently limited knowledge about this enzyme.

Succinyl-CoA synthetase (SCS) exists in the mitochondria of mammals as two different isoforms; one is ATP-specific and the other is GTP-specific. SCS is a heterodimer, and the two isoforms have a common α-subunit, but different β-subunits [1]. The β-subunit determines nucleotide specificity. Mutations in the α-subunit or the ATP-specific β-subunit can cause encephalomyopathy due to mitochondrial DNA depletion, along with lactic acidosis and methylmalonic aciduria (reviewed in [2]). The reaction catalyzed by SCS, succinyl-CoA+ NDP + Pi⇌succinate +CoA + NTP, is reversible, and the direction depends on the relative concentrations of substrates and products. Only after all substrate-binding sites are discovered can the catalytic mechanism of SCS be fully understood. Structures of SCS with ADP, GDP, GTP, Pi and CoA have been determined, but the succinate-binding site, or the binding site for the succinyl-portion of succinyl-CoA, is still unknown. Succinate is predicted to bind to the conserved sequence Gly-Gly-Ile-Val (327β-330β) located in a loop of the β-subunit of GTP-specific SCS. Crystals of other complexes with pig GTP-specific SCS have diffracted well, so we are crystallizing this enzyme in complex with succinate. Initially, plasmid containing the genes encoding pig GTP-specific SCS was transformed into E coli. After overproducing the desired protein with a 6-His tag on the C-terminus of the α-subunit, three different purification columns were used to obtain the GTP-SCS protein at high purity. Succinate was then co-crystallized with GTP-SCS under conditions containing polyethylene glycol 3350, magnesium formate and HEPES, pH 7.0.

Succinyl-CoA synthetase (SCS) catalyzes the reversible phosphorylation/dephosphorylation reaction:???rm succinyl ?hbox ?-?CoA+NDP+P_i?leftrightarrow succinate+CoA+NTP??where N denotes adenosine or guanosine. In the course of the reaction, an essential histidine residue is transiently phosphorylated. We have crystallized and solved the structure of the GTP-specific isoform of SCS from pig heart (EC 6.2.1.4) in both the dephosphorylated and phosphorylated forms. The structures were refined to 2.1 A resolution. In the dephosphorylated structure, the enzyme is stabilized via coordination of a phosphate ion by the active-site histidine residue and the two "power" helices, one contributed by each subunit of the alphabeta-dimer. Small changes in the conformations of residues at the amino terminus of the power helix contributed by the alpha-subunit allow the enzyme to accommodate either the covalently bound phosphoryl group or the free phosphate ion. Structural comparisons are made between the active sites in these two forms of the enzyme, both of which can occur along the catalytic path. Comparisons are also made with the structure of Escherichia coli SCS. The domain that has been shown to bind ADP in E. coli SCS is more open in the pig heart, GTP-specific SCS structure.

Succinyl-CoA synthetase (SCS) catalyzes the following reversible reaction via a phosphorylated histidine intermediate (His 246alpha): succinyl-CoA + P(i) + NDP succinate + CoA + NTP (N denotes adenosine or guanosine). To determine the structure of the enzyme with nucleotide bound, crystals of phosphorylated Escherichia coli SCS were soaked in successive experiments adopting progressive strategies. In the first experiment, 1 mM ADP (>15 x K(d)) was added; Mg(2+) ions were omitted to preclude the formation of an insoluble precipitate with the phosphate and ammonium ions. X-ray crystallography revealed that the enzyme was dephosphorylated, but the nucleotide did not remain bound to the enzyme (R(working) = 17.2%, R(free) = 22.8% for data to 2.9 A resolution). Catalysis requires Mg(2+) ions; hence, the "true" nucleotide substrate is probably an ADP-Mg(2+) complex. In the successful experiment, the phosphate buffer was exchanged with MOPS, the concentration of sulfate ions was lowered, and the concentrations of ADP and Mg(2+) ions were increased to 10.5 and 50 mM, respectively. X-ray diffraction data revealed an ADP-Mg(2+) complex bound in the ATP-grasp fold of the N-terminal domain of each beta-subunit (R(working) = 19.1%, R(free) = 24.7% for data to 3.3 A resolution). We describe the specific interactions of the nucleotide-Mg(2+) complex with SCS, compare these results with those for other proteins containing the ATP-grasp fold, and present a hypothetical model of the histidine-containing loop in the "down" position where it can interact with the nucleotide approximately 35 A from where His 246alpha is seen in both phosphorylated and dephosphorylated SCS.