Molecular mechanism for the regulation of human mitochondrial NAD(P)+-dependent malic enzyme by ATP and fumarate

The regulation of human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) by ATP and fumarate may be crucial for the metabolism of glutamine for energy production in rapidly proliferating tissues and tumors. Here we report the crystal structure at 2.2 A resolution of m-NAD-ME in complex with ATP, Mn2+, tartronate, and fumarate. Our structural, kinetic, and mutagenesis studies reveal unexpectedly that ATP is an active-site inhibitor of the enzyme, despite the presence of an exo binding site. The structure also reveals the allosteric binding site for fumarate in the dimer interface. Mutations in this binding site abolished the activating effects of fumarate. Comparison to the structure in the absence of fumarate indicates a possible molecular mechanism for the allosteric function of this compound.     

Dual functional roles of ATP in the human mitochondrial malic enzyme

Human mitochondrial malic enzyme is a regulatory enzyme with ATP as an inhibitor. Structural studies reveal that the enzyme has two ATP-binding sites, one at the NAD(+)-binding site in the active center and the other at the exo site in the tetramer interface. Inhibition of the enzyme activity is due to the competition between ATP and NAD(+) for the nucleotide-binding site at the active center with an inhibition constant of 81 microM. Binding of the ATP molecule at the exo site, on the other hand, is important for the maintenance of the quaternary structural integrity. The enzyme exists in solution at neutral pH and at equilibrium of the dimer and tetramer with a dissociation constant (K(TD)) of 0.67 microM. ATP, at a physiological concentration, shifts the equilibrium toward tetramer and decreases the K(TD) by many orders of magnitude. Mutation of a single residue Arg542 at the tetrameric interfacial exo site resulted in dimeric mutants. ATP thus has dual functional roles in the mitochondrial malic enzyme.     

Structural characteristics of the nonallosteric human cytosolic malic enzyme

Human cytosolic NADP⁺-dependent malic enzyme (c-NADP-ME) is neither a cooperative nor an allosteric enzyme, whereas mitochondrial NAD(P)⁺-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by fumarate. This study examines the molecular basis for the different allosteric properties and quaternary structural stability of m-NAD(P)-ME and c-NADP-ME. Multiple residues corresponding to the fumarate-binding site were mutated in human c-NADP-ME to correspond to those found in human m-NAD(P)-ME. Additionally, the crystal structure of the apo (ligand-free) human c-NADP-ME conformation was determined. Kinetic studies indicated no significant difference between the wild-type and mutant enzymes in K_m,NADP, K_m,malate, and k_cat. A chimeric enzyme, [51-105]_c-NADP-ME, was designed to include the putative fumarate-binding site of m-NAD(P)-ME at the dimer interface of c-NADP-ME; however, this chimera remained nonallosteric. In addition to fumarate activation, the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME is quite different; c-NADP-ME is a stable tetramer, whereas m-NAD(P)-ME exists in equilibrium between a dimer and a tetramer. The quaternary structures for the S57K/N59E/E73K/S102D and S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G mutants of c-NADP-ME are tetrameric, whereas the K57S/E59N/K73E/D102S m-NAD(P)-ME quadruple mutant is primarily monomeric with some dimer formation. These results strongly suggest that the structural features near the fumarate-binding site and the dimer interface are highly related to the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME. In this study, we attempt to delineate the structural features governing the fumarate-induced allosteric activation of malic enzyme.     

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases.

BACKGROUND: Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate and CO2 with the concomitant reduction of NAD(P)+ to NAD(P)H. They are widely distributed in nature and have important biological functions. Human mitochondrial NAD(P)+-dependent malic enzyme (mNAD-ME) may have a crucial role in the metabolism of glutamine for energy production in rapidly dividing cells and tumors. Moreover, this isoform is unique among malic enzymes in that it is a cooperative enzyme, and its activity is controlled allosterically. RESULTS: The crystal structure of human mNAD-ME has been determined at 2.5 A resolution by the selenomethionyl multiwavelength anomalous diffraction method and refined to 2.1 A resolution. The structure of the monomer can be divided into four domains; the active site of the enzyme is located in a deep cleft at the interface between three of the domains. Three acidic residues (Glu255, Asp256 and Asp279) were identified as ligands for the divalent cation that is required for catalysis by malic enzymes. CONCLUSIONS: The structure reveals that malic enzymes belong to a new class of oxidative decarboxylases. The tetramer of the enzyme appears to be a dimer of dimers. The active site of each monomer is located far from the tetramer interface. The structure also shows the binding of a second NAD+ molecule in a pocket 35 A away from the active site. The natural ligand for this second binding site may be ATP, an allosteric inhibitor of the enzyme.     

Effects of protein-ligand associations on the subunit interactions of phosphofructokinase from B. stearothermophilus

Differences between the crystal structures of inhibitor-bound and uninhibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphoenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits simultaneously containing two different extrinsic fluorophores in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel fluorescence correlation spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor binding because binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated.     

The crystal structures of chloramphenicol phosphotransferase reveal a novel inactivation mechanism

Chloramphenicol (Cm), produced by the soil bacterium Streptomyces venezuelae, is an inhibitor of bacterial ribosomal peptidyltransferase activity. The Cm-producing streptomycete modifies the primary (C-3) hydroxyl of the antibiotic by a novel Cm-inactivating enzyme, chloramphenicol 3-O-phosphotransferase (CPT). Here we describe the crystal structures of CPT in the absence and presence of bound substrates. The enzyme is dimeric in a sulfate-free solution and tetramerization is induced by ammonium sulfate, the crystallization precipitant. The tetrameric quaternary structure exhibits crystallographic 222 symmetry and has ATP binding pockets located at a crystallographic 2-fold axis. Steric hindrance allows only one ATP to bind per dimer within the tetramer. In addition to active site binding by Cm, an electron-dense feature resembling the enzyme's product is found at the other subunit interface. The structures of CPT suggest that an aspartate acts as a general base to accept a proton from the 3-hydroxyl of Cm, concurrent with nucleophilic attack of the resulting oxyanion on the gamma-phosphate of ATP. Comparison between liganded and substrate-free CPT structures highlights side chain movements of the active site's Arg136 guanidinium group of >9 A upon substrate binding.     

Functional roles of ATP-binding residues in the catalytic site of human mitochondrial NAD(P)+-dependent malic enzyme

Human mitochondrial NAD(P)+-dependent malic enzyme is inhibited by ATP. The X-ray crystal structures have revealed that two ATP molecules occupy both the active and exo site of the enzyme, suggesting that ATP might act as an allosteric inhibitor of the enzyme. However, mutagenesis studies and kinetic evidences indicated that the catalytic activity of the enzyme is inhibited by ATP through a competitive inhibition mechanism in the active site and not in the exo site. Three amino acid residues, Arg165, Asn259, and Glu314, which are hydrogen-bonded with NAD+ or ATP, are chosen to characterize their possible roles on the inhibitory effect of ATP for the enzyme. Our kinetic data clearly demonstrate that Arg165 is essential for catalysis. The R165A enzyme had very low enzyme activity, and it was only slightly inhibited by ATP and not activated by fumarate. The values of K(m,NAD) and K(i,ATP) to both NAD+ and malate were elevated. Elimination of the guanidino side chain of R165 made the enzyme defective on the binding of NAD+ and ATP, and it caused the charge imbalance in the active site. These effects possibly caused the enzyme to malfunction on its catalytic power. The N259A enzyme was less inhibited by ATP but could be fully activated by fumarate at a similar extent compared with the wild-type enzyme. For the N259A enzyme, the value of K(i,ATP) to NAD+ but not to malate was elevated, indicating that the hydrogen bonding between ATP and the amide side chain of this residue is important for the binding stability of ATP. Removal of this side chain did not cause any harmful effect on the fumarate-induced activation of the enzyme. The E314A enzyme, however, was severely inhibited by ATP and only slightly activated by fumarate. The values of K(m,malate), K(m,NAD), and K(i,ATP) to both NAD+ and malate for E314A were reduced to about 2-7-folds compared with those of the wild-type enzyme. It can be concluded that mutation of Glu314 to Ala eliminated the repulsive effects between Glu314 and malate, NAD+, or ATP, and thus the binding affinities of malate, NAD+, and ATP in the active site of the enzyme were enhanced.     

Allosteric control of the oligomerization of carbamoyl phosphate synthetase from Escherichia coli

Carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by the metabolites ornithine, IMP, and UMP. Ornithine and IMP function as activators, whereas UMP is an inhibitor. CPS undergoes changes in the state of oligomerization that are dependent on the protein concentration and the binding of allosteric effectors. Ornithine and IMP promote the formation of an (alphabeta)4 tetramer while UMP favors the formation of an (alphabeta)2 dimer. The three-dimensional structure of the (alphabeta)4 tetramer has unveiled two regions of molecular contact between symmetry-related monomeric units. Identical residues within two pairs of allosteric domains interact with one another as do twin pairs of oligomerization domains. There are thus two possible structures for an (alphabeta)2 dimer: an elongated dimer formed at the interface of two allosteric domains and a more compact dimer formed at the interface between two oligomerization domains. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (alphabeta)4 tetramer through disruption of the molecular binding interactions between monomeric units. When Leu-421 (located in the oligomerization domain) was mutated to a glutamate residue, CPS formed an (alphabeta)2 dimer in the presence of ornithine, UMP, or IMP. In contrast, when Asn-987 (located in the allosteric binding domain) was mutated to an aspartate, an (alphabeta) monomer was formed regardless of the presence of any allosteric effectors. These results are consistent with a model for the structure of the (alphabeta)2 dimer that is formed through molecular contact between two pairs of allosteric domains. Apparently, the second interaction, between pairs of oligomerization domains, does not form until after the interaction between pairs of allosteric domains is formed. The binding of UMP to the allosteric domain inhibits the dimerization of the (alphabeta)2 dimer, whereas the binding of either IMP or ornithine to this same domain promotes the dimerization of the (alphabeta)2 dimer. In the oligomerization process, ornithine and IMP must exert a conformational alteration on the oligomerization domain, which is approximately 45 A away from their site of binding within the allosteric domain. No significant dependence of the specific catalytic activity on the protein concentration could be detected, and thus the effects induced by the allosteric ligands on the catalytic activity and the state of oligomerization are unlinked from one another.     

Reversible ligand-induced dissociation of a tryptophan-shift mutant of phosphofructokinase from Bacillus stearothermophilus

The biophysical properties of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus (BsPFK) have been examined. The mutant, designated W179Y/Y164W, has kinetic and thermodynamic properties similar to the wild-type enzyme. A 2-fold decrease in kcat is observed, and the mutant displays a 3-fold smaller K(0.5) for the substrate, fructose-6-phosphate (Fru-6-P), as compared to the wild-type enzyme. The dissociation constant for the inhibitor, phospho(enol)pyruvate (PEP), increases 2-fold, and the coupling parameter, Q(ay), decreases 2-fold. This suggests that while the mutant displays a slightly decreased affinity for PEP, PEP is still an effective inhibitor once bound. The new position of the tryptophan in W179Y/Y164W is approximately 6 A from the Fru-6-P portion of the active site. A 25% decrease in fluorescence intensity is observed upon Fru-6-P binding, and an 80% decrease in fluorescence intensity is observed with PEP binding. In addition, the intrinsic fluorescence polarization increases from 0.327 +/- 0.001 to 0.353 +/- 0.001 upon Fru-6-P binding, but decreases to 0.290 +/- 0.001 when PEP binds. Most notably, the presence of PEP induces dissociation of the tetramer. Dissociation of the tetramer into dimers occurs along the active site interface and can be monitored by the loss in activity or the loss in tryptophan fluorescence that is observed when the enzyme is titrated with PEP. Activity can be protected or recovered by incubating the enzyme with Fru-6-P. Recovery of activity is enzyme concentration dependent, and the rate constant for association is 6.2 +/- 0.3 M(-1) x s(-1). Ultracentrifugation experiments revealed that in the absence of PEP the mutant enzyme exists in an equilibrium between the dimer and tetramer forms with a dissociation constant of 11.8 +/- 0.5 microM, while in the presence of PEP the enzyme exists in equilibrium between the dimer and monomer forms with a dissociation constant of 7.5 +/- 0.02 microM. A 3.1 A crystal structure of the mutant enzyme suggests that the amino acid substitutions have not dramatically altered the tertiary structure of the enzyme. While it is clear that wild-type BsPFK exists as a tetramer under these same conditions, these results suggest that quaternary structural changes probably play an important role in allosteric communication.     

Biophysical Characterization of the Dimer and Tetramer Interface Interactions of the Human Cytosolic Malic Enzyme

The cytosolic NADP⁺-dependent malic enzyme (c-NADP-ME) has a dimer-dimer quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In this study, the urea-induced unfolding process of the c-NADP-ME interface mutants was monitored using fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and enzyme activities. Here, we demonstrate the differential protein stability between dimer and tetramer interface interactions of human c-NADP-ME. Our data clearly demonstrate that the protein stability of c-NADP-ME is affected predominantly by disruptions at the dimer interface rather than at the tetramer interface. First, during thermal stability experiments, the melting temperatures of the wild-type and tetramer interface mutants are 8–10°C higher than those of the dimer interface mutants. Second, during urea denaturation experiments, the thermodynamic parameters of the wild-type and tetramer interface mutants are almost identical. However, for the dimer interface mutants, the first transition of the urea unfolding curves shift towards a lower urea concentration, and the unfolding intermediate exist at a lower urea concentration. Third, for tetrameric WT c-NADP-ME, the enzyme is first dissociated from a tetramer to dimers before the 2 M urea treatment, and the dimers then dissociated into monomers before the 2.5 M urea treatment. With a dimeric tetramer interface mutant (H142A/D568A), the dimer completely dissociated into monomers after a 2.5 M urea treatment, while for a dimeric dimer interface mutant (H51A/D90A), the dimer completely dissociated into monomers after a 1.5 M urea treatment, indicating that the interactions of c-NADP-ME at the dimer interface are truly stronger than at the tetramer interface. Thus, this study provides a reasonable explanation for why malic enzymes need to assemble as a dimer of dimers.     

Dynamic properties of the N-terminal swapped dimer of ribonuclease A

Bovine pancreatic ribonuclease (RNase A) forms two 3-dimensional domain-swapped dimers with different quaternary structures. One dimer is characterized by the swapping of the C-terminal region (C-Dimer) and presents a rather loose structure. The other dimer (N-Dimer) exhibits a very compact structure with exchange of the N-terminal helix. Here we report the results of a molecular dynamics/essential dynamics (MD/ED) study carried out on the N-Dimer. This investigation, which represents the first MD/ED analysis on a three-dimensional domain-swapped enzyme, provides information on the dynamic properties of the active site residues as well as on the global motions of the dimer subunits. In particular, the analysis of the flexibility of the active site residues agrees well with recent crystallographic and site-directed mutagenesis studies on monomeric RNase A, thus indicating that domain swapping does not affect the dynamics of the active sites. A slight but significant rearrangement of N-Dimer quaternary structure, favored by the formation of additional hydrogen bonds at subunit interface, has been observed during the MD simulation. The analysis of collective movements reveals that each subunit of the dimer retains the functional breathing motion observed for RNase A. Interestingly, the breathing motion of the two subunits is dynamically coupled, as they open and close in phase. These correlated motions indicate the presence of active site intercommunications in this dimer. On these bases, we propose a speculative mechanism that may explain negative cooperativity in systems preserving structural symmetry during the allosteric transitions.     

A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure

We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine. Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer. However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-biphosphate (Fru-1,6-P2). The mutation has not significantly changed the catalytic properties of the dimer (Kd NADH, Km pyruvate, Ki oxamate and kcat), but has weakened the binding of Fru-1,6-P2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect. We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-1,6-P2, is important for allosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism.     

Crystal structure of the malic enzyme from Ascaris suum complexed with nicotinamide adenine dinucleotide at 2.3 A resolution

The structure of the Ascaris suum mitochondrial NAD-malic enzyme in binary complex with NAD has been solved to a resolution of 2.3 A by X-ray crystallography. The structure resembles that of the human mitochondrial enzyme determined in complex with NAD [Xu, Y., Bhargava, G., Wu, H., Loeber, G., and Tong, L. (1999) Structure 7, 877-889]. The enzyme is a tetramer comprised of subunits possessing four domains organized in an "open" structure typical of the NAD-bound form. The subunit organization, as in the human enzyme, is a dimer of dimers. The Ascaris enzyme contains 30 additional residues at its amino terminus relative to the human enzyme. These residues significantly increase the interactions that promote tetramer formation and give rise to different subunit-subunit interactions. Unlike the mammalian enzyme, the Ascaris malic enzyme is not regulated by ATP, and no ATP binding site is observed in this structure. Although the active sites of the two enzymes are similar, residues interacting with NAD differ between the two. The structure is discussed in terms of the mechanism and particularly with respect to previously obtained kinetic and site-directed mutagenesis experiments.     

Activation of the hetero-octameric ATP phosphoribosyl transferase through subunit interface rearrangement by a tRNA synthetase paralog

ATP phosphoribosyl transferase (ATP-PRT) joins ATP and 5-phosphoribosyl-1-pyrophosphate (PRPP) in a highly regulated reaction that initiates histidine biosynthesis. The unusual hetero-octameric version of ATP-PRT includes four HisG(S) catalytic subunits based on the periplasmic binding protein fold and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. Here, we present the first structure of a PRPP-bound ATP-PRT at 2.9 A and provide a structural model for allosteric activation based on comparisons with other inhibited and activated ATP-PRTs from both the hetero-octameric and hexameric families. The activated state of the octameric enzyme is characterized by an interstitial phosphate ion in the HisZ-HisG interface and new contacts between the HisZ motif 2 loop and the HisG(S) dimer interface. These contacts restructure the interface to recruit conserved residues to the active site, where they activate pyrophosphate to promote catalysis. Additionally, mutational analysis identifies the histidine binding sites within a region highly conserved between HisZ and the functional HisRS. Through the oligomerization and functional re-assignment of protein domains associated with aminoacylation and phosphate binding, the HisZ-HisG octameric ATP-PRT acquired the ability to initiate the synthesis of a key metabolic intermediate in an allosterically regulated fashion.     

Crystallographic studies on Ascaris suum NAD-malic enzyme bound to reduced cofactor and identification of an effector site

The crystal structure of the mitochondrial NAD-malic enzyme from Ascaris suum, in a quaternary complex with NADH, tartronate, and magnesium has been determined to 2.0-A resolution. The structure closely resembles the previously determined structure of the same enzyme in binary complex with NAD. However, a significant difference is observed within the coenzyme-binding pocket of the active site with the nicotinamide ring of NADH molecule rotating by 198 degrees over the C-1-N-1 bond into the active site without causing significant movement of the other catalytic residues. The implications of this conformational change in the nicotinamide ring to the catalytic mechanism are discussed. The structure also reveals a binding pocket for the divalent metal ion in the active site and a binding site for tartronate located in a highly positively charged environment within the subunit interface that is distinct from the active site. The tartronate binding site, presumably an allosteric site for the activator fumarate, shows striking similarities and differences with the activator site of the human NAD-malic enzyme that has been reported recently. Thus, the structure provides additional insights into the catalytic as well as the allosteric mechanisms of the enzyme.     

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase has been determined at 2.7 A resolution. Its first 120 amino acids form a central five-stranded, beta-pleated sheet surrounded by five alpha helices. In one of the four dyad-related dimers, the two active site Ser-10 residues are 19 A apart, perhaps close enough to contact and become covalently linked to the DNA at the recombination site. This dimer also forms the only closely packed tetramer found in the crystal. The subunit interface at a second dyad-related dimer is more extensive and more highly conserved among the homologous recombinases; however, its active site Ser-10 residues are more than 30 A apart. Side chains, identified by mutations that eliminate catalysis but not DNA binding, are located on the subunit surface near the active site serine and at the interface between a third dyad-related pair of subunits of the tetramer.     

Structural and Kinetic Studies of the Allosteric Transition in Sulfolobus solfataricus Uracil Phosphoribosyltransferase: Permanent Activation by Engineering of the C-Terminus

Uracil phosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl-α-1-diphosphate (PRPP) and uracil to uridine monophosphate (UMP) and diphosphate (PP_i). The tetrameric enzyme from Sulfolobus solfataricus has a unique type of allosteric regulation by cytidine triphosphate (CTP) and guanosine triphosphate (GTP). Here we report two structures of the activated state in complex with GTP. One structure (refined at 2.8-Å resolution) contains PRPP in all active sites, while the other structure (refined at 2.9-Å resolution) has PRPP in two sites and the hydrolysis products, ribose-5-phosphate and PP_i, in the other sites. Combined with three existing structures of uracil phosphoribosyltransferase in complex with UMP and the allosteric inhibitor cytidine triphosphate (CTP), these structures provide valuable insight into the mechanism of allosteric transition from inhibited to active enzyme. The regulatory triphosphates bind at a site in the center of the tetramer in a different manner and change the quaternary arrangement. Both effectors contact Pro94 at the beginning of a long β-strand in the dimer interface, which extends into a flexible loop over the active site. In the GTP-bound state, two flexible loop residues, Tyr123 and Lys125, bind the PP_i moiety of PRPP in the neighboring subunit and contribute to catalysis, while in the inhibited state, they contribute to the configuration of the active site for UMP rather than PRPP binding. The C-terminal Gly216 participates in a hydrogen-bond network in the dimer interface that stabilizes the inhibited, but not the activated, state. Tagging the C-terminus with additional amino acids generates an endogenously activated enzyme that binds GTP without effects on activity.     

The molecular mechanism for the tetrameric association of glycogen phosphorylase promoted by protein phosphorylation.

The allosteric transition of glycogen phosphorylase promoted by protein phosphorylation is accompanied by the association of a pair of functional dimers to form a tetramer. The conformational changes within the dimer that lead to the creation of a protein recognition surface have been analyzed from a comparison of the crystal structures of T-state dimeric phosphorylase b and R-state tetrameric phosphorylase a. Regions of the structure that participate in the tetramer interface are situated within structural subdomains. These include the glycogen storage subdomain, the C-terminal subdomain and the tower helix. The subdomains undergo concerted conformational transitions on conversion from the T to the R state (overall r.m.s. shifts between 1 and 1.7 A) and, together with the quaternary conformational change within the functional dimer, create the tetramer interface. The glycogen storage subdomain and the C-terminal subdomain are distinct from those regions that contribute to the dimer interface, but shifts in the subdomains are correlated with the allosteric transitions that are mediated by the dimer interface. The structural properties of the tetramer interface are atypical of an oligomeric protein interface and are more similar to protein recognition surfaces observed in protease inhibitors and antibody-protein antigen complexes. There is a preponderance of polar and charged residues at the tetramer interface and a high number of H-bonds per surface area (one H-bond per 130 A2). In addition, the surface area made inaccessible at the interface is relatively small (1,142 A2 per subunit on dimer to tetramer association compared with 2,217 A2 per subunit on monomer-to-dimer association).     

Domain motions and quaternary packing of phosphofructokinase-2 from Escherichia coli studied by small angle x-ray scattering and homology modeling

The binding of MgATP and fructose-6-phosphate to phosphofructokinase-2 from Escherichia coli induces conformational changes that result in significant differences in the x-ray-scattering profiles compared with the unligated form of the enzyme. When fructose- 6-phosphate binds to the active site of the enzyme, the pair distribution function exhibits lower values at higher distances, indicating a more compact structure. Upon binding of MgATP to the allosteric site of the enzyme, the intensity at lower angles increases as a consequence of tetramer formation, but differences along higher angles also suggest changes at the tertiary structure level. We have used homology modeling to build the native dimeric form of phosphofructokinase-2 and fitted the experimental scattering curves by using rigid body movements of the domains in the model, similar to those observed in known homologous structures. The best fit with the experimental data of the unbound protein was achieved with open conformations of the domains in the model, whereas domain closure improves the agreement with the scattering of the enzyme-fructose-6-phosphate complex. Using the same approach, we utilized the scattering curve of the phosphofructokinase-2-MgATP complex to model the arrangement and conformation of dimers in the tetramer. We observed that, along with tetramerization, binding of MgATP to the allosteric site induces domain closure. Additionally, we used the scattering data to restore the low resolution structure of phosphofructokinase-2 (free and bound forms) by an ab initio procedure. Based on these findings, a proposal is made to account for the inhibitory effect of MgATP on the enzymatic activity.     

A model for the Bacillus subtilis formylglycinamide ribonucleotide amidotransferase multiprotein complex

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), ATP, and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway. PurL exists in two forms: large PurL (lgPurL) is a single chain, multidomain enzyme of about 1300 amino acids, whereas small PurL (smPurL) contains about 800 amino acids but requires two additional gene products, PurS and PurQ, for activity. smPurL contains the ATP and FGAR binding sites, PurQ is a glutaminase, and the function of PurS is just now becoming understood. We determined the structure of Bacillus subtilis PurS in two different crystal forms P2(1) and C2 at 2.5 and 2.0 A resolution, respectively. PurS forms a tight dimer with a central six-stranded beta-sheet flanked by four helices. In both the P2(1) and the C2 crystal forms, the quaternary structure of PurS is a tetramer. The concave faces of the PurS dimers interact via the C-terminal region to form a twelve-stranded beta-barrel with a hydrophilic core. We used the structure of PurS together with the structure of lgPurL from Salmonella typhimurium to construct a model of the PurS/smPurL/PurQ complex. The HisH (glutaminase) domain of imidazole glycerol phosphate synthetase was used as an additional model of PurQ. The model shows stoichiometry of 2PurS/smPurL/PurQ using a PurS dimer or 4PurS/2smPurL/2PurQ using a PurS tetramer. Both models place key conserved residues at the ATP/FGAR binding site and at a structural ADP binding site. The homology model is consistent with biochemical studies on the reconstituted complex.