GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme.

How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.

Structural studies of human muscle FBPase.

Muscle fructose-1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) and inorganic phosphate, regulates glucose homeostasis by controlling the glyconeogenic pathway. FBPase requires divalent cations, such as Mg2+, Mn2+, or Zn2+, for its catalytic activity; however, calcium ions inhibit the muscle isoform of FBPase by interrupting the movement of the catalytic loop. It has been shown that residue E69 in this loop plays a key role in the sensitivity of muscle FBPase towards calcium ions. The study presented here is based on five crystal structures of wild-type human muscle FBPase and its E69Q mutant in complexes with the substrate and product of the enzymatic reaction, namely F1,6BP and F6P. The ligands are bound in the active site of the studied proteins in the same manner and have excellent definition in the electron density maps. In all studied crystals, the homotetrameric enzyme assumes the same cruciform quaternary structure, with the κ angle, which describes the orientation of the upper dimer with respect to the lower dimer, of -85o. This unusual quaternary arrangement of the subunits, characteristic of the R-state of muscle FBPase, is also observed in solution by small-angle X-ray scattering (SAXS).

Cloning, purification and characterisation of cytosolic fructose-1,6-bisphosphatase from mung bean (Vigna radiata).

To improve the crop yield and quality, the cytosolic fructose-1,6-bisphosphatase (cFBPase) from mung bean (Vigna radiata), a rate-limiting enzyme in gluconeogenesis, was cloned, purified, and structurally characterised. To function it required Mg2+ and Mn2+ at 0.01-10 mM. The Michaelis-Menton constant and adenosine monophosphate (AMP) inhibitory constant (Ki) were 7.96 and 111.09 µM, respectively. The functional site residues of AMP binding (Arg30, Asp32, and Phe33) and the active site residues (Asn218 and Met251) were tested via site-directed mutagenesis and molecular docking. Asn218 and Met251 were replaced by Tyr and Leu, respectively. The M251L mutant showed enhanced substrate affinity and activity, resulting from decreased binding energy (-2.58 kcal·mol-1) and molecular distance (4.2 Å). AMP binding site mutations changed the enzyme activities, indicating a connection between the binding and active sites. Furthermore, Ki and docking analysis revealed that Asp32 plays a key role in maintaining the AMP binding conformation.

Structural and biochemical characterization of the class II fructose-1,6-bisphosphatase from Francisella tularensis.

The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4 Šresolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P21, unit-cell parameters a = 76.30, b = 100.17, c = 92.02 Å, ß = 90.003°) with four chains in the asymmetric unit. Chain A had two coordinated Mg2+ ions in its active center, which is distinct from previous findings, and is presumably deactivated by their presence. The structure revealed an approximate 222 (D2) symmetry homotetramer analogous to that previously described for MtFBPaseII, which is formed by a crystallographic dyad and which differs from the exact tetramer found in EcFBPaseII at a 222 symmetry site in the crystal. Instead, the approximate homotetramer is very similar to that found in the dual phosphatase from Synechocystis, even though no allosteric effector was found in FtFBPase. The amino-acid sequence and folding of the active site of FtFBPaseII result in structural characteristics that are more similar to those of the previously published EcFBPaseII than to those of MtFBPaseII. The kinetic parameters of native FtFBPaseII were found to be in agreement with published studies. Kinetic analyses of the Thr89Ser and Thr89Ala mutations in the active site of the enzyme are consistent with the previously proposed mechanism for other class II bisphosphatases. The Thr89Ala variant enzyme was inactive but the Thr89Ser variant was partially active, with an approximately fourfold lower Km and Vmax than the native enzyme. The structural and functional insights derived from the structure of FtFBPaseII will provide valuable information for the design of specific inhibitors.

Discovery of N-Arylsulfonyl-Indole-2-Carboxamide Derivatives as Potent, Selective, and Orally Bioavailable Fructose-1,6-Bisphosphatase Inhibitors-Design, Synthesis, In Vivo Glucose Lowering Effects, and X-ray Crystal Complex Analysis.

Liver fructose-1,6-bisphosphatase (FBPase) is a key enzyme in the gluconeogenesis pathway. Inhibiting FBPase activity represents a potential treatment for type 2 diabetes mellitus. A series of novel N-arylsulfonyl-4-arylamino-indole-2-carboxamide derivatives have been disclosed as FBPase inhibitors. Through extensive structure-activity relationship investigations, a promising candidate molecule Cpd118 [sodium (7-chloro-4-((3-methoxyphenyl)amino)-1-methyl-1H-indole-2-carbonyl] [(4-methoxyphenyl)sulfonyl)amide] has been identified with high inhibitory activity against human liver FBPase (IC50, 0.029 ± 0.006 µM) and high selectivity relative to the other six AMP-binding enzymes. Importantly, Cpd118 produced significant glucose-lowering effects on both type 2 diabetic KKAy mice and ZDF rats as demonstrated by substantial reductions in the fasting and postprandial blood glucose levels, as well as the HbA1c level. Furthermore, Cpd118 elicited a favorable pharmacokinetic profile with an oral bioavailability of 99.1%. Moreover, the X-ray crystal structure of the Cpd118-FBPase complex was resolved, which revealed a unique binding mode and provided a structural basis for its high potency and selectivity.

Fructose-1, 6-bisphosphatase 1 interacts with NF-κB p65 to regulate breast tumorigenesis via PIM2 induced phosphorylation.

Rationale: Fructose-1, 6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor and could mediate the activities of multiple transcriptional factors via its non-canonical functions. However, the underlying mechanism of posttranscriptional modification on the non-canonical functions of FBP1 remains elusive. Methods: We employed immunoaffinity purification to identify binding partner(s) and used co-immunoprecipitation to verify their interactions. Kinase reaction was used to confirm PIM2 could phosphorylate FBP1. Overexpression or knockdown proteins were used to assess the role in modulating p65 protein stability. Mechanistic analysis was involved in protein degradation and polyubiquitination assays. Nude mice and PIM2-knockout mice was used to study protein functions in vitro and in vivo. Results: Here, we identified Proviral Insertion in Murine Lymphomas 2 (PIM2) as a new binding partner of FBP1, which could phosphorylate FBP1 on Ser144. Surprisingly, phosphorylated FBP1 Ser144 abrogated its interaction with NF-κB p65, promoting its protein stability through the CHIP-mediated proteasome pathway. Furthermore, phosphorylation of FBP1 on Ser144 increased p65 regulated PD-L1 expression. As a result, phosphorylation of FBP1 on Ser144 promoted breast tumor growth in vitro and in vivo. Moreover, the levels of PIM2 and pSer144-FBP1 proteins were positively correlated with each other in human breast cancer and PIM2 knockout mice. Conclusions: Our findings revealed that phosphorylation noncanonical FBP1 by PIM2 was a novel regulator of NF-κB pathway, and highlights PIM2 inhibitors as breast cancer therapeutics.

Performance Evaluation of Docking Programs- Glide, GOLD, AutoDock & SurflexDock, Using Free Energy Perturbation Reference Data: A Case Study of Fructose-1, 6-bisphosphatase-AMP Analogs.

BACKGROUND: The accurate ranking of analogs of lead molecules with respect to their estimated binding free energies to drug targets remains highly challenging in molecular docking due to small relative differences in their free energy values. METHODS: Free energy perturbation (FEP) method, which provides the most accurate relative binding free energy values were earlier used to calculate free energies of many ligands for several important drug targets including Fructose-1,6-BisphosPhatase (FBPase). The availability of abundant structural and experimental binding affinity data for FBPase inhibitors provided an ideal system to evaluate four widely used docking programs, AutoDock, Glide, GOLD and SurflexDock, distinct from earlier comparative evaluation studies. RESULTS: The analyses suggested that, considering various parameters such as docking pose, scoring and ranking accuracy, sensitivity analysis and newly introduced relative ranking score, Glide provided reasonably consistent results in all respects for the system studied in the present work. Whereas GOLD and AutoDock also demonstrated better performance, AutoDock results were found to be significantly superior in terms of scoring accuracy compared to the rest. CONCLUSION: Present analysis serves as a useful guide for researchers working in the field of lead optimization and for developers in upgradation of the docking programs.

Enhancing the yield of human erythropoietin in Aspergillus niger by introns and CRISPR-Cas9.

Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L-1 by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein.

Evolution of Substrates and Components of the Pro/N-Degron Pathway.

Gid4, a subunit of the ubiquitin ligase GID, is the recognition component of the Pro/N-degron pathway. Gid4 targets proteins in particular through their N-terminal (Nt) proline (Pro) residue. In Saccharomyces cerevisiae and other Saccharomyces yeasts, the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 bear Nt-Pro and are conditionally destroyed by the Pro/N-degron pathway. However, in mammals and in many non-Saccharomyces yeasts, for example, in Kluyveromyces lactis, these enzymes lack Nt-Pro. We used K. lactis to explore evolution of the Pro/N-degron pathway. One question to be addressed was whether the presence of non-Pro Nt residues in K. lactis Fbp1, Icl1, and Mdh2 was accompanied, on evolutionary time scales (S. cerevisiae and K. lactis diverged ∼150 million years ago), by a changed specificity of the Gid4 N-recognin. We used yeast-based two-hybrid binding assays and protein-degradation assays to show that the non-Pro (Ala) Nt residue of K. lactis Fbp1 makes this enzyme long-lived in K. lactis. We also found that the replacement, through mutagenesis, of Nt-Ala and the next three residues of K. lactis Fbp1 with the four-residue Nt-PTLV sequence of S. cerevisiae Fbp1 sufficed to make the resulting "hybrid" Fbp1 a short-lived substrate of Gid4 in K. lactis. We consider a blend of quasi-neutral genetic drift and natural selection that can account for these and related results. To the best of our knowledge, this work is the first study of the ubiquitin system in K. lactis, including development of the first protein-degradation assay (based on the antibiotic blasticidin) suitable for use with this organism.

Phosphofructokinase-1 and fructose bisphosphatase-1 in canine liver and kidney.

In healthy individuals, plasma glucose levels are maintained within a normal range. During fasting, endogenous glucose is released either through glycogenolysis or gluconeogenesis. Gluconeogenesis involves the formation of glucose-6-phosphate from a variety of precursors followed by its subsequent hydrolysis to glucose. Gluconeogenesis occurs in the liver and the kidney. In order to compare gluconeogenesis in canine liver and kidney, the activity and expression of the rate limiting enzymes that catalyze the fructose-6-phosphate and fructose 1,6-bisphosphate steps, namely, phosphofructokinase-1 (PFK-1) (glycolysis) and fructose bisphosphatase-1 (FBP-1) (gluconeogenesis), were examined. Healthy male and female beagle dogs aged 1-2 years were euthanized humanely, and samples of their liver and kidney were obtained for analysis. The levels of PFK-1 and FBP-1 in canine liver and kidney were assessed by enzymatic assays, Western blotting, and RT-qPCR. Enzyme assays showed that, in dogs, the kidney had higher specific activity of PFK-1 and FBP-1 than the liver. Western blotting and RT-qPCR data demonstrated that of the three different subunits (PFK-M, PFK-L, and PFK-P) the PFK-1 in canine liver mainly comprised PFK-L, whereas the PFK-1 in the canine kidney comprised all three subunits. As a result of these differences in the subunit composition of PFK-1, glucose metabolism might be regulated differently in the liver and kidney.

Fructose-1,6-bisphosphatase: From a glucose metabolism enzyme to multifaceted regulator of a cell fate.

Fructose-1,6-bisphosphatase (FBPase) is one of the ancient, evolutionarily conserved enzymes of carbohydrate metabolism. It has been described for a first time in 1943, however, for the next half a century mostly kinetic and structural parameters of animal FBPases have been studied. Discovery of ubiquitous expression of the muscle isozyme of FBPase, thus far considered to merely regulate glycogen synthesis from carbohydrate precursors, and its nuclear localisation in several cell types has risen new interest in the protein, resulting in numerous publications revealing complex functions/properties of FBPase. This review summarises the current knowledge of FBPase in animal cells providing evidence that the enzyme merits the name of moonlighting protein.

Characterization of recombinant fructose-1,6-bisphosphatase gene mutations: evidence of inhibition/activation of FBPase protein by gene mutation.

Specific residues of the highly regulated fructose-1,6-bisphosphatase (FBPase) enzyme serve as important contributors to the catalytic activity of the enzyme. Previous clinical studies exploring the genetic basis of hypoglycemia revealed two significant mutations in the coding region of the FBPase gene in patients with hypoglycemia, linking the AMP-binding site to the active site of the enzyme. In the present study, a full kinetic analysis of similar mutants was performed. Kinetic results of mutants Y164A and M177A revealed an approximate two to three-fold decrease in inhibitory constants (Ki's) for natural inhibitors AMP and fructose-2,6-bisphosphate (F2,6-BP) compared with the Wild-type enzyme (WT). A separate mutation (M248D) was performed in the active site of the enzyme to investigate whether the enzyme could be activated. This mutant displayed an approximate seven-fold increase in Ki for F2,6-BP. Interfacial mutants L56A and L73A exhibited an increase in Ki for F2,6-BP by approximately five-fold. Mutations in the AMP-binding site (K112A and Y113A) demonstrated an eight to nine-fold decrease in AMP inhibition. Additionally, mutant M248D displayed a four-fold decrease in its apparent Michelis constant (Km), and a six-fold increase in catalytic efficiency (CE). The importance-and medical relevance-of specific residues for FBPase structural/functional relationships in both the catalytic site and AMP-binding site is discussed.

In silico screening of a novel scaffold for fructose-1,6-bisphosatase (FBPase) inhibitors.

Fructose-1, 6-bisphosphatase (FBPase) has been regarded as an attractive drug target to control blood glucose against Type 2 diabetes (T2D). In this study, by using the strategy of pharmacophore-based virtual screening, a novel scaffold inhibitor targeted the AMP allosteric site of human liver FBPase were screened, their inhibitory activities were further tested. The experimental results showed that compound H27 exhibited high inhibitory activities with the IC50 value of 5.3 µM. Therefore, compound H27 was chosen as the probe molecule, it's possible binding conformation targeted into FBPase was identified by using DOX2.0 strategy. The importance of key residues (T27, T31, K112 and R140) in allosteric site of FBPase for the binding inhibitors were validated by mutation experiments. The agreement between theory and experiment suggest that the interactional information of FBPase and inhibitors (H27) were reliable. On basis of these rational interactional information, the compound H29 was further designed to exhibit more potential FBPase inhibition (IC50 = 2.5 µM).

Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase.

Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its antihyperglycemic action primarily through lowering hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5'-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose-2,6-bisphosphate (F-2,6-P2), and knock-in (KI) of this mutant in mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The antihyperglycemic effect of metformin in high-fat diet-fed diabetic FBP1-KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action for metformin and provide further evidence that molecular targeting of FBP1 can have antihyperglycemic effects.

Structures of the Mycobacterium tuberculosis GlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition.

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6 Šresolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus. This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis (MtFBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of MtFBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of MtFBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.

Structures of Leishmania Fructose-1,6-Bisphosphatase Reveal Species-Specific Differences in the Mechanism of Allosteric Inhibition.

The gluconeogenic enzyme fructose-1,6-bisphosphatase has been proposed as a potential drug target against Leishmania parasites that cause up to 20,000-30,000 deaths annually. A comparison of three crystal structures of Leishmania major fructose-1,6-bisphosphatase (LmFBPase) along with enzyme kinetic data show how AMP acts as an allosteric inhibitor and provides insight into its metal-dependent reaction mechanism. The crystal structure of the apoenzyme form of LmFBPase is a homotetramer in which the dimer of dimers adopts a planar conformation with disordered "dynamic loops". The structure of LmFBPase, complexed with manganese and its catalytic product phosphate, shows the dynamic loops locked into the active sites. A third crystal structure of LmFBPase complexed with its allosteric inhibitor AMP shows an inactive form of the tetramer, in which the dimer pairs are rotated by 18° relative to each other. The three structures suggest an allosteric mechanism in which AMP binding triggers a rearrangement of hydrogen bonds across the large and small interfaces. Retraction of the "effector loop" required for AMP binding releases the side chain of His23 from the dimer-dimer interface. This is coupled with a flip of the side chain of Arg48 which ties down the key catalytic dynamic loop in a disengaged conformation and also locks the tetramer in an inactive rotated T-state. The structure of the effector site of LmFBPase shows different structural features compared with human FBPases, thereby offering a potential and species-specific drug target.

Enzymatic Characterization of Fructose 1,6-Bisphosphatase II from Francisella tularensis, an Essential Enzyme for Pathogenesis.

The glpX gene from Francisella tularensis encodes for the class II fructose 1,6-bisphosphatase (FBPaseII) enzyme. The glpX gene has been verified to be essential in F. tularensis, and the inactivation of this gene leads to impaired bacterial growth on gluconeogenic substrates. In the present work, we have complemented a ∆glpX mutant of Escherichia coli with the glpX gene of F. tularensis (FTF1631c). Our complementation work independently verifies that the glpX gene (FTF1631c) in F. tularensis is indeed an FBPase and supports the growth of the ΔglpX E. coli mutant on glycerol-containing media. We have performed heterologous expression and purification of the glpX encoded FBPaseII in F. tularensis. We have confirmed the function of glpX as an FBPase and optimized the conditions for enzymatic activity. Mn2+ was found to be an absolute requirement for activity, with no other metal substitutions rendering the enzyme active. The kinetic parameters for this enzyme were found as follows: Km 11 µM, Vmax 2.0 units/mg, kcat 1.2 s-1, kcat/Km 120 mM-1 s-1, and a specific activity of 2.0 units/mg. Size exclusion data suggested an abundance of a tetrameric species in solution. Our findings on the enzyme's properties will facilitate the initial stages of a structure-based drug design program targeting this essential gene of F. tularensis.

Pcal_0111, a highly thermostable bifunctional fructose-1,6-bisphosphate aldolase/phosphatase from Pyrobaculum calidifontis.

Pyrobaculum calidifontis genome harbors an open reading frame Pcal_0111 annotated as fructose bisphosphate aldolase. Although the gene is annotated as fructose bisphosphate aldolase, it exhibits a high homology with previously reported fructose-1,6-bisphosphate aldolase/phosphatase from Thermoproteus neutrophilus. To examine the biochemical properties of Pcal_0111, we have cloned and expressed the gene in Escherichia coli. Purified recombinant Pcal_0111 catalyzed both phosphatase and aldolase reactions with specific activity values of 4 U and 1.3 U, respectively. These values are highest among the fructose 1,6-bisphosphatases/aldolases characterized from archaea. The enzyme activity increased linearly with the increase in temperature until 100 °C. Recombinant Pcal_0111 is highly stable with a half-life of 120 min at 100 °C. There was no significant change in the circular dichroism spectra of the protein up to 90 °C. The enzyme activity was not affected by AMP but strongly inhibited by ATP with an IC50 value of 0.75 mM and mildly by ADP. High thermostability and inhibition by ATP make Pcal_0111 a unique fructose 1,6-bisphosphatase/aldolase.

Triple-Stage Mass Spectrometry Unravels the Heterogeneity of an Endogenous Protein Complex.

Protein complexes often represent an ensemble of different assemblies with distinct functions and regulation. This increased complexity is enabled by the variety of protein diversification mechanisms that exist at every step of the protein biosynthesis pathway, such as alternative splicing and post transcriptional and translational modifications. The resulting variation in subunits can generate compositionally distinct protein assemblies. These different forms of a single protein complex may comprise functional variances that enable response and adaptation to varying cellular conditions. Despite the biological importance of this layer of complexity, relatively little is known about the compositional heterogeneity of protein complexes, mostly due to technical barriers of studying such closely related species. Here, we show that native mass spectrometry (MS) offers a way to unravel this inherent heterogeneity of protein assemblies. Our approach relies on the advanced Orbitrap mass spectrometer capable of multistage MS analysis across all levels of protein organization. Specifically, we have implemented a two-step fragmentation process in the inject flatapole device, which was converted to a linear ion trap, and can now probe the intact protein complex assembly, through its constituent subunits, to the primary sequence of each protein. We demonstrate our approach on the yeast homotetrameric FBP1 complex, the rate-limiting enzyme in gluconeogenesis. We show that the complex responds differently to changes in growth conditions by tuning phosphorylation dynamics. Our methodology deciphers, on a single instrument and in a single measurement, the stoichiometry, kinetics, and exact position of modifications, contributing to the exposure of the multilevel diversity of protein complexes.

Identification of a potential proton donor to the linking oxygen atom in a three-metal ion assisted catalysis pathway catalyzed by Fructose-1, 6-bisphosphatase.

In this paper, the dephosphorylation mechanism of FBP to F6P catalyzed by the Fructose-1, 6-bisphosphatase (St-Fbp) from Sulfolobus tokodaii was studied using quantum mechanical/molecular mechanical (QM/MM) approach. Based on the experimental results, total five possible catalytic mechanisms (path1-path4') were designed. The most possible dephosphorylation reaction follows a two-step mechanism (path2): a dephosphorylation process (with D12 being an base of W6 and residue K133 being the proton donor of the linking FBP:O4) and a proton exchange process (between K133 and the water W1). Furthermore, the three-step of path4 is also possible: a dephosphorylation process (with D54 being the base of W6 and residue K133 being the proton donor of the linking FBP:O4) and two proton exchange processes (first between residues D54 and D12 then between K133 and the water W1). The relative low energy of this pathway suggests that D54 might also be a base except D12. Our calculations indicate that K133 is the preferred proton donor during the breaking of the phosphate bond O4-P1, with the W1 being an alternative proton donor to access to a more stable product. Findings here give a new insight into the understanding of catalytic mechanism of FBPase.