Structural basis of negative cooperativity in transthyretin

A comparison of the AC and BD binding sites of transthyretin (TTR) was made in terms of the interatomic distances between the Ca atoms of equivalent amino acids, measured across the tetramer channel in each binding site. The comparison of the channel diameter for apo TTR from different sources revealed that in the unliganded transthyretin tetramers the distances between the A, D and H beta-strands are consistently larger, while the distances between the G beta-strands are smaller in one site than in the other. These differences might be described to have a 'wave' character. An analogous analysis performed for transthyretin complexes reveals that the shape of the plot is similar, although the amplitudes of the changes are smaller. The analysis leads us to a model of the changes in the binding sites caused by ligand binding. The sequence of events includes ligand binding in the first site, followed by a slight collapse of this site and concomitant opening of the second site, binding of the second molecule and collapse of the second site. The following opening of the first, already occupied site upon ligand binding in the second site is smaller because of the bridging interactions already formed by the first ligand. This explains the negative cooperativity (NC) effect observed for many ligands in transthyretin.     

Structural basis for the reaction mechanism of UDP-glucose pyrophosphorylase

UDP-glucose pyrophosphorylases (UGPase; EC 2.7.7.9) catalyze the conversion of UTP and glucose-1-phosphate to UDP-glucose and pyrophosphate and vice versa. Prokaryotic UGPases are distinct from their eukaryotic counterparts and are considered appropriate targets for the development of novel antibacterial agents since their product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharides and capsular polysaccharides. In this study, the crystal structures of UGPase from Helicobacter pylori (HpUGPase) were determined in apo- and UDP-glucose/Mg²⁺-bound forms at 2.9 Å and 2.3 Å resolutions, respectively. HpUGPase is a homotetramer and its active site is located in a deep pocket of each subunit. Magnesium ion is coordinated by Asp130, two oxygen atoms of phosphoryl groups, and three water molecules with octahedral geometry. Isothermal titration calorimetry analyses demonstrated that Mg²⁺ ion plays a key role in the enzymatic activity of UGPase by enhancing the binding of UGPase to UTP or UDP-glucose, suggesting that this reaction is catalyzed by an ordered sequential Bi Bi mechanism. Furthermore, the crystal structure explains the specificity for uracil bases. The current structural study combined with functional analyses provides essential information for understanding the reaction mechanism of bacterial UGPases, as well as a platform for the development of novel antibacterial agents.     

Structural basis for subunit assembly in UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae

UDP-glucose is the universal activated form of glucose, employed in all organisms for glucosyl transfer reactions and as precursor for various activated carbohydrates. In animal and fungal metabolism, UDP-glucose is required for utilization of galactose and for the synthesis of glycogen, the major carbohydrate storage polymer. The formation of UDP-glucose is catalyzed by UDP-glucose pyrophosphorylase (UGPase), which is highly conserved among eukaryotes. Here, we present the crystal structure of yeast UGPase, Ugp1p. Both in solution and in the crystal, Ugp1p forms homooctamers, which represent the enzymatically active form of the protein. Ugp1p subunits consist of three domains, with the active site presumably located in the central SpsA GnT I core (SGC) domain. The association in the octamer is mediated by contacts between left-handed beta-helices in the C-terminal domains, forming a toroidal solenoid structure in the core of the complex. The catalytic domains attached to this scaffold core do not directly contact each other, consistent with simple Michaelis-Menten kinetics found for Ugp1p. Conservation of hydrophobic residues at the subunit interfaces suggests that all fungal and animal homologs form this quarternary structure arrangement in contrast to monomeric plant UGPases, which have charged residues at these positions. Implications of this oligomeric arrangement for regulation of UGPase activity in fungi and animals are discussed.     

Structural basis of affinity maturation and intramolecular cooperativity in a protein-protein interaction

Although protein-protein interactions are involved in nearly all cellular processes, general rules for describing affinity and selectivity in protein-protein complexes are lacking, primarily because correlations between changes in protein structure and binding energetics have not been well determined. Here, we establish the structural basis of affinity maturation for a protein-protein interaction system that we had previously characterized energetically. This model system exhibits a 1500-fold affinity increase. Also, its affinity maturation is restricted by negative intramolecular cooperativity. With three complex and six unliganded variant X-ray crystal structures, we provide molecular snapshots of protein interface remodeling events that span the breadth of the affinity maturation process and present a comprehensive structural view of affinity maturation. Correlating crystallographically observed structural changes with measured energetic changes reveals molecular bases for affinity maturation, intramolecular cooperativity, and context-dependent binding.     

Radiochemical assay for UDP-glucose dehydrogenase

A rapid radioisotopic assay was developed for the enzyme UDP-glucose dehydrogenase. The assay is based on formation of UDP-glucuronate-¹⁴C from UDP-glucose-¹⁴C. The enzyme product was rapidly separated from unreacted substrate on anion-exchange paper after hydrolysis of the sugar nucleotides to give the sugar acid and neutral sugar. The assay was developed with the enzyme from lily pollen, but it should be useful for any biological system.     

Inhibitory effects of Aphanizomenon flos-aquae constituents on human UDP-glucose dehydrogenase activity

Objective: The purpose of this study was to investigate the in vitro inhibitory effects of the edible microalga Aphanizomenon flos-aquae (AFA) on human UDP-α-d-glucose 6-dehydrogenase (UGDH) activity, a cytosolic enzyme involved both in tumor progression and in phytochemical bioavailability.

Methods: Both the hydrophilic and ethanolic AFA extracts as well as the constitutive active principles phycocyanin (PC), phycocyanobilin (PCB) and mycosporine-like amino acids (MAAs) were tested.

Results: Among AFA components, PCB presented the strongest inhibitory effect on UGDH activity, acting as a competitive inhibitor with respect to UDP-glucose and a non-competitive inhibitor with respect to NAD⁺. In preliminary experiments, AFA PCB was also effective in reducing the colony formation capacity of PC-3 prostate cancer cells and FTC-133 thyroid cancer cells.

Conclusions: Overall, these findings confirmed that AFA and its active principles are natural compounds with high biological activity. Further studies evaluating the effects of AFA PCB in reducing tumor cell growth and phytochemical glucuronidation are encouraged.     

A microassay for UDP-glucose dehydrogenase

An assay for UDP-glucuronic acid [J. Singh, L. R. Schwarz, and F. J. Wiebel, Biochem. J. 189, 369–372 (1980)] has been utilized for determining UDP-glucose dehydrogenase activity. The assay for UDP-glucuronic acid, a product of UDP-glucose dehydrogenase, is based on the fluorometric determination of -glucuronosyl benzo(a)pyrene. This compound is formed from UDP-glucuronic acid and 3-hydroxybenzo(a)pyrene in a reaction catalyzed by the glycuronosyl transferase of guinea pig microsomes. Unreacted 3-hydroxybenzo(a)pyrene is removed by extraction with chloroform-methanol, and the amount of gluconosylbenzo(a)pyrene formed is determined fluorometrically. Because this assay for UDP-glucose dehydrogenase is about 500 times more sensitive than spectrophotometric assays, it can be used to measure the amount of enzyme extractable from milligram quantities of connective tissue. Some kinetic properties of UDP-glucose dehydrogenase extracted from rabbit tissue have been determined. No evidence of different forms of the enzyme in rabbit liver, cartilage, or corneal stroma was found.     

Recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase: Structural basis for enhanced stability

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) is bound to the fibrous sheath of the sperm flagellum through the hydrophobic N-terminal domain of the enzyme molecule. Expression of human GAPDS in E.coli cells yields inactive and insoluble protein. Presumably, the N-terminal domain prevents correct folding of the full-length recombinant enzyme. To obtain GAPDS in a soluble and active form, a recombinant enzyme lacking in 68 amino acids of the N-terminal domain (dN-GAPDS) was expressed in E.coli cells. Purified dN-GAPDS was shown to be a protein of 9.3 nm in diameter (by dynamic light scattering), which is close to the size of the muscle tetrameric glyceraldehyde-3-phosphate dehydrogenase (8.6 nm). The catalytic properties of the protein differed a little from those of the muscle glyceraldehyde-3-phoshate dehydrogenase. However, compared to muscle glyceraldehyde-3-phoshate dehydrogenase, dN-GAPDS exhibited enhanced thermostability (the transition midpoints values are 60.8 and 67.4 °C, respectively) and was much more resistant towards action of guanidine hydrochloride (inactivation constants are 2.45 ± 0.018 and 0.118 ± 0.008 min^? 1, respectively). The enhanced stability of dN-GAPDS is likely to be related to some specific features of the GAPDS structure compared to that of the muscle enzyme: 1) reduced number of solvent-exposed salt bridges; 2) 2 additional buried salt bridges; and 3) 6 additional proline residues in GAPDS meeting the “proline rule”. It is assumed that high stability of the sperm-specific GAPDS is of importance for the efficiency of fertilization.     

Structural basis for altered activity of M- and H-isozyme forms of human lactate dehydrogenase

Lactate dehydrogenase (LDH) interconverts pyruvate and lactate with concomitant interconversion of NADH and NAD(+). Although crystal structures of a variety of LDH have previously been described, a notable absence has been any of the three known human forms of this glycolytic enzyme. We have now determined the crystal structures of two isoforms of human LDH-the M form, predominantly found in muscle; and the H form, found mainly in cardiac muscle. Both structures have been crystallized as ternary complexes in the presence of the NADH cofactor and oxamate, a substrate-like inhibitor. Although each of these isoforms has different kinetic properties, the domain structure, subunit association, and active-site regions are indistinguishable between the two structures. The pK(a) that governs the K(M) for pyruvate for the two isozymes is found to differ by about 0.94 pH units, consistent with variation in pK(a) of the active-site histidine. The close similarity of these crystal structures suggests the distinctive activity of these enzyme isoforms is likely to result directly from variation of charged surface residues peripheral to the active site, a hypothesis supported by electrostatic calculations based on each structure. Proteins 2001;43:175-185.     

Importance of Gly-13 for the coenzyme binding of human UDP-glucose dehydrogenase

UDP-glucose dehydrogenase (UGDH) is the unique pathway enzyme furnishing in vertebrates UDP-glucuronate for numerous transferases. In this report, we have identified an NAD(+)-binding site within human UGDH by photoaffinity labeling with a specific probe, [(32)P]nicotinamide 2-azidoadenosine dinucleotide (2N(3) NAD(+)), and cassette mutagenesis. For this work, we have chemically synthesized a 1509-base pair gene encoding human UGDH and expressed it in Escherichia coli as a soluble protein. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of NAD(+) during photolysis, demonstrating a selectivity of the photoprobe for the NAD(+)-binding site. Amino acid sequencing and compositional analysis identified the NAD(+)-binding site of UGDH as the region containing the sequence ICCIGAXYVGGPT, corresponding to Ile-7 through Thr-19 of the amino acid sequence of human UGDH. The unidentified residue, X, can be designated as a photolabeled Gly-13 because the sequences including the glycine residue in question have a complete identity with those of other UGDH species known. The importance of Gly-13 residue in the binding of NAD(+) was further examined with a G13E mutant by cassette mutagenesis. The mutagenesis at Gly-13 had no effects on the expression or stability of the mutant. Enzyme activity of the G13E point mutant was not measurable under normal assay conditions, suggesting an important role for the Gly-13 residue. No incorporation of [(32)P]2N(3)NAD(+) was observed for the G13E mutant. These results indicate that Gly-13 plays an important role for efficient binding of NAD(+) to human UGDH.     

Structural basis of folding cooperativity in model proteins: insights from a microcanonical perspective

Two-state cooperativity is an important characteristic in protein folding. It is defined by a depletion of states that lie energetically between folded and unfolded conformations. There are different ways to test for two-state cooperativity; however, most of these approaches probe indirect proxies of this depletion. Generalized-ensemble computer simulations allow us to unambiguously identify this transition by a microcanonical analysis on the basis of the density of states. Here, we present a detailed characterization of several helical peptides obtained by coarse-grained simulations. The level of resolution of the coarse-grained model allowed to study realistic structures ranging from small α-helices to a de novo three-helix bundle without biasing the force field toward the native state of the protein. By linking thermodynamic and structural features, we are able to show that whereas short α-helices exhibit two-state cooperativity, the type of transition changes for longer chain lengths because the chain forms multiple helix nucleation sites, stabilizing a significant population of intermediate states. The helix bundle exhibits signs of two-state cooperativity owing to favorable helix-helix interactions, as predicted from theoretical models. A detailed analysis of secondary and tertiary structure formation fits well into the framework of several folding mechanisms and confirms features that up to now have been observed only in lattice models.     

Structural basis for flip-flop action of thiamin pyrophosphate-dependent enzymes revealed by human pyruvate dehydrogenase

The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the Calpha-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-A resolution. We propose a model for the flip-flop action of this enzyme through a concerted approximately 2-A shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.     

Thermodynamic basis of site-specific cooperativity

Cooperative phenomena in biological macromolecules arise from the interaction of many distinct subsystems, such as structural domains or binding sites. Cooperative properties of the system as a whole, like protein folding or allosteric transitions, are subject to the restrictions imposed by thermodynamic stability. These restrictions, however, do not apply in the case of individual subsystems open to interactions with the rest of the macromolecule. The site-specific properties of such subsystems can be understood in general thermodynamic terms from those of a multicomponent system under particular conditions. The analogy provides a thermodynamic basis for site-specific Cooperativity. © 1994 John Wiley & Sons, Inc.     

Kinetic cooperativity of human liver alcohol dehydrogenase gamma(2)

Previous studies showed that natural human liver alcohol dehydrogenase gamma exhibits negative cooperativity (substrate activation) with ethanol. Studies with the recombinant gamma(2) isoenzyme now confirm that observation and show that the saturation kinetics with other alcohols are also nonhyperbolic, whereas the kinetics for reactions with NAD(+), NADH, and acetaldehyde are hyperbolic. The substrate activation with ethanol and 1-butanol are explained by an ordered mechanism with an abortive enzyme-NADH-alcohol complex that releases NADH more rapidly than does the enzyme-NADH complex. In contrast, high concentrations of cyclohexanol produce noncompetitive substrate inhibition against varied concentrations of NAD(+) and decrease the maximum velocity to 25% of the value that is observed at optimal concentrations of cyclohexanol. Transient kinetics experiments show that cyclohexanol inhibition is due to a slower rate of dissociation of NADH from the abortive enzyme-NADH-cyclohexanol complex than from the enzyme-NADH complex. Fluorescence quenching experiments confirm that the alcohols bind to the enzyme-NADH complex. The nonhyperbolic saturation kinetics for oxidation of ethanol, cyclohexanol, and 1-butanol are quantitatively explained with the abortive complex mechanism. Physiologically relevant concentrations of ethanol would be oxidized predominantly by the abortive complex pathway.     

Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase

Glutamate dehydrogenase (GDH) catalyzes reversible conversion between glutamate and 2-oxoglutarate using NAD(P)(H) as a coenzyme. Although mammalian GDH is regulated by GTP through the antenna domain, little is known about the mechanism of allosteric activation by leucine. An extremely thermophilic bacterium, Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA (regulatory subunit) and GdhB (catalytic subunit). T. thermophilus GDH is unique in that the enzyme is subject to allosteric activation by leucine. To elucidate the structural basis for leucine-induced allosteric activation of GDH, we determined the crystal structures of the GdhB-Glu and GdhA-GdhB-Leu complexes at 2.1 and 2.6 Å resolution, respectively. The GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits. The GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex. Consistent with the structure, replacement of the amino acid residues of T. thermophilus GDH responsible for leucine binding made T. thermophilus GDH insensitive to leucine. Equivalent amino acid replacement caused a similar loss of sensitivity to leucine in human GDH2, suggesting that human GDH2 also uses the same allosteric site for regulation by leucine.     

Alteration of the quaternary structure of human UDP-glucose dehydrogenase by a double mutation

There are conflicting views for the polymerization process of human UDP-glucose dehydrogenase (UGDH) and no clear evidence has been reported yet. Based on crystal coordinates for Streptococcus pyogenes UGDH, we made double mutant A222Q/S233G. The double mutagenesis had no effects on expression, stability, and secondary structure. Interestingly, A222Q/S233G was a dimeric form and showed an UGDH activity, although it showed increased Km values for substrates. These results suggest that Ala222 and Ser233 play an important role in maintaining the hexameric structure and the reduced binding affinities for substrates are attributable to its altered subunit communication although quaternary structure may not be critical for catalysis.     

Chemical basis of glycine riboswitch cooperativity

The glycine binding riboswitch forms a unique tandem aptamer structure that binds glycine cooperatively. We employed nucleotide analog interference mapping (NAIM) and mutagenesis to explore the chemical basis of glycine riboswitch cooperativity. Based on the interference pattern, at least two sites appear to facilitate cooperative tertiary interactions, namely, the minor groove of the P1 helix from aptamer 1 and the major groove of the P3a helix from both aptamers. Mutation of these residues altered both the cooperativity and binding affinity of the riboswitch. The data support a model in which the P1 helix of the first aptamer participates in a tertiary interaction important for cooperativity, while nucleotides in the P1 helix of the second aptamer interface with the expression platform. These data have direct analogy to well-characterized mutations in hemoglobin, which provides a framework for considering cooperativity in this RNA-based system.