Pyruvate carboxylase.
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Acetyl Coenzyme A↗
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Results for “Allosteric Regulation”
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Inversion of the allosteric response of Escherichia coli glucosamine-6-P deaminase to N-acetylglucosamine 6-P, by single amino acid replacements.
Amino acid replacements in the active site of glucosamine-6-P deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) involving the residues D141 and E148 produce atypical allosteric kinetics. These residues are located in the chain segment 139-156 which is part of the active site and which also forms several intersubunit contacts close to the allosteric site. In the D141N and E148Q mutant forms of this deaminase, there is an inversion of the effect of its physiological allosteric effector, N-acetylglucosamine 6-P, which becomes an inhibitor at substrate concentrations above a critical value. For both mutants, this particular point appears at low substrate concentration and the inhibition by the allosteric activator is the dominant effect in velocity versus substrate curves. These effects are analyzed as a particular case of the concerted allosteric model, assuming that the R state, the conformer displaying the higher affinity for the substrate, is the less catalytic state, thus producing an inverted allosteric response.
Aldose-Ketose Isomerases↗
Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates.
The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Knock down of cytosolic phospholipase A2: an antisense oligonucleotide having a nuclear localization binds a C-terminal motif of glyceraldehyde-3-phosphate dehydrogenase.
We have previously shown that an antisense, effective in the knock down of cytosolic phospholipase A2 (cPLA2), localizes mainly in the nucleus of human endothelial cells and monocytes and that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is involved in its nuclear localization. In this study, we clarify how GAPDH participates in the nuclear localization of this antisense oligodeoxynucleotide (ODN) directed against cPLA2 mRNA. A central TAAAT motif providing specificity and high affinity binding was assumed to interact with the enzyme Rossmann fold region on the basis of competition to this site by NAD+. To asses whether the TAAAT motif interacts directly with the enzyme Rossmann fold region, we evaluated the binding to GAPDH of different oligonucleotides and the effect of competitors such as NAD+, NADH, mononucleotides, DNA, polyribonucleic acids and polyanions. We found that the dissociation constant for TAAAT containing oligonucleotides was three--to fivefold higher with respect to oligo not containing this motif. By covalently linking 32P-labeled cPLA2p(N)16 to GAPDH and after executing hydrolysis with hydroxylamine, the labeling was exclusively found in the C-terminal domain (aa 286-334). These results indicate that the antisense oligonucleotide interacts with a site not having a defined function but which can be negatively allosterically regulated when NAD+ or polynucleotides are bound to Rossmann fold.
Amino Acid Motifs↗
First-time crystallization and preliminary X-ray crystallographic analysis of a bacterial-archaeal type UMP kinase, a key enzyme in microbial pyrimidine biosynthesis.
UMP phosphorylation, a key step for pyrimidine nucleotide biosynthesis, is catalyzed in bacteria by UMP kinase (UMPK), an enzyme specific for UMP that is dissimilar to the eukaryotic UMP/CMP kinase or to other nucleoside monophosphate kinases. UMPK is allosterically regulated and participates in pyrimidine-triggered gene repression. As first step towards determining UMPK structure, the putative UMPK-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus was cloned and overexpressed in Escherichia coli. The protein product was purified and confirmed to be a genuine UMPK. It was crystallized at 294 K in hanging drops by the vapor diffusion technique using 3.5-4 M Na formate. Cubic 0.2-mm crystals diffracted synchrotron X-rays to 2.4-angstroms resolution. Space group was I23 (a=b=c=144.95 angstroms), and the asymmetric unit contained two monomers, with 52% solvent content. The self-rotation function suggests that the enzyme is hexameric, which agrees with biochemical studies on bacterial UMPKs.
Catalysis↗
Identification of a Zn2+-binding site on the dopamine D2 receptor.
Zinc (II) modulates the function of many integral membrane proteins. To identify the Zn(2+)-binding site responsible for allosteric modulation of the D(2) dopamine receptor, we first demonstrated that the binding site is likely located in extracellular loops or in transmembrane regions that are accessible from the extracellular milieu. We mutated every histidine in these regions to alanine; two mutants, H394A and H399A, exhibited a reduced response to Zn(2+). Combined mutation of H394 and H399 caused a larger effect of zinc than did either single mutation. Mutation of other potential Zn(2+)-binding residues predicted to be in proximity to H394 or H399 did not substantially alter the potency of Zn(2+). The double mutant H394A/H399A was similar to D(2) in affinity for [(3)H]spiperone and ability to inhibit cyclic AMP accumulation. We conclude that binding of Zn(2+) to H394 and H399 on the dopamine D(2) receptor contributes to allosteric regulation of antagonist binding.
Amino Acid Substitution↗
Band 3 (AE1, SLC4A1)-mediated transport of stilbenedisulfonates. II: Evidence for transmembrane allosteric interactions between the "primary" stilbenedisulfonate binding site and the stilbenedisulfonate efflux site.
Results from the first paper in this series indicated that the "primary" stilbenedisulfonate (PSD) site was not located on the DBDS (4, 4'-dibenzamido-2, 2'-stilbenedisulfonate) transport pathway into magnesium resealed ghosts (MRSG). Rather, transport correlated with DBDS binding to the "second" class of proton-activated binding sites located on the membrane domain of band 3 [Biochem. J. 388 (2005) 343]. Here we report the discovery that reversible binding of extracellular H2DIDS (4, 4'-diisothiocyanatodihydro-2, 2'-stilbenedisulfonate) to the PSD site causes a greater than 5-fold acceleration in the rate of DBDS efflux from pre-loaded MRSG at physiological pH. Pre-labeling all of the PSD sites with H2DIDS inhibited the acceleration effect completely, thus confirming mediation by band 3. Acceleration of DBDS efflux could be mimicked by establishing an externally directed proton gradient (acidic inside, pH 7.4 outside). Under these conditions, addition of extracellular H2DIDS neither accelerated DBDS efflux further nor was proton-induced acceleration inhibited. The results of this paper support the view that the PSD binding site on band 3 is an allosteric regulatory site which is not located on the SD transport pathway. We propose a model where H2DIDS binding to the PSD site modulates activity at the "second" class of sites by raising the pK for transport of DBDS into the physiological pH range.
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
6H,13H-Pyrazino[1,2-a;4,5-a']diindole analogs: probing the pharmacophore for allosteric ligands of muscarinic M2 receptors.
A series of 6H,13H-pyrazino[1,2-a;4,5-a']diindole analogs was synthesized in order to probe the pharmacophore hypothesis for allosteric ligands of muscarinic M(2) receptors. The 3D structure of the novel ring system was determined by means of NMR spectroscopy and X-ray diffraction revealing a totally flat geometry. Low binding affinities for the [(3)H]N-methylscopolamine-occupied M(2) receptors (reflected by EC(50,diss)) indicated that the spatial arrangement of the pharmacophore elements (two aromatic rings flanked by two cationic centers) incorporated in the bisquaternary analogs 5 and 6 is unfavorable for strong ligand-receptor interactions. Due to the structural similarity of the novel compounds to neuromuscular-blocking agents, their affinities (reflected by K(i)) to the muscle type of nicotinic acetylcholine receptors were also determined. The dimethyl and diallyl analogs 5 and 6 exhibited rather high affinities to the muscle type of nicotinic acetylcholine receptors, suggesting a pronounced neuromuscular-blocking activity. Compound 5 showed a 34-fold higher affinity for the muscle type nAChR than for the allosteric site of M(2) receptors.
Allosteric Regulation↗
Challenges in the development of mGluR5 positive allosteric modulators: the discovery of CPPHA.
This Letter describes, for the first time, the synthesis and SAR, developed through an iterative analog library approach, that led to the discovery of the positive allosteric modulator (PAM) of the metabotropic glutamate receptor mGluR5 CPPHA. Binding to a unique allosteric binding site distinct from other mGluR5 PAMs, CPPHA has been the focus of numerous pharmacology studies by several laboratories.
Allosteric Regulation↗
Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons.
Tetramethylpyrazine (TMP) is one of the alkaloids contained in Ligustrazine which has been used in traditional Chinese medicine as an analgesic for injury and dysmenorrhea. ATP can elicit the sensation of pain. This study observed the effects of TMP on ATP-activated current (IATP) in rat DRG neurons. TMP (0.1-1 mM) concentration-dependently inhibited ATP (100 microM)-activated current in rat DRG neurons. The inhibitory time of ATP (100 microM)-activated current appeared at 15 s after preapplication of TMP and reached its peak at about 45 s. The dose-response curves for IATP in the absence and presence of 1 mM TMP showed that TMP (1 mM) shifted the concentration-response curve of IATP downward markedly and the two EC50 values were very close (75 vs. 82 microM), while the threshold value remained unchanged. Therefore, the inhibitory effect of TMP on IATP may be noncompetitive. TMP did not alter the reversal potential (0 mV) of ATP-activated current, indicating that the site of TMP action is on or near the exterior surface of channel protein and not within the channel pore. Externally applied TMP (1 mM) increases the inhibitory effect of chelerythrine (PKC inhibitor) contained in pipette solution on IATP. The site of TMP action may be the binding of TMP to an allosteric site on the large extracellular region of ATP receptor-ion channel complex (P2X receptors) or PKC site of the N-terminus of P2X receptors. The mechanism of TMP action may be the allosteric regulation via acting on the large extracellular region of ATP receptor-ion channel complex (P2X receptors) and promoting the phosphorylation of PKC site of the N-terminus of P2X receptors.
Action Potentials↗
Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine.
ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.
Adenosine Diphosphate↗
Aptamers meet allostery.
Engineered RNAs have demonstrated remarkable properties of molecular recognition and allosteric function. Liu and colleagues now report the isolation and in vivo function of a ligand-dependent RNA-based transcription factor that opens wide the door for allosterically controllable aptamers.
Allosteric Regulation↗
Engineering switches, genetically.
Ostermeier, Guntas, and Mitchel describe a new approach to design enzymes that are allosterically regulated by an unrelated ligand . The resulting protein, constructed by nonhomologous recombination and genetic screens, displays switch-like behavior.
Binding Sites↗
Structural biochemistry of ATP-driven dimerization and DNA-stimulated activation of SMC ATPases.
Structural maintenance of chromosome (SMC) proteins play a central role in higher-order chromosome structure in all kingdoms of life. SMC proteins consist of a long coiled-coil domain that joins an ATP binding cassette (ABC) ATPase domain on one side and a dimerization domain on the other side. SMC proteins require ATP binding or hydrolysis to promote cohesion and condensation, which is suggested to proceed via formation of SMC rings or assemblies. To learn more about the role of ATP in the architecture of SMC proteins, we report crystal structures of nucleotide-free and ATP bound P. furiosus SMC ATPase domains. ATP dimerizes two SMC ATPase domains by binding to opposing Walker A and signature motifs, indicating that ATP binding can directly assemble SMC proteins. DNA stimulates ATP hydrolysis in the engaged SMC ABC domains, suggesting that ATP hydrolysis can be allosterically regulated. Structural and mutagenesis data identify an SMC protein conserved-arginine finger that is required for DNA stimulation of the ATPase activity and directly connects a putative DNA interaction site to ATP. Our results suggest that stimulation of the SMC ATPase activity may be a specific feature to regulate the ATP-driven assembly and disassembly of SMC proteins.
Adenosine Triphosphatases↗
The Cool-2/alpha-Pix protein mediates a Cdc42-Rac signaling cascade.
BACKGROUND: Cloned-out of library-2 (Cool-2)/PAK-interactive exchange factor (alpha-Pix) was identified through its ability to bind the Cdc42/Rac target p21-activated kinase (PAK) and has been implicated in certain forms of X-linked mental retardation as well as in growth factor- and chemoattractant-coupled signaling pathways. We recently found that the dimeric form of Cool-2 is a specific guanine nucleotide exchange factor (GEF) for Rac, whereas monomeric Cool-2 is a GEF for Cdc42 as well as Rac. However, unlike many GEFs, Cool-2 binds to activated forms of Cdc42 and Rac. Thus, we have investigated the functional consequences of these interactions. RESULTS: We show that the binding of activated Cdc42 to the Cool-2 dimer markedly enhances its ability to associate with GDP bound Rac1, resulting in a significant activation of Rac-GEF activity. While the Rac-specific GEF activity of Cool-2 is mediated through the Dbl homology (DH) domain from one monomer and the Pleckstrin homology domain from the other, activated Cdc42 interacts with the DH domain, most likely opposite the DH domain binding site for GDP bound Rac. Activated Rac also binds to Cool-2; however, it strongly inhibits the GEF activity of dimeric Cool-2. CONCLUSIONS: We provide evidence for novel mechanisms of allosteric regulation of the Rac-GEF activity of the Cool-2 dimer, involving stimulatory effects by Cdc42 and feedback inhibition by Rac. These findings demonstrate that by serving as a target for GTP bound Cdc42 and a GEF for Rac, Cool-2 mediates a GTPase cascade where the activation of Cdc42 is translated into the activation of Rac.
Animals↗
Screening for positive allosteric modulators of biological targets.
The benefit of using positive allosteric modulators of protein function in the therapy of human diseases is becoming more apparent. The advantage of positive allosteric modulators is that they can possess specificity and selectivity profiles as well as concentration-independent limits on activity that can significantly reduce off-target effects in vivo. However, many current screening paradigms are not designed to discover positive allosteric modulators, and modulators that are discovered serendipitously can be overlooked during the hit-picking process. The conditions needed to discover positive allosteric modulators in a HTS are reasonable and simple to implement, generally requiring consideration of the ligand concentration in a screen. Other considerations in the screening for positive allosteric modulators can be derived from the analysis of simple kinetic schemes that describe the interactions of ligands and modulators with different protein targets.
Allosteric Regulation↗
Sites of positive allosteric modulation by neurosteroids on ionotropic gamma-aminobutyric acid receptor subunits.
Neurosteroids are known as allosteric modulators of ionotropic gamma-aminobutyric acid (GABA) receptors. Here, we investigated sites of positive allosteric modulation by allotetrahydrodeoxycorticosterone (5alpha-THDOC) at GABA receptors using the technique of chimeragenesis and the Xenopus oocyte expression system. Our findings have demonstrated that the region from transmembrane segment (TM) 4 to the C-terminus of the GABA(A) receptor alpha1 subunit is crucial for the action of 5alpha-THDOC, but insufficient for the action of another neurosteroid allopregnanolone, suggesting that a specific region critical for neurosteroid action at GABA receptors exists in the domain between TM4 and the C-terminus of GABA receptor subunits.
Allosteric Regulation↗
A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum.
Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.
Corynebacterium↗