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PubMed2002

Allosteric negative regulation of smt O/P binding of the zinc sensor, SmtB, by metal ions: a coupled equilibrium analysis.

The Synechococcus PCC 7942 smt operon is responsible for cellular resistance to excess zinc and consists of two divergently transcribed genes, smtB and smtA. SmtB is the Zn(II)-sensing metal-regulated repressor of the system and binds to a 12-2-12 imperfect inverted repeat in the smtA O/P region. Using fluorescence anisotropy to monitor SmtB-smt O/P multiple equilibria, we show that four SmtB homodimers bind to a 40 bp oligonucleotide containing a single 12-2-12 inverted repeat. The binding affinities of the first two dimers are very tight (K(int) = 2.9 x 10(9) M(-1)) with the affinities of the third and fourth dimers lower by approximately 10- and approximately 30-fold, respectively. A single monomer equivalent of Zn(II), Cd(II), or Co(II) promotes disassembly of the oligomeric complex to a mixture of (P(2)).D and (P(2))(2).D SmtB dimer-DNA complexes with the intrinsic affinity of all SmtB homodimers for DNA greatly reduced by approximately 500-2000-fold. Substitution or derivatization of cysteines which comprise the alpha3N metal binding site (Cys14 and Cys61) [VanZile, M. L., et al. (2002) Biochemistry 41, 9765-9775] has no effect on allosteric negative regulation by Zn(II); in contrast, H106Q SmtB, harboring a single zinc-liganding substitution in the alpha5 metal binding site, is refractory to zinc-induced disassembly of SmtB-DNA complexes. The alpha5 metal binding sites are therefore regulatory for Zn(II) sensing in vitro and in vivo, while the high-affinity alpha3N sites play some other role. This finding for SmtB is the opposite of that previously determined for Staphylococcus aureus pI258 CadC, a Pb(II)/Cd(II)/Bi(III) sensor [Busenlehner, L. S., et al. (2002) J. Mol. Biol. 319, 685-701], thus providing insight into the origin of functional metal ion selectivity in this family of metal sensor proteins.

Allosteric Regulation↗
PubMed1977

[A mathematical model of the pyruvate oxidation in liver mitochondria. 1. Regulation of the Krebs cycle by adenine and pyridine nucleotides].

A mathematical model is proposed to describe the behavior of the pyruvate metabolic reactions, Krebs cycle and oxidative phosphorylation over a wide range of changes in the pyruvate influx rate and the activities of ATPase and NADH-reoxidating dehydrogenase. The role of adenine and pyridine nucleotides in various allosteric regulations of the Krebs cycle enzymes is discussed. The accumulation of ATP and NADH has been shown to proceed in definite succession, which makes the allosteric regulation of the Krebs cycle enzymes successive too. First "works" the inhibition by ATP, then by NADH. It has been shown that the properties of the model are in qualitative agreement with the experimental data (Garber A., Hanson R. [1]) on pyruvate oxidation by mitochondria from guinea pig liver, when allosteric regulation of isocitrate dehydrogenase by adenine nucleotides is taken into account.

Adenine Nucleotides↗
PubMed1999

Caspase-9 and APAF-1 form an active holoenzyme.

Autocatalytic activation of initiator caspases is the link between pro-apoptotic signals and the destruction machinery of apoptosis. Activation of caspase-9, which mediates oncogene and drug-induced apoptosis, requires binding to the protein APAF-1. We found that the proteolytic activity of caspase-9 in a complex with APAF-1 is several orders of magnitude higher than that of the free enzyme. Thus, this complex functions as a holoenzyme in which caspase-9 is the catalytic subunit and APAF-1 its allosteric regulator. We argue that caspase-9 is activated by allosteric regulation and suggest that this mechanism is common for other initiator caspases.

Allosteric Regulation↗
PubMed1996

[Structure, regulation, and denaturation of muscle glycogen phosphorylase b].

The review summarizes data on structure, allosteric regulation, and denaturation of muscle glycogen phosphorylase b. Specific attention is paid to correlations between the structure and function of phosphorylase b and molecular mechanism of its allosteric regulation. Chemical and thermal denaturation of phosphorylase b is reviewed.

Allosteric Regulation↗
PubMed2005

Quantifying allosteric effects in proteins.

In allosteric regulation, protein activity is altered when ligand binding causes changes in the protein conformational distribution. Little is known about which aspects of protein design lead to effective allosteric regulation, however. To increase understanding of the relation between protein structure and allosteric effects, we have developed theoretical tools to quantify the influence of protein-ligand interactions on probability distributions of reaction rates and protein conformations. We define the rate divergence, Dk, and the allosteric potential, Dx, as the Kullback-Leibler divergence between either the reaction-rate distributions or protein conformational distributions with and without the ligand bound. We then define Dx as the change in the conformational distribution of the combined protein/ligand system, derive Dx in the harmonic approximation, and identify contributions from 3 separate terms: the first term, D[stackxomega], results from changes in the eigenvalue spectrum; the second term, D[stackxDeltax], results from changes in the mean conformation; and the third term, Dxv, corresponds to changes in the eigenvectors. Using normal modes analysis, we have calculated these terms for a natural interaction between lysozyme and the ligand tri-N-acetyl-D-glucosamine, and compared them with calculations for a large number of simulated random interactions. The comparison shows that interactions in the known binding-site are associated with large values of Dxv. The results motivate using allosteric potential calculations to predict functional binding sites on proteins, and suggest the possibility that, in Nature, effective ligand interactions occur at intrinsic control points at which binding induces a relatively large change in the protein conformational distribution.

Allosteric Regulation↗
PubMed2003

Insights into the evolution of allosteric properties. The NADH binding site of hexameric type II citrate synthases.

Study of the hexameric and allosterically regulated citrate synthases (type II CS) provides a rare opportunity to gain not only an understanding of a novel allosteric mechanism but also insight into how such properties can evolve from an unregulated structural platform (the dimeric type I CS). To address both of these issues, we have determined the structure of the complex of NADH (a negative allosteric effector) with the F383A variant of type II Escherichia coli CS. This variant was chosen because its kinetics indicate it is primarily in the T or inactive allosteric conformation, the state that strongly binds to NADH. Our structural analyses show that the six NADH binding sites in the hexameric CS complex are located at the interfaces between dimer units such that most of each site is formed by one subunit, but a number of key residues are drawn from the adjacent dimer. This arrangement of interactions serves to explain why NADH allosteric regulation is a feature only of hexameric type II CS. Surprisingly, in both the wild-type enzyme and the NADH complex, the two subunits of each dimer within the hexameric conformation are similar but not identical in structure, and therefore, while the general characteristics of NADH binding interactions are similar in each subunit, the details of these are somewhat different between subunits. Detailed examination of the observed NADH binding sites indicates that both direct charged interactions and the overall cationic nature of the sites are likely responsible for the ability of these sites to discriminate between NADH and NAD(+). A particularly novel characteristic of the complex is the horseshoe conformation assumed by NADH, which is strikingly different from the extended conformation found in its complexes with most proteins. Sequence homology studies suggest that this approach to binding NADH may arise out of the evolutionary need to add an allosteric regulatory function to the base CS structure. Comparisons of the amino acid sequences of known type II CS enzymes, from different Gram-negative bacteria taxonomic groups, show that the NADH-binding residues identified in our structure are strongly conserved, while hexameric CS molecules that are insensitive to NADH have undergone key changes in the sequence of this part of the protein.

Allosteric Regulation↗
PubMed2005

Crystal structure of potato tuber ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase catalyzes the first committed and rate-limiting step in starch biosynthesis in plants and glycogen biosynthesis in bacteria. It is the enzymatic site for regulation of storage polysaccharide accumulation in plants and bacteria, being allosterically activated or inhibited by metabolites of energy flux. We report the first atomic resolution structure of ADP-glucose pyrophosphorylase. Crystals of potato tuber ADP-glucose pyrophosphorylase alpha subunit were grown in high concentrations of sulfate, resulting in the sulfate-bound, allosterically inhibited form of the enzyme. The N-terminal catalytic domain resembles a dinucleotide-binding Rossmann fold and the C-terminal domain adopts a left-handed parallel beta helix that is involved in cooperative allosteric regulation and a unique oligomerization. We also report structures of the enzyme in complex with ATP and ADP-glucose. Communication between the regulator-binding sites and the active site is both subtle and complex and involves several distinct regions of the enzyme including the N-terminus, the glucose-1-phosphate-binding site, and the ATP-binding site. These structures provide insights into the mechanism for catalysis and allosteric regulation of the enzyme.

Adenosine Diphosphate↗
PubMed2001

Allosteric phenomena in cytochrome P450-catalyzed monooxygenations.

Allosteric regulation of monooxygenase activity is shown to occur with diverse cytochrome P450 isoforms and is characterized by kinetic patterns deviating from the Michaelis-Menten model. Homotropic and heterotropic phenomena are encountered in both substrate activation and productive coupling of the electron donors NADPH-cytochrome P450 reductase and cytochrome b5, and the lipid environment of the system also appears to play a role as an effector. Circumstantial analysis reveals the components of the electron transfer chain to be mutually beneficial in interactions with each other depending on the substrate used and type of cytochrome P450 operative. It is noteworthy that association of diatomic gaseous ligands may be amenable to allosteric regulation as well. Thus, dioxygen binding to cytochrome P450 displays nonhyperbolic kinetic profiles in the presence of certain substrates; the latter, together with redox proteins such as cytochrome b5, can exert efficient control of the abortive breakdown of the oxyferrous intermediates formed. Similarly, substrates may modulate the structural features of the access channel for solutes such as carbon monoxide in specific cytochrome P450 isozymes to either facilitate or impair ligand diffusion to the heme iron. The in vivo importance of allosteric regulation of enzyme activity is discussed in detail.

Allosteric Regulation↗
PubMed2004

Three-dimensional structure of the R115E mutant of T4-bacteriophage 2'-deoxycytidylate deaminase.

2'-Deoxycytidylate deaminase (dCD) converts deoxycytidine 5'-monophosphate (dCMP) to deoxyuridine 5'-monophosphate and is a major supplier of the substrate for thymidylate synthase, an important enzyme in DNA synthesis and a major target for cancer chemotherapy. Wild-type dCD is allosterically regulated by the end products of its metabolic pathway, deoxycytidine 5'-triphosphate and deoxythymidine 5'-triphosphate, which act as an activator and an inhibitor, respectively. The first crystal structure of a dCD, in the form of the R115E mutant of the T4-bacteriophage enzyme complexed with the active site inhibitor pyrimidin-2-one deoxyribotide, has been determined at 2.2 A resolution. This mutant of dCD is active, even in the absence of the allosteric regulators. The molecular topology of dCD is related to that of cytidine deaminase (CDA) but with modifications for formation of the binding site for the phosphate group of dCMP. The enzyme has a zinc ion-based mechanism that is similar to that of CDA. A second zinc ion that is present in bacteriophage dCD, but absent in mammalian dCD and CDA, is important for the structural integrity of the enzyme and for the binding of the phosphate group of the substrate or inhibitor. Although the R115E mutant of dCD is a dimer in solution, it crystallizes as a hexamer, mimicking the natural state of the wild-type enzyme. Residues 112 and 115, which are known to be important for the binding of the allosteric regulators, are found in a pocket that is at the intersubunit interfaces in the hexamer but distant from the substrate-binding site. The substrate-binding site is composed of residues from a single protein molecule and is sequestered in a deep groove. This groove is located at the outer surface of the hexamer but ends at the subunit interface that also includes residue 115. It is proposed that the absence of subunit interactions at this interface in the dimeric R115E mutant renders the substrate-binding site accessible. In contrast, for the wild-type enzyme, binding of dCTP induces an allosteric effect that affects the subunit interactions and results in an increase in the accessibility of the binding site.

Allosteric Regulation↗
PubMed1996

Regulation of D2 dopamine receptors by amiloride and amiloride analogs.

Allosteric regulation of rat D2 dopamine receptors by amiloride and amiloride analogs has been studied by investigating their ability to accelerate the dissociation of [3H]spiperone from the receptors expressed in Ltk- cells. The amiloride analogs were more potent and produced a greater maximal effect on the rate of [3H]spiperone dissociation than did amiloride. [3H]Spiperone dissociation was biphasic and could be resolved into contributions from fast and slow rates in the absence and presence of the modulators. Methylisobutylamiloride accelerated both the fast and slow rates of dissociation and modulated the proportions of the two rates. The association of [3H]spiperone in the absence of modulators was also biphasic. The combination of the two sets of association and dissociation rate constants gave very similar equilibrium dissociation constants, and this was confirmed in equilibrium binding experiments that were consistent with a single binding site. It is proposed that there are two binding states for [3H]spiperone that can be distinguished kinetically but not in equilibrium binding experiments. The proportions of these states are differentially modulated with the use of sodium ions and magnesium ions, whereas GTP has no significant effect. Allosteric regulation of [3H]spiperone binding by methylisobutylamiloride could also be observed in saturation and inhibition binding experiments. These effects can be accounted for in a model in which the modulator binds to the competitive site of the receptor and to an allosteric site on the receptor from which it exerts negatively cooperative effects on [3H]spiperone binding to the competitive site and positively cooperative effects on the binding of the modulator to the competitive site.

Amiloride↗
PubMed2006

Structural insights into homo- and heterotropic allosteric coupling in the zinc sensor S. aureus CzrA from covalently fused dimers.

The Zn(II)/Co(II)-sensing transcriptional repressor, Staphylococcus aureus CzrA, is a homodimer containing a symmetry-related pair of subunit-bridging tetrahedral N(3)O metal sensor coordination sites. A metal-induced quaternary structural change within the homodimer is thought to govern the biological activity of this and other metal sensor proteins. Here, we exploit covalent (Gly(4)Ser)(n)() linkers of variable length in "fused" CzrAs, where n = 1 (designated 5L-fCzrA), 2 (10L-fCzrA), or 3 (15L-fCzrA), as molecular rulers designed to restrict any quaternary structural changes that are associated with metal binding and metal-mediated allosteric regulation of DNA binding to varying degrees. While 15L-fCzrA exhibits properties most like homodimeric CzrA, shortening the linker in 10L-fCzrA abolishes negative homotropic cooperativity of Zn(II) binding and reduces DNA binding affinity of the apoprotein significantly. Decreasing the linker length further in 5L-fCzrA effectively destroys one metal site altogether and further reduces DNA binding affinity. However, Zn(II) negatively regulates DNA binding of all fCzrAs, with allosteric coupling free energies (DeltaG(1)(c)) of 4.6, 3.1, and 2.7 kcal mol(-1) for 15L-, 10L-, and 5L-fCzrAs, respectively. Introduction of a single nonliganding H97N substitution into either the N-terminal or C-terminal protomer domain in 10L-fCzrA results in DeltaG(1)(c) = 2.6 kcal mol(-1) or approximately 83% that of 10L-fCzrA; in contrast, homodimeric H97N CzrA gives DeltaG(1)(c) = 0. (1)H-(15)N HSQC spectra acquired for wt-, 10L-fCzrA and H97N 10L-fCzrA in various Zn(II) ligation states reveal that the allosteric change of the protomer domains within the fused dimer is independent and not concerted. Thus, occupancy of a single metal site by Zn(II) introduces asymmetry into the CzrA homodimer that leads to significant allosteric regulation of DNA binding.

Bacterial Proteins↗
PubMed1996

Long-term rod dark adaptation in man. Threshold measurements, rhodopsin regeneration and allosteric sensitivity regulation. An evaluation.

Recent evidence strongly suggests that the relationship between threshold elevation (T) and fraction of bleached rhodopsin (B), obtained during a major, middle period of long-term rod dark adaptation in man, is well described by a power function, i.e., T = k.Bn, where k is a multiplicative constant and n is the exponent. Due primarily to the low reliability of measurements of rhodopsin regeneration, however, the exponent n of the power function cannot, at present, be given an exact value. Available information indicates that the value of the exponent ranges between 2.4 and 4. Implications of this uncertainty are discussed within the framework of the allosteric, tetrameric model of rod dark adaptation. It is concluded that this model in its simplest form may only offer a first approximation of the real system implicated in the process.

Allosteric Regulation↗
PubMed2006

Networks for the allosteric control of protein kinases.

The allosteric regulation of protein kinases serves as an efficient strategy for molecular communication, event coupling and interconversion between catalytic states. Recent co-crystal structures have revealed novel ways in which kinases control activity and substrate specificity following phosphorylation, dimerization, or binding to regulatory proteins, substrates and scaffolds. In addition, hydrogen exchange coupled with mass spectrometry is emerging as a complementary strategy to probe the solution behavior of kinases; recent results have shown that allosteric regulation may involve transitions in protein motions as well as structural rearrangements.

Allosteric Regulation↗
PubMed1983

[Oxygen-dissociation properties and the regulation of the oxygen affinity of a hemoglobin polymer in the bull].

Oxygen-dissociation properties and some aspects of allosteric regulation of bovine hemoglobin (Hb) and its polymerized form (HbP) have been examined. It has been shown that bovine HbP as well as bovine Hb does not require the presence of organic phosphates for normal functioning and has gas transport characteristics which are similar to those of the whole human blood. Pyridoxal-5'-phosphate (PP) works as an allosteric regulator of bovine HbP only in the absence of Cl- ions and vice versa. However, in covalent binding of PP to HbP both the effectors (PP and Cl- ions) can work simultaneously and independently. Thus, by varying the Hb/PP ratio it is possible to prepare HbP solutions with preset P50 values within the range 30-55 mm Hg. The results can be used in simulation of gas transport characteristics of artificial oxygen carriers.

Allosteric Regulation↗
PubMed2006

NMR dynamic studies suggest that allosteric activation regulates ligand binding in chicken liver bile acid-binding protein.

Apo chicken liver bile acid-binding protein has been structurally characterized by NMR. The dynamic behavior of the protein in its apo- and holo-forms, complexed with chenodeoxycholate, has been determined via (15)N relaxation and steady state heteronuclear (15)N((1)H) nuclear Overhauser effect measurements. The dynamic parameters were obtained at two pH values (5.6 and 7.0) for the apoprotein and at pH 7.0 for the holoprotein, using the model free approach. Relaxation studies, performed at three different magnetic fields, revealed a substantial conformational flexibility on the microsecond to millisecond time scales, mainly localized in the C-terminal face of the beta-barrel. The observed dynamics are primarily caused by the protonation/deprotonation of a buried histidine residue, His(98), located on this flexible face. A network of polar buried side chains, defining a spine going from the E to J strand, is likely to provide the long range connectivity needed to communicate motion from His(98) to the EF loop region. NMR data are accompanied by molecular dynamics simulations, suggesting that His(98) protonation equilibrium is the triggering event for the modulation of a functionally important motion, i.e. the opening/closing at the protein open end, whereas ligand binding stabilizes one of the preexisting conformations (the open form). The results presented here, complemented with an analysis of proteins belonging to the intracellular lipid-binding protein family, are consistent with a model of allosteric activation governing the binding mechanism. The functional role of this mechanism is thoroughly discussed within the framework of the mechanism for the enterohepatic circulation of bile acids.

Allosteric Regulation↗
PubMed2002

Single amino acid substitution (G456A) in the vicinity of the GTP binding domain of human housekeeping glutamate dehydrogenase markedly attenuates GTP inhibition and abolishes the cooperative behavior of the enzyme.

Human glutamate dehydrogenase (GDH) exists in two isoforms encoded by the GLUD1 and GLUD2 genes, respectively. Although the two enzymes share in their mature form all but 15 of their 505 amino acids, they differ markedly in their allosteric regulation. To identify the structural basis for these allosteric characteristics, we performed site-directed mutagenesis on the human GLUD1 gene at sites that differ from the GLUD2 gene using a cloned GLUD1 cDNA. Results showed that substitution of Ala for Gly-456, but not substitution of His for Arg-470 or Ser for Asn-498, renders the enzyme markedly resistant to GTP inhibition (IC(50) = 2.80 microm) as compared with the wild type GLUD1-derived GDH (IC(50) = 0.19 microm). The G456A mutation abolished the cooperative behavior of the enzyme, as revealed by the GTP inhibitory curves. The catalytic and kinetic properties of the G456A mutant and its activation by ADP were comparable with those of the wild type GDH. Gly-456 lies in a very tightly packed region of the GDH molecule, and its replacement by Ala may lead to steric clashes with neighboring amino acids. These, in turn, may affect the conformational state of the protein that is essential for the allosteric regulation of the enzyme by GTP.

Adenosine Diphosphate↗
PubMed2006

Ribonucleotide reductases.

Ribonucleotide reductases (RNRs) transform RNA building blocks to DNA building blocks by catalyzing the substitution of the 2'OH-group of a ribonucleotide with a hydrogen by a mechanism involving protein radicals. Three classes of RNRs employ different mechanisms for the generation of the protein radical. Recent structural studies of members from each class have led to a deeper understanding of their catalytic mechanism and allosteric regulation by nucleoside triphosphates. The main emphasis of this review is on regulation of RNR at the molecular and cellular level. Conformational transitions induced by nucleotide binding determine the regulation of substrate specificity. An intricate interplay between gene activation, enzyme inhibition, and protein degradation regulates, together with the allosteric effects, enzyme activity and provides the appropriate amount of deoxynucleotides for DNA replication and repair. In spite of large differences in the amino acid sequences, basic structural features are remarkably similar and suggest a common evolutionary origin for the three classes.

Allosteric Regulation↗
PubMed2006

Mining for allosteric information: natural mutations and positional sequence conservation in pyruvate kinase.

Although the amino acid sequences and the structures of pyruvate kinase (PYK) isozymes are highly conserved, allosteric regulations differ. This suggests that amino acids with low conservation play important roles in the allosteric mechanism. The current work exploits a 'natural screen'- the 122 point mutations identified in the human gene encoding the erythrocyte PYK isozyme and associated with nonspherocytic hemolytic anemia - to learn what amino acid positions in PYK may be important for allosteric regulations. In addition to the mutations, we consider the conservation of each amino acid position across 241 PYK sequences. Three groups of residue positions have been created, those with: (1) no disease causing mutation identified; (2) a disease causing mutation identified and high conservation across isozymes; and (3) a disease causing mutation identified and low conservation. Mutations at positions not identified in the natural screen are likely to be tolerated with minimal loss of function. Mutations at highly conserved positions are more likely to disrupt properties common to all PYK isozymes (e.g., structure, catalysis). Residues in the third group are likely to be involved in roles that are necessary for function but not common to all isozymes (e.g., allostery). Many of the Group 3 residues are located in the C-domain and to a lesser extent the A domain.

Allosteric Regulation↗