Hybrid Tetramers of Porcine Liver Fructose-1,6-bisphosphatase Reveal Multiple Pathways of Allosteric Inhibition

Nelson, Scott W.; Honzatko, Richard B.; Fromm, Herbert J.

doi:10.1074/jbc.m112304200

Cited by 24 publications

(26 citation statements)

References 43 publications

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“…Similar studies utilizing hybrid oligomers have been reported for L-lactate dehydrogenase from Bifidobacterium longum (11,12) and for porcine fructose 1,6-biphosphatase (13). Although these enzymes are homo-tetramers like PGDH, they differ significantly in the arrangement of their substrate and effector binding sites.…”

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confidence: 49%

Hybrid Tetramers Reveal Elements of Cooperativity in Escherichia colid-3-Phosphoglycerate Dehydrogenase

Grant

2003

Journal of Biological Chemistry

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D-3-Phosphoglycerate dehydrogenase from Escherichia coli is a tetramer of identical subunits that is inhibited when L-serine binds at allosteric sites between subunits. Co-expression of two genes, the native gene containing a charge difference mutation and a gene containing a mutation that eliminates serine binding, produces hybrid tetramers that can be separated by ion exchange chromatography. Activity in the hybrid tetramer with only a single intact serine binding site is inhibited by ϳ58% with a Hill coefficient of 1. Thus, interaction at a single regulatory domain interface does not, in itself, lead to the positive cooperativity of inhibition manifest in the native enzyme. Tetramers with only two intact serine binding sites purify as a mixture that displays a maximum inhibition level that is less than that of native enzyme, suggesting the presence of a population of tetramers that are unable to be fully inhibited. Differential analysis of this mixture supports the conclusion that it contains two forms of the tetramer. One form contains two intact serine binding sites at the same interface and is not fully inhibitable. The second form is a fully inhibitable population that has one serine binding site at each interface. Overall, the hybrid tetramers show that the positive cooperativity observed for serine binding is mediated across the nucleotide binding domain interface, and the negative cooperativity is mediated across the regulatory domain interface. That is, they reveal a pattern in which the binding of serine at one interface leads to negative cooperativity of binding of a subsequent serine at the same interface and positive cooperativity of binding of a subsequent serine to the opposite interface. This trend is propagated to subsequent binding sites in the tetramer such that the negative cooperativity that is originally manifest at one interface is decreased by subsequent binding of ligand at the opposite interface.

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confidence: 49%

Hybrid Tetramers Reveal Elements of Cooperativity in Escherichia colid-3-Phosphoglycerate Dehydrogenase

Grant

2003

Journal of Biological Chemistry

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“…However, when applied to the study of allosteric systems, this approach fails to reveal the individual contribution to protein function of all possible hybrid protein particles. In only a handful of cases reported in the literature, one particular hybrid species was separated from all possible combinations, yielding valuable insight into the function of the allosteric protein under study (37,38). The indirect nature of such a hybrid/chimeric approach, combined with the complexity in functional data interpretation, call for a rigorous and direct method for assessing the contribution of intersubunit interactions to cooperativity in protein function.…”

Section: Discussionmentioning

confidence: 99%

Direct analysis of cooperativity in multisubunit allosteric proteins

Zandany

Ovadia

Orr

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Allosteric regulation of protein function is a fundamental phenomenon of major importance in many cellular processes. Such regulation is often achieved by ligand-induced conformational changes in multimeric proteins that may give rise to cooperativity in protein function. At the heart of allosteric mechanisms offered to account for such phenomenon, involving either concerted or sequential conformational transitions, lie changes in intersubunit interactions along the ligation pathway of the protein. However, structurefunction analysis of such homooligomeric proteins by means of mutagenesis, although it provides valuable indirect information regarding (allosteric) mechanisms of action, it does not define the contribution of individual subunits nor interactions thereof to cooperativity in protein function, because any point mutation introduced into homooligomeric proteins will be present in all subunits. Here, we present a general strategy for the direct analysis of cooperativity in multisubunit proteins that combines measurement of the effects on protein function of all possible combinations of mutated subunits with analysis of the hierarchy of intersubunit interactions, assessed by using high-order double-mutant cyclecoupling analysis. We show that the pattern of high-order intersubunit coupling can serve as a discriminative criterion for defining concerted versus sequential conformational transitions underlying protein function. This strategy was applied to the particular case of the voltage-activated potassium channel protein (Kv) to provide compelling evidence for a concerted all-or-none activation gate opening of the Kv channel pore domain. An direct and detailed analysis of the contribution of high-order intersubunit interactions to cooperativity in the function of an allosteric protein has not previously been presented. activation gate ͉ allosteric enzymes ͉ double-mutant cycles ͉ voltage-activated potassium channels ͉ non-additivity A llosteric regulation of protein function is often achieved by changes in protein conformation induced by the binding of substrate or other ligand molecules (1). In multisubunit proteins, such conformational changes may give rise to cooperativity in ligand binding. Several mechanistic models, in particular the Monod-Wymann-Changeux (MWC) model (2) and the Koshland-Némethy-Filmer (KNF) model (3), were developed in the 1960s to describe cooperativity in ligand binding. In both models, cooperativity is explained by binding-induced conformational changes in the subunits of the protein that may be concerted (MWC), sequential (KNF), or a combination of both (4). No matter the type of conformational change, cooperativity in ligand binding in all allosteric models is manifested by changes in intersubunit interactions along the ligation pathway of the protein (1).Despite the general acceptance of intersubunit interactions playing a fundamental role in determining the magnitude of cooperativity in ligand binding by an allosteric enzyme, structure-function analysis of such proteins throu...

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“…In the presence of substrate and metal cofactors and in the absence of AMP or Fru-2,6-P 2 , the porcine enzyme is in the R-state, defined by a subunit pair rotation angle of 0°. Transition to the T-state (subunit pair rotation of 15°) by a sequential allosteric mechanism requires a minimum of two bound AMP molecules (66). By binding to an R-state subunit, AMP drives a shearing motion of helices H1 and H2, disrupting intra-and intersubunit hydrogen bonds, the end result of which is an R-state AMP complex of high energy (27).…”

Section: Discussionmentioning

confidence: 99%

“…Models of porcine FBPase, as usual lacking electron density for residues at the N terminus, begin with Asn 9 and go to the C-terminal residue at position 337. Only residues [63][64][65][66][67][68][69][70] are not in strong electron density. These residues are part of the dynamic loop, which is in the disengaged conformation in both the Mg 2ϩ -and Zn 2ϩ -bound complexes of the porcine enzyme.…”

Section: Fru-26-p 2 -Bound Structures Of Porcine Fbpase (Protein Datmentioning

confidence: 99%

Structures of Mammalian and Bacterial Fructose-1,6-bisphosphatase Reveal the Basis for Synergism in AMP/Fructose 2,6-Bisphosphate Inhibition

Hines

Chen

Nix

et al. 2007

Journal of Biological Chemistry

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Fructose-1,6-bisphosphatase (FBPase) operates at a control point in mammalian gluconeogenesis, being inhibited synergistically by fructose 2,6-bisphosphate (Fru-2,6-P 2 ) and AMP. AMP and Fru-2,6-P 2 bind to allosteric and active sites, respectively, but the mechanism responsible for AMP/Fru-2,6-P 2 synergy is unclear. Demonstrated here for the first time is a global conformational change in porcine FBPase induced by Fru-2,6-P 2 in the absence of AMP. The Fru-2,6-P 2 complex exhibits a subunit pair rotation of 13°from the R-state (compared with the 15°rotation of the T-state AMP complex) with active site loops in the disengaged conformation. A three-state thermodynamic model in which Fru-2,6-P 2 drives a conformational change to a T-like intermediate state can account for AMP/Fru-2,6-P 2 synergism in mammalian FBPases. AMP and Fru-2,6-P 2 are not synergistic inhibitors of the Type I FBPase from Escherichia coli, and consistent with that model, the complex of E. coli FBPase with Fru-2,6-P 2 remains in the R-state with dynamic loops in the engaged conformation. Evidently in porcine FBPase, the actions of AMP at the allosteric site and Fru-2,6-P 2 at the active site displace engaged dynamic loops by distinct mechanisms, resulting in similar quaternary end-states. Conceivably, Type I FBPases from all eukaryotes may undergo similar global conformational changes in response to Fru-2,6-P 2 ligation.

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Hybrid Tetramers of Porcine Liver Fructose-1,6-bisphosphatase Reveal Multiple Pathways of Allosteric Inhibition

Cited by 24 publications

References 43 publications

Hybrid Tetramers Reveal Elements of Cooperativity in Escherichia colid-3-Phosphoglycerate Dehydrogenase

Hybrid Tetramers Reveal Elements of Cooperativity in Escherichia colid-3-Phosphoglycerate Dehydrogenase

Direct analysis of cooperativity in multisubunit allosteric proteins

Structures of Mammalian and Bacterial Fructose-1,6-bisphosphatase Reveal the Basis for Synergism in AMP/Fructose 2,6-Bisphosphate Inhibition

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