Antje Brüser

Structure and allosteric regulation of eukaryotic 6-phosphofructokinases.

Abstract Although the crystal structures of prokaryotic 6-phosphofructokinase, a key enzyme of glycolysis, have been available for almost 25 years now, structural information about the more complex and highly regulated eukaryotic enzymes is still lacking until now. This review provides an overview of the current knowledge of eukaryotic 6-phosphofructokinase based on recent crystal structures, kinetic analyses and site-directed mutagenesis data with special focus on the molecular architecture and the structural basis of allosteric regulation.

The Activation Mechanism of Glycoprotein Hormone Receptors with Implications in the Cause and Therapy of Endocrine Diseases

Glycoprotein hormones (GPHs) are the main regulators of the pituitary-thyroid and pituitary-gonadal axes. Selective interaction between GPHs and their cognate G protein-coupled receptors ensure specificity in GPH signaling. The mechanisms of how these hormones activate glycoprotein hormone receptors (GPHRs) or how mutations and autoantibodies can alter receptor function were unclear. Based on the hypothesis that GPHRs contain an internal agonist, we systematically screened peptide libraries derived from the ectodomain for agonistic activity on the receptors. We show that a peptide (p10) derived from a conserved sequence in the C-terminal part of the extracellular N terminus can activate all GPHRs in vitro and in GPHR-expressing tissues. Inactivating mutations in this conserved region or in p10 can inhibit activation of the thyroid-stimulating hormone receptor by autoantibodies. Our data suggest an activation mechanism where, upon extracellular ligand binding, this intramolecular agonist isomerizes and induces structural changes in the 7-transmembrane helix domain, triggering G protein activation. This mechanism can explain the pathophysiology of activating autoantibodies and several mutations causing endocrine dysfunctions such as Graves disease and hypo- and hyperthyroidism. Our findings highlight an evolutionarily conserved activation mechanism of GPHRs and will further promote the development of specific ligands useful to treat Graves disease and other dysfunctions of GPHRs.

Molecular architecture and structural basis of allosteric regulation of eukaryotic phosphofructokinases

Eukaryotic ATP‐dependent 6‐phospho‐fructokinases (Pfks) differ from their bacterial counterparts in a much more complex structural organization and allosteric regulation. Pichia pastoris Pfk (PpPfk) is, with ~1 MDa, the most complex and probably largest eukaryotic Pfk. We have determined the crystal structure of full‐length PpPfk to 3.05 Å resolution in the T state. PpPfk forms a (αβγ)4 dodec‐amer of D2 symmetry with dimensions of 161 × 157 × 233 Å mainly via interactions of the α chains. The N‐terminal domains of the a and β chains have folds that are distantly related to glyoxalase I, but the active sites are no longer functional. Interestingly, these domains located at the 2 distal ends of this protein along the long 2‐fold axis form a (aβ)2 dimer as does the core Pfk domains;however, the domains are swapped across the tetramerization interface. In PpPfk, the unique γ subunit participates in oligomerization of the αβ chains. This modulator protein was acquired from an ancient S‐adenosylmethionine‐dependent methyl‐transferase. The identification of novel ATP binding sites, which do not correspond to the bacterial catalytic or effector binding sites, point to marked structural and functional differences between bacterial and eukaryotic Pfks.—Strater, N., Marek, S., Kuettner, E. B., Kloos, M., Keim, A., Brüser, A., Kirch‐berger, J., Schoneberg, T. Molecular architecture and structural basis of allosteric regulation of eukary‐otic phosphofructokinases. FASEB J. 25, 89–98 (2011). www.fasebj.org

What are they waiting for?—Tethered agonism in G protein-coupled receptors

In classical pharmacology agonists bind to their respective receptors by specific interaction and induce structural changes followed by cellular responses. However, some G protein-coupled receptor (GPCRs), such as rhodopsin and protease-activated receptors (PARs), have their agonists already covalently bound and are parts of the receptor proteins, respectively. Recent studies add adhesion GPCRs and glycoprotein hormone receptors (GPHRs) to the group of GPCRs activated by integral agonists. In contrast to rhodopsin and PARs, adhesion GPCRs and GPHRs exhibit large ectodomains (ECDs) which bind a number of different proteins and other extracellular molecules. It seems that these large size ECDs are required to integrate a multitude of extracellular signals, such as protein ligand binding, cell-cell contacts and even mechanical forces, into uniform intracellular signals. Upon extracellular ligand binding, the intramolecular agonist of those receptors is exposed or isomerizes and induces structural changes in the 7-transmembrane helix domain triggering G-protein activation. The existence of activating structures integrated in receptor molecules challenges our current pharmacological definition of an agonist. We summarized and discussed the specifics of tethered agonist pharmacology which add a number of new features of the already broad signaling abilities of GPCRs and may find useful applications in designer GPCRs.

Functional Linkage of Adenine Nucleotide Binding Sites in Mammalian Muscle 6-Phosphofructokinase *

Background: Crystal structures of 6-phosphofructokinases revealed nucleotide binding sites with unknown functional relevance. Results: Function of two allosteric nucleotide binding sites was determined by mutagenesis and kinetic studies and revealed reciprocal linkage of both. Conclusion: Activity of mammalian Pfk is regulated by structurally linked new allosteric sites. Significance: Reciprocal linkage between allosteric binding sites evolved convergent in prokaryotes and eukaryotes. 6-Phosphofructokinases (Pfk) are homo- and heterooligomeric, allosteric enzymes that catalyze one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk revealed several adenine nucleotide binding sites. Herein, we determined the functional relevance of two adenine nucleotide binding sites through site-directed mutagenesis and enzyme kinetic studies. Subsequent characterization of Pfk mutants allowed the identification of the activating (AMP, ADP) and inhibitory (ATP, ADP) allosteric binding sites. Mutation of one binding site reciprocally influenced the allosteric regulation through nucleotides interacting with the other binding site. Such reciprocal linkage between the activating and inhibitory binding sites is in agreement with current models of allosteric enzyme regulation. Because the allosteric nucleotide binding sites in eukaryotic Pfk did not evolve from prokaryotic ancestors, reciprocal linkage of functionally opposed allosteric binding sites must have developed independently in prokaryotic and eukaryotic Pfk (convergent evolution).

Prostaglandin E 2 glyceryl ester is an endogenous agonist of the nucleotide receptor P2Y 6

Cyclooxygenase-2 catalyses the biosynthesis of prostaglandins from arachidonic acid but also the biosynthesis of prostaglandin glycerol esters (PG-Gs) from 2-arachidonoylglycerol. Previous studies identified PG-Gs as signalling molecules involved in inflammation. Thus, the glyceryl ester of prostaglandin E2, PGE2-G, mobilizes Ca2+ and activates protein kinase C and ERK, suggesting the involvement of a G protein-coupled receptor (GPCR). To identify the endogenous receptor for PGE2-G, we performed a subtractive screening approach where mRNA from PGE2-G response-positive and -negative cell lines was subjected to transcriptome-wide RNA sequencing analysis. We found several GPCRs that are only expressed in the PGE2-G responder cell lines. Using a set of functional readouts in heterologous and endogenous expression systems, we identified the UDP receptor P2Y6 as the specific target of PGE2-G. We show that PGE2-G and UDP are both agonists at P2Y6, but they activate the receptor with extremely different EC50 values of ~1 pM and ~50 nM, respectively. The identification of the PGE2-G/P2Y6 pair uncovers the signalling mode of PG-Gs as previously under-appreciated products of cyclooxygenase-2.

Crystal structure of human platelet phosphofructokinase-1 locked in an activated conformation

Phosphofructokinase-1 (Pfk) acts as the main control point of flux through glycolysis. It is involved in complex allosteric regulation and Pfk mutations have been linked to cancer development. Whereas the 3D structure and structural basis of allosteric regulation of prokaryotic Pfk has been studied in great detail, our knowledge about the molecular basis of the allosteric behaviour of the more complex mammalian Pfk is still very limited. To characterize the structural basis of allosteric regulation, the subunit interfaces and the functional consequences of modifications in Taruis disease and cancer, we analysed the physiological homotetramer of human platelet Pfk at up to 2.67 Å resolution in two crystal forms. The crystallized enzyme is permanently activated by a deletion of the 22 C-terminal residues. Complex structures with ADP and fructose-6-phosphate (F6P) and with ATP suggest a role of three aspartates in the deprotonation of the OH-nucleophile of F6P and in the co-ordination of the catalytic magnesium ion. Changes at the dimer interface, including an asymmetry observed in both crystal forms, are the primary mechanism of allosteric regulation of Pfk by influencing the F6P-binding site. Whereas the nature of this conformational switch appears to be largely conserved in bacterial, yeast and mammalian Pfk, initiation of these changes differs significantly in eukaryotic Pfk.

Crystallization and preliminary crystallographic analysis of human muscle phosphofructokinase, the main regulator of glycolysis.

Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great detail, knowledge of the molecular basis of the allosteric behaviour of the far more complex mammalian Pfks is still very limited. The human muscle isozyme was expressed heterologously in yeast cells and purified using a five-step purification protocol. Protein crystals suitable for diffraction experiments were obtained by the vapour-diffusion method. The crystals belonged to space group P6222 and diffracted to 6.0 Å resolution. The 3.2 Å resolution structure of rabbit muscle Pfk (rmPfk) was placed into the asymmetric unit and optimized by rigid-body and group B-factor refinement. Interestingly, the tetrameric enzyme dissociated into a dimer, similar to the situation observed in the structure of rmPfk.

Altered allosteric regulation of muscle 6-phosphofructokinase causes Tarui disease.

Tarui disease is a glycogen storage disease (GSD VII) and characterized by exercise intolerance with muscle weakness and cramping, mild myopathy, myoglobinuria and compensated hemolysis. It is caused by mutations in the muscle 6-phosphofructokinase (Pfk). Pfk is an oligomeric, allosteric enzyme which catalyzes one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk displayed several allosteric adenine nucleotide binding sites. Functional studies revealed a reciprocal linkage between the activating and inhibitory allosteric binding sites. Herein, we showed that Asp(543)Ala, a naturally occurring disease-causing mutation in the activating binding site, causes an increased efficacy of ATP at the inhibitory allosteric binding site. The reciprocal linkage between the activating and inhibitory binding sites leads to reduced enzyme activity and therefore to the clinical phenotype. Pharmacological blockage of the inhibitory allosteric binding site or highly efficient ligands for the activating allosteric binding site may be of therapeutic relevance for patients with Tarui disease.