Oliver Anderka

The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin‐arginine translocase

The Rieske [2Fe−2S] protein (ISP) is an essential subunit of cytochrome bc1 complexes in mitochondrial and bacterial respiratory chains. Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram‐negative soil bacterium, is inserted into the cytoplasmic membrane via the twin‐arginine translocation (Tat) pathway. Here, we provide experimental evidence that membrane integration of the bacterial ISP indeed relies on the Tat translocon. We show that targeting of the ISP depends on the twin‐arginine motif. A strict requirement is established particularly for the second arginine residue (R16); conservative replacement of the first arginine (R15K) still permits substantial ISP transport. Comparative sequence analysis reveals characteristics common to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor. These differences include an elevated hydrophobicity of the h‐region compared with generic Tat signals and the absence of an otherwise well‐conserved ‘+5’‐consensus motif lysine residue. Substitution of the +5 lysine (Y20K) compromises ISP export and/or cytochrome bc1 stability to some extent and points to a specific role for this deviation from the canonical Tat motif. EPR spectroscopy confirms cytosolic insertion of the [2Fe−2S] cofactor. Mutation of an essential cofactor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway.

Direct Demonstration of Half-of-the-sites Reactivity in the Dimeric Cytochrome bc1 Complex ENZYME WITH ONE INACTIVE MONOMER IS FULLY ACTIVE BUT UNABLE TO ACTIVATE THE SECOND UBIQUINOL OXIDATION SITE IN RESPONSE TO LIGAND BINDING AT THE UBIQUINONE REDUCTION SITE

We previously proposed that the dimeric cytochrome bc1 complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc1 monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289–22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc1 complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10–16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c1 by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.

Surf1, Associated with Leigh Syndrome in Humans, Is a Heme-binding Protein in Bacterial Oxidase Biogenesis

Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with Kd values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool.

Biophysical Characterization of the Interaction between Hepatic Glucokinase and Its Regulatory Protein IMPACT OF PHYSIOLOGICAL AND PHARMACOLOGICAL EFFECTORS

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic β-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, ΔH = 15.6 kcal/mol, TΔS = 25.7 kcal/mol, ΔCp = –354 cal mol–1 K–1). With koff = 1.3 × 10–2 s–1, the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.

Influence of Heparin Mimetics on Assembly of the FGF-FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM6), octasaccharide (HM8), and decasaccharide (HM10), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM8 and HM10 are significantly more potent than HM6 in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM8 relative to HM6 in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM8 to bind and dimerize FGF2. Notably FGF2·HM8 exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.

Thermodynamic characterization of allosteric glycogen phosphorylase inhibitors.

Glycogen phosphorylase (GP) is a validated target for the treatment of type 2 diabetes. Here we describe highly potent GP inhibitors, AVE5688, AVE2865, and AVE9423. The first two compounds are optimized members of the acyl urea series. The latter represents a novel quinolone class of GP inhibitors, which is introduced in this study. In the enzyme assay, both inhibitor types compete with the physiological activator AMP and act synergistically with glucose. Isothermal titration calorimetry (ITC) shows that the compounds strongly bind to nonphosphorylated, inactive GP (GPb). Binding to phosphorylated, active GP (GPa) is substantially weaker, and the thermodynamic profile reflects a coupled transition to the inactive (tense) conformation. Crystal structures confirm that the three inhibitors bind to the AMP site of tense state GP. These data provide the first direct evidence that acyl urea and quinolone compounds are allosteric inhibitors that selectively bind to and stabilize the inactive conformation of the enzyme. Furthermore, ITC reveals markedly different thermodynamic contributions to inhibitor potency that can be related to the binding modes observed in the cocrystal structures. For AVE5688, which occupies only the lower part of the bifurcated AMP site, binding to GPb (Kd = 170 nM) is exclusively enthalpic (Delta H = -9.0 kcal/mol, TDelta S = 0.3 kcal/mol). The inhibitors AVE2865 (Kd = 9 nM, Delta H = -6.8 kcal/mol, TDelta S = 4.2 kcal/mol) and AVE9423 (Kd = 24 nM, Delta H = -5.9 kcal/mol, TDelta S = 4.6 kcal/mol) fully exploit the volume of the binding pocket. Their pronounced binding entropy can be attributed to the extensive displacement of solvent molecules as well as to ionic interactions with the phosphate recognition site.

Characterization of mutations in crucial residues around the Qo binding site of the cytochrome bc1 complex from Paracoccus denitrificans

The protonation state of residues around the Qo binding site of the cytochrome bc1 complex from Paracoccus denitrificans and their interaction with bound quinone(s) was studied by a combined electrochemical and FTIR difference spectroscopic approach. Site‐directed mutations of two groups of conserved residues were investigated: (a) acidic side chains located close to the surface and thought to participate in a water chain leading up to the heme bL edge, and (b) residues located in the vicinity of this site. Interestingly, most of the mutants retain a high degree of catalytic activity. E295Q, E81Q and Y297F showed reduced stigmatellin affinity. On the basis of electrochemically induced FTIR difference spectra, we suggest that E295 and D278 are protonated in the oxidized form or that their mutation perturbs protonated residues. Mutations Y302, Y297, E81 and E295, directly perturb signals from the oxidized quinone and of the protein backbone. By monitoring the interaction with the inhibitor stigmatellin for the wild‐type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as protonated acidic residues and the backbone were observed, as well as difference signals arising from the redox active inhibitor itself and the replaced quinone. The infrared difference spectra of the above Qo site mutations in the presence of stigmatellin confirm the previously established role of E295 as a direct interaction partner in the enzyme from P. denitrificans as well. The protonated residue E295 is proposed to change the hydrogen‐bonding environment upon stigmatellin binding in the oxidized form, and is deprotonated in the reduced form. Of the residues located close to the surface, D278 remains protonated and unperturbed in the oxidized form but its frequency shifts in the reduced form. The mechanistic implications of our observations are discussed, together with previous inhibitor binding data, and referred to the published X‐ray structures.

The state of association of the cytochrome bc1 complex from Paracoccus denitrificans in solutions of dodecyl maltoside

The cytochrome bc1 complex is an intrinsic membrane protein of many respiratory chains. We studied the association state of solubilized cytochrome bc1 from Paracoccus denitrificans by sedimentation equilibrium experiments in the analytical ultracentrifuge. Since the stability of the solubilized complex was only maintained in solutions containing low concentrations of the nonionic detergent n-dodecyl-β-d-maltoside (DDM), this detergent was also applied in the ultracentrifuge experiments. DDM is an unfavorable detergent in ultracentrifuge studies owing to its high density (ρ = 1.23 g/ml), which strongly complicates “density-matching”, the standard method for eliminating the contribution of protein-bound detergent to protein molar mass. Use of high sucrose concentrations for matching the DDM density is basically possible but probably induces significant changes in the protein’s partial specific volume, -v; therefore we tried to decrease the necessary sucrose concentration by using mixtures of sucrose and 95% D2O/ 5% H2O (v/v). The effect of the solvent on -v was controlled by studying a related protein of known -v, cytochrome c oxidase, under identical conditions. In sedimentation equilibrium experiments the cytochrome bc1 complex behaved as an ideal homogeneous compound in the presence of 0.02% (w/v) DDM. Its molar mass was determined to be (240,000 ± 30,000) g/mol (mean and maximum error, respectively). Since the calculated mass of the protein protomer is 117,000 g/mol, the solubilized complex represents a dimer. Measurements in water-containing buffers, in the absence of sucrose, showed that the amount of DDM bound by the complex was (0.86 ± 0.12) g/g protein. A dimeric structure was already established for the much larger mitochondrial cytochrome bc1 complexes that had been crystallized. In the case of two other bacterial complexes experimental evidence points in the direction of dimers as well.