Mark L. Richter

Broad Specificity of Mammalian Adenylyl Cyclase for Interaction with 2,3-Substituted Purine- and Pyrimidine Nucleotide Inhibitors

Membrane adenylyl cyclases (mACs) play an important role in signal transduction and are therefore potential drug targets. Earlier, we identified 2′,3′-O-(N-methylanthraniloyl) (MANT)-substituted purine nucleotides as a novel class of highly potent competitive mAC inhibitors (Ki values in the 10 nM range). MANT nucleotides discriminate among various mAC isoforms through differential interactions with a binding pocket localized at the interface between the C1 and C2 domains of mAC. In this study, we examine the structure/activity relationships for 2′,3′-substituted nucleotides and compare the crystal structures of mAC catalytic domains (VC1:IIC2) bound to MANT-GTP, MANT-ATP, and 2′,3′-(2,4,6-trinitrophenyl) (TNP)-ATP. TNP-substituted purine and pyrimidine nucleotides inhibited VC1:IIC2 with moderately high potency (Ki values in the 100 nM range). Elongation of the linker between the ribosyl group and the MANT group and substitution of N-adenine atoms with MANT reduces inhibitory potency. Crystal structures show that MANT-GTP, MANT-ATP, and TNP-ATP reside in the same binding pocket in the VC1:IIC2 protein complex, but there are substantial differences in interactions of base, fluorophore, and polyphosphate chain of the inhibitors with mAC. Fluorescence emission and resonance transfer spectra also reflect differences in the interaction between MANT-ATP and VC1:IIC2 relative to MANT-GTP. Our data are indicative of a three-site mAC pharmacophore; the 2′,3′-O-ribosyl substituent and the polyphosphate chain have the largest impact on inhibitor affinity and the nucleotide base has the least. The mAC binding site exhibits broad specificity, accommodating various bases and fluorescent groups at the 2′,3′-O-ribosyl position. These data should greatly facilitate the rational design of potent, isoform-selective mAC inhibitors.

Structural Analysis of the Regulatory Dithiol-containing Domain of the Chloroplast ATP Synthase γ Subunit

The γ subunit of the F1 portion of the chloroplast ATP synthase contains a critically placed dithiol that provides a redox switch converting the enzyme from a latent to an active ATPase. The switch prevents depletion of intracellular ATP pools in the dark when photophosphorylation is inactive. The dithiol is located in a special regulatory segment of about 40 amino acids that is absent from the γ subunits of the eubacterial and mitochondrial enzymes. Site-directed mutagenesis was used to probe the relationship between the structure of the γ regulatory segment and its function in ATPase regulation via its interaction with the inhibitory ϵ subunit. Mutations were designed using a homology model of the chloroplast γ subunit based on the analogous structures of the bacterial and mitochondrial homologues. The mutations included (a) substituting both of the disulfide-forming cysteines (Cys199 and Cys205) for alanines, (b) deleting nine residues containing the dithiol, (c) deleting the region distal to the dithiol (residues 224-240), and (d) deleting the entire segment between residues 196 and 241 with the exception of a small spacer element, and (e) deleting pieces from a small loop segment predicted by the model to interact with the dithiol domain. Deletions within the dithiol domain and within parts of the loop segment resulted in loss of redox control of the ATPase activity of the F1 enzyme. Deleting the distal segment, the whole regulatory domain, or parts of the loop segment had the additional effect of reducing the maximum extent of inhibition obtained upon adding the ϵ subunit but did not abolish ϵ binding. The results suggest a mechanism by which the γ and ϵ subunits interact with each other to induce the latent state of the enzyme.

Gamma–epsilon Interactions Regulate the Chloroplast ATP Synthase

Current literature on the structure and function of the chloroplast ATP synthase is reviewed with an emphasis on the roles of the γ and ε subunits. Together these two subunits are thought to couple, via rotation, the proton motive force to nucleotide synthesis and hydrolysis by the catalytic F1 segment of the enzyme. These two subunits are also responsible for inducing the latent state of the enzyme that is necessary to prevent futile hydrolysis of ATP in the dark when electron transfer and ATP synthesis are inactive. A model is presented to explain how γ and ε interact to achieve the transition between the active and latent states.

The 20 C-terminal amino acid residues of the chloroplast ATP synthase gamma subunit are not essential for activity.

It has been suggested that the last seven to nine amino acid residues at the C terminus of the γ subunit of the ATP synthase act as a spindle for rotation of the γ subunit with respect to the αβ subunits during catalysis (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994)Nature 370, 621–628). To test this hypothesis we selectively deleted C-terminal residues from the chloroplast γ subunit, two at a time starting at the sixth residue from the end and finishing at the 20th residue from the end. The mutant γ genes were overexpressed in Escherichia coli and assembled with a native α3β3 complex. All the mutant forms of γ assembled as effectively as the wild-type γ. Deletion of the terminal 6 residues of γ resulted in a significant increase (>50%) in the Ca-dependent ATPase activity when compared with the wild-type assembly. The increased activity persisted even after deletion of the C-terminal 14 residues, well beyond the seven residues proposed to form the spindle. Further deletions resulted in a decreased activity to ∼19% of that of the wild-type enzyme after deleting all 20 C-terminal residues. The results indicate that the tip of the γC terminus is not essential for catalysis and raise questions about the role of the C terminus as a spindle for rotation.

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′- O -(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In a recent study (Mol Pharmacol 70:878–886, 2006), we reported that 2′,3′-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5′-triphosphates (TNP-NTPs) are potent inhibitors (Ki values in the 10 nM range) of the purified catalytic subunits VC1 and IIC2 of membranous AC (mAC). The crystal structure of VC1:IIC2 in complex with TNP-ATP revealed that the nucleotide binds to the catalytic site with the TNP-group projecting into a hydrophobic pocket. The aims of this study were to analyze the interaction of TNP-nucleotides with VC1:IIC2 by fluorescence spectroscopy and to analyze inhibition of mAC isoforms, soluble AC (sAC), soluble guanylyl cyclase (sGC), and G-proteins by TNP-nucleotides. Interaction of VC1:IIC2 with TNP-NDPs and TNP-NTPs resulted in large fluorescence increases that were differentially reduced by a water-soluble forskolin analog. TNP-ATP turned out to be the most potent inhibitor for ACV (Ki, 3.7 nM) and sGC (Ki, 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (Ki, 7.1 nM) and ACII (Ki, 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by Gs- and Gi-proteins only with low potencies. Molecular modeling revealed that TNP-GTP and TNP-ATP interact very similarly, but not identically, with VC1:IIC2. Collectively, our data show that TNP-nucleotides are useful fluorescent probes to monitor conformational changes in VC1:IIC2 and that TNP-NTPs are a promising starting point to develop isoform-selective AC and sGC inhibitors. TNP-ATP is the most potent sGC inhibitor known so far.

Reactive oxygen species affect ATP hydrolysis by targeting a highly conserved amino acid cluster in the thylakoid ATP synthase γ subunit.

The vast majority of organisms produce ATP by a membrane-bound rotating protein complex, termed F-ATP synthase. In chloroplasts, the corresponding enzyme generates ATP by using a transmembrane proton gradient generated during photosynthesis, a process releasing high amounts of molecular oxygen as a natural byproduct. Due to its chemical properties, oxygen can be reduced incompletely which generates several highly reactive oxygen species (ROS) that are able to oxidize a broad range of biomolecules. In extension to previous studies it could be shown that ROS dramatically decreased ATP synthesis in situ and affected the CF1 portion in vitro. A conserved cluster of three methionines and a cysteine on the chloroplast γ subunit could be identified by mass spectrometry to be oxidized by ROS. Analysis of amino acid substitutions in a hybrid F1 assembly system indicated that these residues were exclusive catalytic targets for hydrogen peroxide and singlet oxygen, although it could be deduced that additional unknown amino acid targets might be involved in the latter reaction. The cluster was tightly integrated in catalytic turnover since mutants varied in MgATPase rates, stimulation by sulfite and chloroplast-specific γ subunit redox-modulation. Some partial disruptions of the cluster by mutagenesis were dominant over others regarding their effects on catalysis and response to ROS.

Assembled F1-(αβ) and Hybrid F1-α3β3γ-ATPases fromRhodospirillum rubrum α, Wild Type or Mutant β, and Chloroplast γ Subunits DEMONSTRATION OF Mg2+ VERSUSCa2+-INDUCED DIFFERENCES IN CATALYTIC SITE STRUCTURE AND FUNCTION

Refolding together the expressed α and β subunits of the Rhodospirillum rubrumF1(RF1)-ATPase led to assembly of only α1β1 dimers, showing a stable low MgATPase activity. When incubated in the presence of AlCl3, NaF and either MgAD(T)P or CaAD(T)P, all dimers associated into closed α3β3 hexamers, which also gained a low CaATPase activity. Both hexamer ATPase activities exhibited identical rates and properties to the open dimer MgATPase. These results indicate that: a) the hexamer, as the dimer, has no catalytic cooperativity; b) aluminium fluoride does not inhibit their MgATPase activity; and c) it does enable the assembly of RrF1-α3β3 hexamers by stabilizing their noncatalytic α/β interfaces. Refolding of the RrF1-α and β subunits together with the spinach chloroplast F1 (CF1)-γ enabled a simple one-step assembly of two different hybrid RrF1-α3β3/CF1γ complexes, containing either wild type RrF1-β or the catalytic site mutant RrF1β-T159S. They exhibited over 100-fold higher CaATPase and MgATPase activities than the stabilized hexamers and showed very different catalytic properties. The hybrid wild type MgATPase activity was, as that of RrF1 and CF1 and unlike its higher CaATPase activity, regulated by excess free Mg2+ ions, stimulated by sulfite, and inhibited by azide. The hybrid mutant had on the other hand a low CaATPase but an exceptionally high MgATPase activity, which was much less sensitive to the specific MgATPase effectors. All these very different ATPase activities were regulated by thiol modulation of the hybrid unique CF1-γ disulfide bond. These hybrid complexes can provide information on the as yet unknown factors that couple ATP binding and hydrolysis to both thiol modulation and rotational motion of their CF1-γ subunit.

Over-expression and refolding of β-subunit from the chloroplast ATP synthase

We established a bacterial system for high‐level over‐expression of the spinach chloroplast atpB gene which encodes the ATP Synthase β subunit. Upon induction, atpB was expressed as at least 50% to 70% of total cell protein. Although the over‐expressed β polypeptide formed insoluble inclusion bodies, more than fifty percent of it was restored to a functional form by solubilizing the inclusion bodies with 4 M urea and slowly removing the urea by stepwise dialysis. The resulting β subunit exhibited specific and selective nucleotide binding properties identical to those of the native β subunit.

Hybrid Rhodospirillum rubrumF0F1 ATP Synthases Containing Spinach Chloroplast F1 β or α and β Subunits Reveal the Essential Role of the α Subunit in ATP Synthesis and Tentoxin Sensitivity

Trace amounts (∼5%) of the chloroplast α subunit were found to be absolutely required for effective restoration of catalytic function to LiCl-treated chromatophores ofRhodospirillum rubrum with the chloroplast β subunit (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067–7072). To clarify the role of the α subunit in the rebinding of β, restoration of catalytic function, and conferral of sensitivity to the chloroplast-specific inhibitor tentoxin, LiCl-treated chromatophores were analyzed by immunoblotting before and after reconstitution with mixtures of R. rubrum and chloroplast α and β subunits. The treated chromatophores were found to have lost, in addition to most of their β subunits, approximately a third of the α subunits, and restoration of catalytic activity required rebinding of both subunits. The hybrid reconstituted with theR. rubrum α and chloroplast β subunits was active in ATP synthesis as well as hydrolysis, and both activities were completely resistant to tentoxin. In contrast, a hybrid reconstituted with both chloroplast α and β subunits restored only a MgATPase activity, which was fully inhibited by tentoxin. These results indicate that all three copies of the R. rubrum α subunit are required for proton-coupled ATP synthesis, whereas for conferral of tentoxin sensitivity at least one copy of the chloroplast α subunit is required together with the chloroplast β subunit. The hybrid system was further used to examine the effects of amino acid substitution at position 83 of the β subunit on sensitivity to tentoxin.

The chloroplast ATP synthase: Structural changes during catalysis

This article summarizes some of the evidence for the existence of light-driven structural changes in theε andγ subunits of the chlorplast ATP synthase. Formation of a transmembrane proton gradient results in: (1) a change in the position of theε subunit such that it becomes exposed to polyclonal antibodies and to reagents which selectively modifyεLys109; (2) enhanced solvent accessibility of several sulfhydryl residues on theγ subunit; and (3) release/ exchange of tightly bound ADP from the enzyme. These and related experimental observations can, at least partially, be explained in terms of two different bound conformational states of theε subunit. Evidence for structural changes in the enzyme which are driven by light or nucleotide binding is discussed with special reference to the popular rotational model for catalysis.