Calcium-independent calmodulin binding and two-metal-ion catalytic mechanism of anthrax edema factor

Edema factor (EF), a key anthrax exotoxin, has an anthrax protective antigen-binding domain (PABD) and a calmodulin (CaM)-activated adenylyl cyclase domain. Here, we report the crystal structures of CaM-bound EF, revealing the architecture of EF PABD. CaM has N- and C-terminal domains and each domain can bind two calcium ions. Calcium binding induces the conformational change of CaM from closed to open. Structures of the EF-CaM complex show how EF locks the N-terminal domain of CaM into a closed conformation regardless of its calcium-loading state. This represents a mechanism of how CaM effector alters the calcium affinity of CaM and uncouples the conformational change of CaM from calcium loading. Furthermore, structures of EF-CaM complexed with nucleotides show that EF uses two-metal-ion catalysis, a prevalent mechanism in DNA and RNA polymerases. A histidine (H351) further facilitates the catalysis of EF by activating a water to deprotonate 3'OH of ATP. Mammalian adenylyl cyclases share no structural similarity with EF and they also use two-metal-ion catalysis, suggesting the catalytic mechanism-driven convergent evolution of two structurally diverse adenylyl cyclases.     

MD simulations of anthrax edema factor: calmodulin complexes with mutations in the edema factor "switch a" region and docking of 3'-deoxy ATP into the adenylyl cyclase active site of wild-type and mutant edema factor variants

Bacillus anthracis, a spore-forming infectious bacterium, produces an exotoxin, called the edema factor (EF), that functions in part by disrupting internal signalling pathways. When complexed with human host cell calmodulin (CaM), EF becomes an active adenylyl cyclase, producing the internal signal substance cyclic-AMP in an uncontrolled fashion. Recently, the crystal structures for uncomplexed EF and EF:CaM complexes in the presence and absence of a substrate analog (3'-deoxy-ATP), were reported. EF mutational studies have implicated a number of residues important in CaM binding and/or in the generation of the adenylyl cyclase active site, formed by the movements of the EF switch A, B and C regions upon CaM binding. Here we report on the results of molecular dynamics (MD) simulations on two EF:CaM complexes, one containing wild-type EF and the other containing EF in which a cluster of residues in the switch A region (L523, K525, Q526 and V529) have been mutated to alanine. The switch A mutations cause a large increase in the flexibility of the switch C region, the rupture of a number of EF-CaM interactions, an expansion of the carboxyl-terminal domain of CaM, and a change in the Ca(2+) ion binding abilities of the CaM that is in complex with EF. The results indicate the importance of the mutated switch A residues in maintaining a compact EF:CaM complex that appears to be a prerequisite for the generation of a fully-functional adenylyl cyclase active site. The effects of mutating key residues (K346, K353, H577, E588, D590 and N639) in the active site region of EF (to alanine) on the ability of EF to bind the 3'-deoxy-ATP substrate analog were also examined. Active-site residue substitutions at positions 583 (N583A) and 577 (H577A) were found to be particularly disruptive for the placement of the adenine ring moiety into the position found in the x-ray crystal structure of the ligand-protein complex.     

Structure-based inhibitor discovery against adenylyl cyclase toxins from pathogenic bacteria that cause anthrax and whooping cough

Edema factor (EF) and CyaA are adenylyl cyclase toxins secreted by pathogenic bacteria that cause anthrax and whooping cough, respectively. Using the structure of the catalytic site of EF, we screened a data base of commercially available, small molecular weight chemicals for those that could specifically inhibit adenylyl cyclase activity of EF. From 24 compounds tested, we have identified one quinazoline compound, ethyl 5-aminopyrazolo[1,5-a]quinazoline-3-carboxylate, that specifically inhibits adenylyl cyclase activity of EF and CyaA with approximately 20 microm Ki. This compound neither affects the activity of host resident adenylyl cyclases type I, II, and V nor exhibits promiscuous inhibition. The compound is a competitive inhibitor, consistent with the prediction that it binds to the adenine portion of the ATP binding site on EF. EF is activated by the host calcium sensor, calmodulin. Surface plasmon resonance spectroscopic analysis shows that this compound does not affect the binding of calmodulin to EF. This compound is dissimilar from a previously described, non-nucleoside inhibitor of host adenylyl cyclase. It may serve as a lead to design antitoxins to address the role of adenylyl cyclase toxins in bacterial pathogenesis and to fight against anthrax and whooping cough.     

MD Simulations of Anthrax Edema Factor: Calmodulin Complexes with Mutations in the Edema Factor “Switch A” Region and Docking of 3′-deoxy ATP into the Adenylyl Cyclase Active Site of Wild-Type and Mutant Edema Factor Variants

Abstract

Bacillus anthracis, a spore-forming infectious bacterium, produces an exotoxin, called the edema factor (EF), that functions in part by disrupting internal signalling pathways. When complexed with human host cell calmodulin (CaM), EF becomes an active adenylyl cyclase, producing the internal signal substance cyclic-AMP in an uncontrolled fashion. Recently, the crystal structures for uncomplexed EF and EF:CaM complexes in the presence and absence of a substrate analog (3′-deoxy-ATP), were reported. EF mutational studies have implicated a number of residues important in CaM binding and/or in the generation of the adenylyl cyclase active site, formed by the movements of the EF switch A, B and C regions upon CaM binding. Here we report on the results of molecular dynamics (MD) simulations on two EF:CaM complexes, one containing wild-type EF and the other containing EF in which a cluster of residues in the switch A region (L523, K525, Q526 and V529) have been mutated to alanine. The switch A mutations cause a large increase in the flexibility of the switch C region, the rupture of a number of EF-CaM interactions, an expansion of the car-boxyl-terminal domain of CaM, and a change in the Ca²⁺ ion binding abilities of the CaM that is in complex with EF. The results indicate the importance of the mutated switch A residues in maintaining a compact EF:CaM complex that appears to be a prerequisite for the generation of a fully-functional adenylyl cyclase active site. The effects of mutating key residues (K346, K353, H577, E588, D590 and N639) in the active site region of EF (to alanine) on the ability of EF to bind the 3′-deoxy-ATP substrate analog were also examined. Active-site residue substitutions at positions 583 (N583A) and 577 (H577A) were found to be particularly distruptive for the placement of the adenine ring moiety into the position found in the x-ray crystal structure of the ligand-protein complex.     

A-type ATP binding consensus sequences are critical for the catalytic activity of the calmodulin-sensitive adenylyl cyclase from Bacillus anthracis

Analysis of the predicted amino acid sequence of Bacillus anthracis adenylyl cyclase revealed sequences with homology to consensus sequences for A- and B-type ATP binding domains found in many ATP binding proteins. Based on the analysis of nucleotide binding proteins, a conserved basic amino acid residue in the A-type consensus sequence and a conserved acidic amino acid residue in the B-type consensus sequence have been implicated in the binding of ATP. The putative ATP binding sequences in the B. anthracis adenylyl cyclase possess analogous lysine residues at positions 346 and 353 within two A-type consensus sequences and a glutamate residue at position 436 within a B-type consensus sequence. The two A-type consensus sequences overlap each other and have the opposite orientation. To determine whether Lys-346, Lys-353, or Glu-436 of the B. anthracis adenylyl cyclase are crucial for enzyme activity, Lys-346 and Lys-353 were replaced with methionine and Glu-436 with glutamine by oligonucleotide-directed mutagenesis. Furthermore, Lys-346 was also replaced with arginine. The genes encoding the wild type and mutant adenylyl cyclases were placed under the control of the lac promoter for expression in Escherichia coli, and extracts were assayed for adenylyl cyclase activity. In all cases, a 90-kDa polypeptide corresponding to the catalytic subunit of the enzyme was detected in E. coli extracts by rabbit polyclonal antibodies raised against the purified B. anthracis adenylyl cyclase. The proteins with the Lys-346 to methionine or arginine mutations exhibited no adenylyl cyclase activity, indicating that Lys-346 in the A-type ATP binding consensus sequence plays a critical role for enzyme catalysis. Furthermore, the enzyme with the Lys-353 to methionine mutation was also inactive, suggesting that Lys-353 may also directly contribute to enzyme catalysis. In contrast, the protein with the Glu-436 to glutamine mutation retained 75% of enzyme activity, suggesting that Glu-436 in the B-type ATP binding consensus sequence may not be directly involved in enzyme catalysis. It is concluded that Lys-346 and Lys-353 in B. anthracis adenylyl cyclase may interact directly with ATP and contribute to the binding of the nucleotide to the enzyme.     

Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins

Edema factor (EF) and CyaA are calmodulin (CaM)-activated adenylyl cyclase exotoxins involved in the pathogenesis of anthrax and whooping cough, respectively. Using spectroscopic, enzyme kinetic and surface plasmon resonance spectroscopy analyses, we show that low Ca(2+) concentrations increase the affinity of CaM for EF and CyaA causing their activation, but higher Ca(2+) concentrations directly inhibit catalysis. Both events occur in a physiologically relevant range of Ca(2+) concentrations. Despite the similarity in Ca(2+) sensitivity, EF and CyaA have substantial differences in CaM binding and activation. CyaA has 100-fold higher affinity for CaM than EF. CaM has N- and C-terminal globular domains, each binding two Ca(2+) ions. CyaA can be fully activated by CaM mutants with one defective C-terminal Ca(2+)-binding site or by either terminal domain of CaM while EF cannot. EF consists of a catalytic core and a helical domain, and both are required for CaM activation of EF. Mutations that decrease the interaction of the helical domain with the catalytic core create an enzyme with higher sensitivity to Ca(2+)-CaM activation. However, CyaA is fully activated by CaM without the domain corresponding to the helical domain of EF.     

The cytoplasmic tail of the D1A receptor subtype: identification of specific domains controlling dopamine cellular responsiveness

In this study the rat D1A receptor (wild-type, WT) and truncation mutants thereof, are utilized to delineate specific cytoplasmic tail (CT) domains responsible for regulating ligand binding and receptor-mediated adenylyl cyclase activation. In human embryonic kidney (HEK) cells, all truncation mutants of the D1A receptor (Delta425, Delta379, Delta351) display cell surface localization and express at high but different receptor numbers. Binding studies suggest that residues located between Cys(351) and Asp(425) may serve to restrain the agonist binding conformation of the D1A receptor. This contention is supported by the observation that the constitutive activation of Delta351 is significantly increased in comparison with WT, Delta425 and Delta379. Furthermore, we demonstrate that the extent of dopamine-mediated maximal activation of adenylyl cyclase is significantly augmented in cells expressing Delta351 when compared with WT or mutants harboring shorter truncations. These results suggest that in addition to restraining receptor conformation, determinants located downstream of Cys(351) may act as negative regulators of the G protein coupling efficiency and adenylyl cyclase activation. Interestingly, all truncated receptors used in the present study display a decrease in dopamine potency when compared with WT. We show that inhibition of protein kinase A (PKA) activity leads also to a reduction in dopamine potency in cells expressing WT but not Delta351 receptors. These results hint at a potential previously unanticipated role for PKA in facilitating D1A receptor coupling efficiency in HEK cells. Overall, the present study has uncovered specific CT domains involved in regulating discrete aspects of the D1A receptor signaling.     

An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis

The edema factor exotoxin produced by Bacillus anthracis is an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells. A C-terminal 60-kDa fragment corresponding to the catalytic domain of edema factor (EF3) was cloned, overexpressed in Escherichia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetella pertussis and Pseudomonas aeruginosa, is highly resistant to protease digestion. The C-terminal 160-amino acid domain (EF3-C) of EF3 is sensitive to proteolysis in the absence of CaM. The addition of CaM protects EF3-C from being digested by proteases. EF3-N and EF3-C were expressed separately, and both fragments were required to reconstitute full CaM-sensitive enzyme activity. Fluorescence resonance energy transfer experiments using a double-labeled CaM molecule were performed and indicated that CaM adopts an extended conformation upon binding to EF3. This contrasts sharply with the compact conformation adopted by CaM upon binding myosin light chain kinase and CaM-dependent protein kinase type II. Mutations in each of the four calcium binding sites of CaM were examined for their effect on EF3 activation. Sites 3 and 4 were found critical for the activation, and neither the N- nor the C-terminal domain of CaM alone was capable of activating EF3. A genetic screen probing loss-of-function mutations of EF3 and site-directed mutations based on the homology of the edema factor family revealed a conserved pair of aspartate residues and an arginine that are important for catalysis. Similar residues are essential for di-metal-mediated catalysis in mammalian adenylyl cyclases and a family of DNA polymerases and nucleotidyltransferases. This suggests that edema factor may utilize a similar catalytic mechanism.     

Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin

CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C-terminal domain of calmodulin. Four discrete regions of CyaA bind calcium-loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an alpha-helix of CyaA makes extensive contacts with the calcium-induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin-induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA-calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.     

Origin of asymmetry in adenylyl cyclases: structures of Mycobacterium tuberculosis Rv1900c

Rv1900c, a Mycobacterium tuberculosis adenylyl cyclase, is composed of an N-terminal alpha/beta-hydrolase domain and a C-terminal cyclase homology domain. It has an unusual 7% guanylyl cyclase side-activity. A canonical substrate-defining lysine and a catalytic asparagine indispensable for mammalian adenylyl cyclase activity correspond to N342 and H402 in Rv1900c. Mutagenic analysis indicates that these residues are dispensable for activity of Rv1900c. Structures of the cyclase homology domain, solved to 2.4 A both with and without an ATP analog, form isologous, but asymmetric homodimers. The noncanonical N342 and H402 do not interact with the substrate. Subunits of the unliganded open dimer move substantially upon binding substrate, forming a closed dimer similar to the mammalian cyclase heterodimers, in which one interfacial active site is occupied and the quasi-dyad-related active site is occluded. This asymmetry indicates that both active sites cannot simultaneously be catalytically active. Such a mechanism of half-of-sites-reactivity suggests that mammalian heterodimeric adenylyl cyclases may have evolved from gene duplication of a primitive prokaryote-type cyclase, followed by loss of function in one active site.     

Characterization of the calmodulin binding domain of neuromodulin. Functional significance of serine 41 and phenylalanine 42.

Neuromodulin (also designated P-57, GAP-43, B-50) is a major presynaptic substrate for protein kinase C. Phosphorylation of neuromodulin decreases its affinity for calmodulin, suggesting that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons, releasing calmodulin locally in response to phosphorylation by protein kinase C (Alexander, K. A., Cimler, B. M., Meier, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). In the present study, we have constructed and characterized several mutant neuromodulins to demonstrate that the amino acid sequence 39-56 is required for calmodulin binding, and that this domain contains the sole in vitro protein kinase C phosphorylation site at serine 41. We also demonstrate that the adjacent phenylalanine 42, interacts hydrophobically with calmodulin. These hydrophobic interactions may be disrupted by the introduction of negative charge at serine 41, and thereby regulate the neuromodulin/calmodulin binding interactions. The sensitivity of the neuromodulin/calmodulin binding interaction to negative charge at serine 41 was determined by substitution of serine 41 with an aspartate or an asparagine residue. The asparagine mutant retained its affinity for calmodulin-Sepharose while the aspartate mutant did not adsorb to calmodulin-Sepharose. We conclude that protein kinase C phosphorylation of neuromodulin abolishes calmodulin binding by introducing negative charges within the calmodulin binding domain at a position adjacent to the phenylalanine.     

Kinase domain mutants of Bcr-Abl exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to imatinib

Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.     

Molecular modeling of manganese regulation of calmodulin-sensitive adenylyl cyclase from mammalian sperm

The soluble calmodulin-sensitive isoform of adenylyl cyclase isolated from equine sperm is unique because it requires Mn(2+) rather than Mg(2+) for activity. To gain insight into the molecular action of metals on sperm adenylyl cyclase, the kinetics of Mn(2+) and ATP effect was examined. A biphasic response to increases in ATP concentration was observed when metal was held constant. When [Mn(2+)] exceeded [ATP], however, greatly enhanced enzyme activity was observed. The kinetic profiles were consistent with allosteric activation of adenylyl cyclase by Mn(2+). Linear transformation of the data yielded an apparent K(m) for Mn-ATP of 5.8 mM and calculated V(max) of 12 nM cyclic AMP formed/min/mg. Data analysis using calculated equilibrium concentrations of free and complexed reactants provided similar estimates of these kinetic parameters.     

Cysteine substitution mutagenesis and the effects of methanethiosulfonate reagents at P2X2 and P2X4 receptors support a core common mode of ATP action at P2X receptors

The agonist binding site of ATP-gated P2X receptors is distinct from other ATP-binding proteins. Mutagenesis on P2X(1) receptors of conserved residues in mammalian P2X receptors has established the paradigm that three lysine residues, as well as FT and NFR motifs, play an important role in mediating ATP action. In this study we have determined whether cysteine substitution mutations of equivalent residues in P2X(2) and P2X(4) receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate (MTS) compounds. All the mutants (except the P2X(2) K69C and K71C that were expressed, but non-functional) showed a significant decrease in ATP potency, with >300-fold decreases for mutants of the conserved asparagine, arginine, and lysine residues close to the end of the extracellular loop. MTS reagents had no effect at the phenylalanine of the FT motif, in contrast, cysteine mutation of the threonine was sensitive to MTS reagents and suggested a role of this residue in ATP action. The lysine-substituted receptors were sensitive to the charge of the MTS reagent consistent with the importance of positive charge at this position for coordination of the negatively charged phosphate of ATP. At the NFR motif the asparagine and arginine residues were sensitive to MTS reagents, whereas the phenylalanine was either unaffected or showed only a small decrease. These results support a common site of ATP action at P2X receptors and suggest that non-conserved residues also play a regulatory role in agonist action.     

K252a, an indrocarbazole derivative, causes the membrane of myoblasts to enter a fusion-capable state

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in plants and microorganisms. ALS is the target of several structurally diverse classes of herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. The roles of three well-conserved histidine residues (H351, H392, and H487) in tobacco ALS were determined using site-directed mutagenesis. Both H487F and H487L mutations abolished the enzymatic activity as well as the binding affinity for the cofactor FAD. Nevertheless, the mutation of H487F did not affect the secondary structure of the ALS. The K(m) values of H351M, H351Q, and H351F are approximately 18-, 60-, and fivefold higher than that of the wild-type ALS, respectively. Moreover, the K(c) value of H351Q for FAD is about 137-fold higher than that of wALS. Mutants H351M and H351Q showed very strong resistance to Londax (a sulfonylurea) and Cadre (an imidazolinone), whereas mutant H351F was weakly resistant to them. However, the secondary structures of mutants H351M and H351Q appeared to be different from that of wALS. The mutation of H392M did not have any significant effect on the kinetic parameters nor the resistance to ALS-inhibiting herbicides. These results suggest that the His487 residue is located at the active site of the enzyme and is likely involved in the binding of cofactor FAD in tobacco ALS. Mutational analyses of the His351 residue imply that the active site of the ALS is probably close to its binding site of the herbicides, Londax and Cadre.     

Identification of Residues in Transmembrane Regions III and VI that Contribute to the Ligand Binding Site of the Serotonin 5-HT6 Receptor

Abstract: We have examined the ligand binding site of the serotonin 5-HT₆ receptor using site-directed mutagenesis. Replacing the highly conserved Asp¹⁰⁶ in transmembrane region III by asparagine eliminated d -[³H]lysergic acid diethylamide ([³H]LSD) binding to the mutant receptor transiently expressed in HEK293 cells. The potency of 5-HT and LSD to stimulate adenylyl cyclase was reduced by 3,600- and 500-fold, respectively, suggesting that an ionic interaction between the positively charged amino group of 5-HT and D106 is essential for high-affinity binding and important for receptor activation. In addition, basal cyclic AMP levels in cells expressing this mutant were increased. Mutation of a tryptophan residue one helix turn toward the extracellular side of transmembrane region III (Trp¹⁰²) to phenylalanine produced significant changes in the binding affinity and potency of several ligands, consistent with a role of this residue in the formation of the ligand binding site. The exchange of two neighboring residues in the carboxy-terminal half of transmembrane region VI (Ala²⁸⁷ and Asn²⁸⁸) for leucine and serine resulted in a mutant receptor with increased affinities (seven- to 30-fold) for sumatriptan and several ergopeptine ligands. The identification of these interactions will help to improve models of the 5-HT₆ receptor ligand binding site.     

Contribution of a conserved phenylalanine residue to the activity of Escherichia coli uracil DNA glycosylase

Uracil DNA glycosylase (UDG) excises uracil from DNA to initiate repair of this lesion. This important DNA repair enzyme is conserved in viruses, bacteria, and eukaryotes. One residue that is conserved among all the members of the UDG family is a phenylalanine that stacks with uracil when it is flipped out of the DNA helix into the enzyme active site. To determine what contribution this conserved Phe residue makes to the activity of UDG, Phe-77 in the Escherichia coli enzyme was mutated to three different amino acid residues, alanine (UDG-F77A), asparagine (UDG-F77N), and tyrosine (UDG-F77Y). The effects of these mutations were measured on the steady-state and pre-steady-state kinetics of uracil excision in addition to enzyme.DNA binding kinetics. The overall excision activity of each of the mutants was reduced relative to the wild-type enzyme; however, each mutation gave rise to a different kinetic phenotype with different effects on substrate binding and catalysis. The excision activity of UDG-F77N was the most severely compromised, but this enzyme still bound to uracil-containing DNA at about the same rate as wild-type UDG. In contrast, the decrease in the excision activity of UDG-F77A is likely to reflect a greater reduction in uracil-DNA binding than in the catalytic step. Overall, the effects of the mutations on catalysis are best correlated with the polarity of the substituted residue such that an increase in polarity decreases the efficiency of uracil excision.     

ATP modulation of the ligand binding and signal transduction activities of the type C natriuretic peptide receptor guanylate cyclase

The type C natriuretic peptide (CNP)-activated guanylate cyclase (CNP-RGC) is a single-chain transmembrane-spanning protein, containing both CNP binding and catalytic cyclase activities. Upon binding CNP to the extracellular receptor domain, the cytosolic catalytic domain of CNP-RGC is activated, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening signal transduction step which is regulated by ATP binding to the cyclase. This bridges the events of ligand binding and cyclase activation. A defined sequence motif (Gly⁴⁹⁹-Xa-Xa-Xa-Gly⁵⁰³), termed ATP regulatory module (ARM), is critical for this step. The present study shows that ATP not only amplifies the signal transduction step, it also concomitantly reduces the ligand binding activity of CNP-RGC. Reduction in the ligand binding activity is a consequence of the transformation of the high affinity receptor-form to the low affinity receptor-form. A single ARM residue Gly⁴⁹⁹ is critical in the mediation of both ATP effects, signal transduction and ligand binding activity of the receptor. Thus, this residue represents an ATP bimodal switch to turn the CNP signal on and off.     

A fluorimetric assay for real-time monitoring of adenylyl cyclase activity based on terbium norfloxacin

Adenylyl cyclases catalyze the production of the second messenger cyclic AMP from ATP. Until now, there has been no fluorescent adenylyl cyclase assay known that is applicable to high-throughput screening and kinetic determinations that can directly monitor the turnover of the unmodified substrate ATP. In this study, a fluorescence-based assay is described using the Ca(II)- and calmodulin-dependent adenylyl cyclase edema factor (EF) from Bacillus anthracis and Tb(III)-norfloxacin as probe for the enzyme activity. This assay can be used to study enzyme regulators, allows real-time monitoring of adenylyl cyclase activity, and does not substitute ATP by fluorescent derivatives. These derivatives must be judged critically due to their interference on the activity of enzymes. Furthermore, the new assay makes redundant the application of radioactively labeled substrates such as [α-³²P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (K_M), the v₀^max value of ATP turnover, and the IC₅₀ values for three inhibitors of EF by this newly developed fluorescent method.     

Mutagenesis of catalytically important residues of cupin type phosphoglucose isomerase from Archaeoglobus fulgidus

Recently, cupin type phosphoglucose isomerases have been described as a novel protein family representing a separate lineage in the evolution of phosphoglucose isomerases. The importance of eight active site residues completely conserved within the cPGI family has been assessed by site-directed mutagenesis using the cPGI from Archaeoglobus fulgidus (AfcPGI) as a model. The mutants T63A, G79A, G79L, H80A, H80D, H82A, E93A, E93D, Y95F, Y95K, H136A, and Y160F were constructed, purified, and the impact of the respective mutation on catalysis and/or metal ion binding as well as thermostability was analyzed. The variants G79A, G79L, and Y95F exhibited a lower thermostability. The catalytic efficiency of the enzyme was reduced by more than 100-fold in the G79A, G79L, H80A, H80D, E93D, Y95F variants and more than 15-fold in the T63A, H82A, Y95K, Y160F variants, but remained about the same in the H136A variant at Ni2+ saturating conditions. Further, the Ni2+ content of the mutants H80A, H80D, H82A, E93A, E93D and their apparent Ni2+ binding ability was reduced, resulting in an almost complete loss of activity and thus underlining the crucial role of the metal ion for catalysis. Evidence is presented that H80, H82 and E93 play an additional role in catalysis besides metal ion binding. E93 appears to be the key catalytic residue of AfcPGI, as the E93A mutant did not show any catalytic activity at all.