The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid

The universal secondary messenger cAMP is produced by adenylyl cyclases (ACs). Most bacterial and all eukaryotic ACs belong to class III of six divergent classes. A class III characteristic is formation of the catalytic pocket at a dimer interface and the presence of additional regulatory domains. Mycobacterium tuberculosis possesses 15 class III ACs, including Rv1264, which is activated at acidic pH due to pH-dependent structural transitions of the Rv1264 dimer. It has been shown by X-ray crystallography that the N-terminal regulatory and C-terminal catalytic domains of Rv1264 interact in completely different ways in the active and inhibited states. Here, we report an in-depth structural and functional analysis of the regulatory domain of Rv1264. The 1.6 A resolution crystal structure shows the protein in a tight, disk-shaped dimer, formed around a helical bundle, and involving a protein chain crossover. To understand pH regulation, we determined structures at acidic and basic pH values and employed structure-based mutagenesis in the holoenzyme to elucidate regulation using an AC activity assay. It has been shown that regulatory and catalytic domains must be linked in a single protein chain. The new studies demonstrate that the length of the linker segment is decisive for regulation. Several amino acids on the surface of the regulatory domain, when exchanged, altered the pH-dependence of AC activity. However, these residues are not conserved amongst a number of related ACs. The closely related mycobacterial Rv2212, but not Rv1264, is strongly activated by the addition of fatty acids. The structure resolved the presence of a deeply embedded fatty acid, characterised as oleic acid by mass spectrometry, which may serve as a hinge. From these data, we conclude that the regulatory domain is a structural scaffold used for distinct regulatory purposes.     

The effect of HAMP domains on class IIIb adenylyl cyclases from Mycobacterium tuberculosis.

The genes Rv1318c, Rv1319c, Rv1320c and Rv3645 of Mycobacterium tuberculosis are predicted to code for four out of 15 adenylyl cyclases in this pathogen. The proteins consist of a membrane anchor, a HAMP region and a class IIIb adenylyl cyclase catalytic domain. Expression and purification of the isolated catalytic domains yielded adenylyl cyclase activity for all four recombinant proteins. Expression of the HAMP region fused to the catalytic domain increased activity in Rv3645 21-fold and slightly reduced activity in Rv1319c by 70%, demonstrating isoform-specific effects of the HAMP domains. Point mutations were generated to remove predicted hydrophobic protein surfaces in the HAMP domains. The mutations further stimulated activity in Rv3645 eight-fold, whereas the effect on Rv1319c was marginal. Thus HAMP domains can act directly as modulators of adenylyl cyclase activity. The modulatory properties of the HAMP domains were confirmed by swapping them between Rv1319c and Rv3645. The data indicate that in the mycobacterial adenylyl cyclases the HAMP domains do not display a uniform regulatory input but instead each form a distinct signaling unit with its adjoining catalytic domain.     

The adenylyl and guanylyl cyclase superfamily

New structures solved in 1997 revealed that the adenylyl cyclase core consists of a pair of catalytic domains arranged in a wreath. Homologous catalytic domains are arranged in diverse adenylyl and guanylyl cyclases as symmetric homodimers or pseudosymmetric heterodimers. The kinship of the adenylyl and guanylyl cyclases has been confirmed by the structure-based interconversion of their nucleotide specificities. Catalysis is activated when two metal-binding aspartate residues on one domain are juxtaposed with a key aspargine—arginine pair on the other. Allosteric activators of mammalian adenylyl cyclase, forskolin and the stimulatory G protein α subunit, promote the catalytically optimal juxtaposition of the two domains.     

CyaG, a novel cyanobacterial adenylyl cyclase and a possible ancestor of mammalian guanylyl cyclases

A novel gene encoding an adenylyl cyclase, designated cyaG, was identified in the filamentous cyanobacterium Spirulina platensis. The predicted amino acid sequence of the C-terminal region of cyaG was similar to the catalytic domains of Class III adenylyl and guanylyl cyclases. The N-terminal region next to the catalytic domain of CyaG was similar to the dimerization domain, which is highly conserved among guanylyl cyclases. As a whole, CyaG is more closely related to guanylyl cyclases than to adenylyl cyclases in its primary structure. The catalytic domain of CyaG was expressed in Escherichia coli and partially purified. CyaG showed adenylyl cyclase (but not guanylyl cyclase) activity. By site-directed mutagenesis of three amino acid residues (Lys(533), Ile(603), and Asp(605)) within the purine ring recognition site of CyaG to Glu, Arg, and Cys, respectively, CyaG was transformed to a guanylyl cyclase that produced cGMP instead of cAMP. Thus having properties of both cyclases, CyaG may therefore represent a critical position in the evolution of Class III adenylyl and guanylyl cyclases.     

A structural basis for the role of nucleotide specifying residues in regulating the oligomerization of the Rv1625c adenylyl cyclase from M. tuberculosis

The Rv1625c Class III adenylyl cyclase from Mycobacterium tuberculosis is a homodimeric enzyme with two catalytic centers at the dimer interface, and shows sequence similarity with the mammalian adenylyl and guanylyl cyclases. Mutation of the substrate-specifying residues in the catalytic domain of Rv1625c, either independently or together, to those present in guanylyl cyclases not only failed to confer guanylyl cyclase activity to the protein, but also severely abrogated the adenylyl cyclase activity of the enzyme. Biochemical analysis revealed alterations in the behavior of the mutants on ion-exchange chromatography, indicating differences in the surface-exposed charge upon mutation of substrate-specifying residues. The mutant proteins showed alterations in oligomeric status as compared to the wild-type enzyme, and differing abilities to heterodimerize with the wild-type protein. The crystal structure of a mutant has been solved to a resolution of 2.7A. On the basis of the structure, and additional biochemical studies, we provide possible reasons for the altered properties of the mutant proteins, as well as highlight unique structural features of the Rv1625c adenylyl cyclase.     

Molecular details of cAMP generation in mammalian cells: a tale of two systems

The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.     

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.     

Class III nucleotide cyclases in bacteria and archaebacteria: lineage-specific expansion of adenylyl cyclases and a dearth of guanylyl cyclases

The Class III nucleotide cyclases are found in bacteria, eukaryotes and archaebacteria. Our survey of the bacterial and archaebacterial genome and plasmid sequences identified 193 Class III cyclase genes in only 29 species, of which we predict the majority to be adenylyl cyclases. Interestingly, several putative cyclase genes were found to have non-conserved substrate specifying residues. Ancestors of the eukaryotic C1-C2 domain containing soluble adenylyl cyclases as well as the protist guanylyl cyclases were found in bacteria. Diverse domains were fused to the cyclase domain and phylogenetic analysis indicated that most proteins within a single cluster have similar domain compositions, emphasising the ancient evolutionary origin and versatility of the cyclase domain.     

Adenylyl cyclase Rv1264 from Mycobacterium tuberculosis has an autoinhibitory N-terminal domain

Mycobacterium tuberculosis contains 15 class III adenylyl cyclase genes. The gene Rv1264 is predicted to be composed of two distinct protein modules. The C terminus seems to code for a catalytic domain belonging to a subfamily of adenylyl cyclase isozymes mostly found in Gram-positive bacteria. The expressed protein was shown to function as a homodimeric adenylyl cyclase (1 micromol of cAMP x mg(-1) x min(-1)). In analogy to the structure of the mammalian adenylyl cyclase catalyst, six amino acids were targeted by point mutations and found to be essential for catalysis. The N-terminal region represents a novel protein domain, the occurrence of which is restricted to several adenylyl cyclases present in Gram-positive bacteria. The purified full-length enzyme was 300-fold less active than the catalytic domain alone. Thus, the N-terminal domain appeared to be autoinhibitory. The N-terminal domain contains three prominent polar amino acid residues (Asp(107), Arg(132), and Arg(191)) that are invariant in all seven sequences of this domain currently available. Mutation of Asp(107) to Ala relaxed the inhibition and resulted in a 6-fold increase in activity of the Rv1264 holoenzyme, thus supporting the role of this domain as a potential novel regulator of adenylyl cyclase activity.     

Adenylyl cyclase Rv0386 from Mycobacterium tuberculosis H37Rv uses a novel mode for substrate selection

Class III adenylyl cyclases usually possess six highly conserved catalytic residues. Deviations in these canonical amino acids are observed in several putative adenylyl cyclase genes as apparent in several bacterial genomes. This suggests that a variety of catalytic mechanisms may actually exist. The gene Rv0386 from Mycobacterium tuberculosis codes for an adenylyl cyclase catalytic domain fused to an AAA-ATPase and a helix-turn-helix DNA-binding domain. In Rv0386, the standard substrate, adenine-defining lysine-aspartate couple is replaced by glutamine-asparagine. The recombinant adenylyl cyclase domain was active with a V(max) of 8 nmol cAMP.mg(-1).min(-1). Unusual for adenylyl cyclases, Rv0386 displayed 20% guanylyl cyclase side-activity with GTP as a substrate. Mutation of the glutamine-asparagine pair either to alanine residues or to the canonical lysine-aspartate consensus abolished activity. This argues for a novel mechanism of substrate selection which depends on two non-canonical residues. Data from individual and coordinated point mutations suggest a model for purine definition based on an amide switch related to that previously identified in cyclic nucleotide phosphodiesterases.     

Structure-functional organization of adenylyl cyclases of unicellular eukaryotes and molecular mechanisms of their regulation

Adenylyl cyclases, the enzymes which catalyze the formation of the second messenger cAMP, are presently known to exist in yeast and related fungi, the amoeba Dictyostelium discoideum, flagellates, plasmodium, and infusoria. However, their structure-functional organization and molecular mechanisms of regulation differ considerably. Thus, in flagellates, tens of structurally similar adenylyl cyclase one-pass transmembrane proteins performing receptor functions have been discovered. In the amoeba D. discoideum, three types of adenylyl cyclases were detected, which differ by their topology, domain organization, and sensitivity to regulatory molecules and physical factors, one of which, adenylyl cyclase-A (AC-A), is similar to mammalian membrane-bound adenylyl cyclases and regulated by extracellular cAMP. Yeasts, in turn, have been shown to possess adenylyl cyclases that do not have transmembrane domains, but are able to form intermolecular complexes stabilized by interactions between repeated regions enriched in leucine residues. The data presented in this review indicate that the main molecular mechanisms underlying the actions of vertebrate adenylyl cyclases evolved as early as in the unicellular organisms and fungi. The structures and functions of adenylyl cyclases of the lower eukaryotes are much more diverse, which might be due both to the peculiarities of their life cycles and to the development at the initial stages of evolution of different models for the functioning and regulation of cAMP-dependent signaling cascades.     

Guanylyl cyclases with the topology of mammalian adenylyl cyclases and an N-terminal P-type ATPase-like domain in Paramecium, Tetrahymena and Plasmodium.

We cloned a guanylyl cyclase of 280 kDa from the ciliate Paramecium which has an N-terminus similar to that of a P-type ATPase and a C-terminus with a topology identical to mammalian adenylyl cyclases. Respective signature sequence motifs are conserved in both domains. The cytosolic catalytic C1a and C2a segments of the cyclase are inverted. Genes coding for topologically identical proteins with substantial sequence similarities have been cloned from Tetrahymena and were detected in sequences from Plasmodium deposited by the Malaria Genome Project. After 99 point mutations to convert the Paramecium TAA/TAG-Gln triplets to CAA/CAG, together with partial gene synthesis, the gene from Paramecium was heterologously expressed. In Sf9 cells, the holoenzyme is proteolytically processed into the two domains. Immunocytochemistry demonstrates expression of the protein in Paramecium and localizes it to cell surface membranes. The data provide a novel structural link between class III adenylyl and guanylyl cyclases and imply that the protozoan guanylyl cyclases evolved from an ancestral adenylyl cyclase independently of the mammalian guanylyl cyclase isoforms. Further, signal transmission in Ciliophora (Paramecium, Tetrahymena) and in the most important endoparasitic phylum Apicomplexa (Plasmodium) is, quite unexpectedly, closely related.     

Regulation of adenylyl cyclases by a region outside the minimally functional cytoplasmic domains.

The highly conserved topological structure of G protein-activated adenylyl cyclases seems unnecessary because the soluble cytoplasmic domains retain regulatory and catalytic properties. Yet, we previously isolated a constitutively active mutant of the Dictyostelium discoideum adenylyl cyclase harboring a single point mutation in the region linking the cytoplasmic and membrane domains (Leu-394). We show here that multiple amino acid substitutions at Leu-394 also display constitutive activity. The constitutive activity of these mutants is not dependent on G proteins or cytosolic regulators, although some of the mutants can be activated to higher levels than wild type. Combining a constitutive mutation such as L394T with K482N, a point mutation that renders the enzyme insensitive to regulators, restores an enzyme with wild type properties of low basal activity and the capacity to be activated by G proteins. Thus regions located outside the cytoplasmic loops of adenylyl cyclases are not only important in the acquisition of an activated conformation, they also have impact on other regions within the catalytic core of the enzyme.     

Internal symmetry of the mirror type and repeats in molecules of the membrane-bound forms of adenylate cyclase]

The analysis of mirror type internal symmetry distribution in primary structures of different types of mammalian membrane-bound adenylyl cyclases was made. The transmembrane domain clusters determining enzyme topology in membrane, a highly conservative region of cytoplasmic domains forming both catalytic and regulatory centres of adenylyl cyclases, and the functionally important regions in variable parts of their molecules (in particular, calmodulin binding regions) are shown to have symmetrical structures. These data are in conformity with a hypothesis put forward by the authors: the centres of internal symmetry may commonly either coincide with sites responsible for protein biological activity, or be spaced in the immediate vicinity of these sites. In different types of adenylyl cyclases long repeating sequences were identified. The segmentary structures were established for some enzyme subdomains. The regions containing repeats usually displayed a symmetrical structure which confirms a positive correlation between internal symmetry of amino acid sequence and its repeat distribution.     

Mycobacterial adenylyl cyclases: biochemical diversity and structural plasticity

The conversion of adenine and guanine nucleoside triphosphates to cAMP and cGMP is carried out by nucleotide cyclases, which vary in their primary sequence and are therefore grouped into six classes. The class III enzymes encompass all eukaryotic adenylyl and guanylyl cyclase, and several bacterial and archaebacterial cyclases. Mycobacterial nucleotide cyclases show distinct biochemical properties and domain fusions, and we review here biochemical and structural studies on these enzymes from Mycobacterium tuberculosis and related bacteria. We also present an in silico analysis of nucleotide cyclases found in completely sequenced mycobacterial genomes. It is clear that this group of enzymes demonstrates the tinkering in the class III cyclase domain during evolution, involving subtle structural changes that retain the overall catalytic function and fine tune their activities.     

The Evolution of Guanylyl Cyclases as Multidomain Proteins: Conserved Features of Kinase-Cyclase Domain Fusions

Guanylyl cyclases (GCs) are enzymes that generate cyclic GMP and regulate different physiologic and developmental processes in a number of organisms. GCs possess sequence similarity to class III adenylyl cyclases (ACs) and are present as either membrane-bound receptor GCs or cytosolic soluble GCs. We sought to determine the evolution of GCs using a large-scale bioinformatic analysis and found multiple lineage-specific expansions of GC genes in the genomes of many eukaryotes. Moreover, a few GC-like proteins were identified in prokaryotes, which come fused to a number of different domains, suggesting allosteric regulation of nucleotide cyclase activity. Eukaryotic receptor GCs are associated with a kinase homology domain (KHD), and phylogenetic analysis of these proteins suggest coevolution of the KHD and the associated cyclase domain as well as a conservation of the sequence and the size of the linker region between the KHD and the associated cyclase domain. Finally, we also report the existence of mimiviral proteins that contain putative active kinase domains associated with a cyclase domain, which could suggest early evolution of the fusion of these two important domains involved in signal transduction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sequence homologies between guanylyl cyclases and structural analogies to other signal-transducing proteins

The cyclic GMP-forming enzyme guanylyl cyclase exists in cytosolic and in membrane-bound forms differing in structure and regulations. Determination of the primary structures of the guanylyl cyclases revealed that the cytosolic enzyme form consists of two similar subunits and that membrane-bound guanylyl cyclases represent enzyme forms in which the catalytic part is located in an intracellular, C-terminal domain and is regulated by an extracelluar, N-terminal receptor domain. A domain of 250 amino acids conserved in all guanylyl cyclases appears to be required for the formation of cyclic nucleotide, as this homologous domain is also found in the cytosolic regions of the adenylyl cyclase. The general structures of guanylyl cyclases shows similarities with other signal transducing enzymes such as protein-tyrosine phosphatases and protein-tyrosine kinases. which also exist in cytosolic and receptor-linked forms.     

Characterization and crystallization of a minimal catalytic core domain from mammalian type II adenylyl cyclase.

Adenylyl cyclases play a pivotal role in signal transduction by carrying out the regulated synthesis of cyclic AMP. The nine cloned mammalian adenylyl cyclases all share two conserved regions of sequence, C1 and C2, which are homologous to each other and are together responsible for catalytic activity. Recombinant C1 and C2 domains catalyze the synthesis of cyclic AMP when they are mixed and activated by forskolin, and C2 domains alone also manifest reduced levels of forskolin-stimulated enzyme activity. Using limited proteolysis and mass spectrometry, we have mapped the boundaries of a minimal stable and active C2 catalytic domain to residues 871-1090 of type II adenylyl cyclase. We report the properties and crystallization of this trimmed domain, termed IIC2-delta 4. Crystals belong to space group P4n2(1)2, where n = 1 or 3; a = b = 81.3, and c = 180.5 A; and there are two molecules per asymmetric unit related by an approximate body centering operation. Flash-frozen crystals diffract anisotropically to 2.2 A along the c* direction and to 2.8 A along the a* and b* directions using synchrotron radiation.     

A survey of nucleotide cyclases in actinobacteria: unique domain organization and expansion of the class III cyclase family in Mycobacterium tuberculosis

Cyclic nucleotides are well-known second messengers involved in the regulation of important metabolic pathways or virulence factors. There are six different classes of nucleotide cyclases that can accomplish the task of generating cAMP, and four of these are restricted to the prokaryotes. The role of cAMP has been implicated in the virulence and regulation of secondary metabolites in the phylum Actinobacteria, which contains important pathogens, such as Mycobacterium tuberculosis, M. leprae, M. bovis and Corynebacterium, and industrial organisms from the genus Streptomyces. We have analysed the actinobacterial genome sequences found in current databases for the presence of different classes of nucleotide cyclases, and find that only class III cyclases are present in these organisms. Importantly, prominent members such as M. tuberculosis and M. leprae have 17 and 4 class III cyclases, respectively, encoded in their genomes, some of which display interesting domain fusions seen for the first time. In addition, a pseudogene corresponding to a cyclase from M. avium has been identified as the only cyclase pseudogene in M. tuberculosis and M. bovis. The Corynebacterium and Streptomyces genomes encode only a single adenylyl cyclase each, both of which have corresponding orthologues in M. tuberculosis. A clustering of the cyclase domains in Actinobacteria reveals the presence of typical eukaryote-like, fungi-like and other bacteria-like class III cyclase sequences within this phylum, suggesting that these proteins may have significant roles to play in this important group of organisms.