Jürgen U. Linder

The HAMP Domain Structure Implies Helix Rotation in Transmembrane Signaling

HAMP domains connect extracellular sensory with intracellular signaling domains in over 7500 proteins, including histidine kinases, adenylyl cyclases, chemotaxis receptors, and phosphatases. The solution structure of an archaeal HAMP domain shows a homodimeric, four-helical, parallel coiled coil with unusual interhelical packing, related to the canonical packing by rotation of the helices. This suggests a model for the mechanism of signal transduction, in which HAMP alternates between the observed conformation and a canonical coiled coil. We explored this mechanism in vitro and in vivo using HAMP domain fusions with a mycobacterial adenylyl cyclase and an E. coli chemotaxis receptor. Structural and functional studies show that the equilibrium between the two forms is dependent on the side-chain size of residue 291, which is alanine in the wild-type protein.

The class III adenylyl cyclases: multi-purpose signalling modules

cAMP serves as a second messenger in virtually all organisms. The most wide-spread class of cAMP-generating enzymes are the class III adenylyl cyclases. Most class III adenylyl cyclases are multi-domain proteins. The catalytic domains exclusively work as dimers, catalysis proceeds at the dimer interface, so that both monomers provide catalytic residues to each catalytic center. Inspection of amino acid sequence profiles suggests a division of the class III adenylyl cyclases in to four subclasses, class IIIa-IIId. Genome projects and postgenomic analysis have provided novel aspects in terms of catalysis and regulation. Alterations in the canonical catalytic residues occur in all four subclasses suggesting a plasticity of the catalytic mechanisms. The vast variety of additional, probably regulatory modules found in class III adenylyl cyclases obviously reflects a large collection of regulatory inputs the catalytic domains have adapted to. The large versatility of class III adenylyl cyclase catalytic domains remains a major scientific challenge.

A GAF‐domain‐regulated adenylyl cyclase from Anabaena is a self‐activating cAMP switch

The gene cyaB1 from the cyanobacterium Anabaena sp. PCC 7120 codes for a protein consisting of two N‐terminal GAF domains (GAF‐A and GAF‐B), a PAS domain and a class III adenylyl cyclase catalytic domain. The catalytic domain is active as a homodimer, as demonstrated by reconstitution from complementary inactive point mutants. The specific activity of the holoenyzme increased exponentially with time because the product cAMP activated dose dependently and nucleotide specifically (half‐maximally at 1 μM), identifying cAMP as a novel GAF domain ligand. Using point mutants of either the GAF‐A or GAF‐B domain revealed that cAMP activated via the GAF‐B domain. We replaced the cyanobacterial GAF domain ensemble in cyaB1 with the tandem GAF‐A/GAF‐B assemblage from the rat cGMP‐stimulated phosphodiesterase type 2, and converted cyaB1 to a cGMP‐stimulated adenylyl cyclase. This demonstrated the functional conservation of the GAF domain ensemble since the divergence of bacterial and eukaryotic lineages >2 billion years ago. In cyanobacteria, cyaB1 may act as a cAMP switch to stabilize committed developmental decisions.

Adenylyl cyclase Rv1625c of Mycobacterium tuberculosis: a progenitor of mammalian adenylyl cyclases

The gene Rv1625c from Mycobacterium tuberculosis encodes a membrane‐anchored adenylyl cyclase corresponding to exactly one‐half of a mammalian adenylyl cyclase. An engineered, soluble form of Rv1625c was expressed in Escherichia coli. It formed a homodimeric cyclase with two catalytic centers. Amino acid mutations predicted to affect catalysis resulted in inactive monomers. A single catalytic center with wild‐type activity could be reconstituted from mutated monomers in stringent analogy to the mammalian heterodimeric cyclase structure. The proposed existence of supramolecular adenylyl cyclase complexes was established by reconstitution from peptide‐linked, mutation‐inactivated homodimers resulting in pseudo‐trimeric and ‐tetrameric complexes. The mycobacterial holoenzyme was expressed successfully in E.coli and mammalian HEK293 cells, i.e. its membrane targeting sequence was compatible with the bacterial and eukaryotic machinery for processing and membrane insertion. The membrane‐anchored mycobacterial cyclase expressed in E.coli was purified to homogeneity as a first step toward the complete structural elucidation of this important protein. As the closest progenitor of the mammalian adenylyl cyclase family to date, the mycobacterial cyclase probably was spread by horizontal gene transfer.

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.

Paramecium genome survey : a pilot project

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

Adenylyl cyclases from Plasmodium, Paramecium and Tetrahymena are novel ion channel/enzyme fusion proteins.

In Paramecium, cAMP formation is stimulated by a potassium conductance, which is an intrinsic property of the adenylyl cyclase. We cloned a full-length cDNA and several gDNA fragments from Paramecium and Tetrahymena coding for adenylyl cyclases with a novel domain composition. A putative N-terminal ion channel domain contains a canonical S4 voltage-sensor and a canonical potassium pore-loop located C-terminally after the last transmembrane span on the cytoplasmic side. The adenylyl cyclase catalyst is C-terminally located. DNA microinjection of a green fluorescent protein (GFP)-tagged construct into the macronucleus of Paramecium resulted in ciliary localization of the expressed protein. An identical gene coding for an ion-channel adenylyl cyclase was cloned from the malaria parasite Plasmodium falciparum. Expression of the catalytic domain of the latter in Sf9 cells yielded an active homodimeric adenylyl cyclase. The occurrence of this highly unique subtype of adenylyl cyclase appears to be restricted to ciliates and apicomplexa.

Guanylyl cyclases in unicellular organisms

Guanylyl cyclases in eukaryotic unicells were biochemically investigated in the ciliates Paramecium and Tetrahymena, in the malaria parasite Plasmodium and in the ameboid Dictyostelium. In ciliates guanylyl cyclase activity is calcium-regulated suggesting a structural kinship to similarly regulated membrane-bound guanylyl cyclases in vertebrates. Yet, cloning of ciliate guanylyl cyclases revealed a novel combination of known modular building blocks. Two cyclase homology domains are inversely arranged in a topology of mammalian adenylyl cyclases, containing two cassettes of six transmembrane spans. In addition the protozoan guanylyl cyclases contain an N-terminal P-type ATPase-like domain. Sequence comparisons indicate a compromised ATPase function. The adopted novel function remains enigmatic to date. The topology of the guanylyl cyclase domain in all protozoans investigated is identical. A recently identified Dictyostelium guanylyl cyclase lacks the N-terminal P-type ATPase domain. The close functional relation of Paramecium guanylyl cyclases to mammalian adenylyl cyclases has been established by heterologous expression, respective point mutations and a series of active mammalian adenylyl cyclase/Paramecium guanylyl cyclase chimeras. The unique structure of protozoan guanylyl cyclases suggests that unexpectedly they do not share a common guanylyl cyclase ancestor with their vertebrate congeners but probably originated from an ancestral mammalian-type adenylyl cyclase.

Fatty acid regulation of adenylyl cyclase Rv2212 from Mycobacterium tuberculosis H37Rv

Adenylyl cyclase Rv2212 from Mycobacterium tuberculosis has a domain composition identical to the pH‐sensing isoform Rv1264, an N‐terminal regulatory domain and a C‐terminal catalytic domain. The maximal velocity of Rv2212 was the highest of all 10 mycobacterial cyclases investigated to date (3.9 µmol cAMP·mg−1·min−1), whereas ATP substrate affinity was low (SC50 = 2.1 mm ATP). Guanylyl cyclase side activity was absent. The activities and kinetics of the holoenzyme and of the catalytic domain alone were similar, i.e. in distinct contrast to the Rv1264 adenylyl cyclase, in which the N‐terminal domain is autoinhibitory. Unsaturated fatty acids strongly stimulated Rv2212 activity by increasing substrate affinity. In addition, fatty acids greatly enhanced the pH sensitivity of the holoenzyme, thus converting Rv2212 to a pH sensor adenylyl cyclase. Fatty acid binding to Rv2212 was modelled by homology to a recent structure of the N‐terminal domain of Rv1264, in which a fatty acid‐binding pocket is defined. Rv2212 appears to integrate three cellular parameters: ATP concentration, presence of unsaturated fatty acids, and pH. These regulatory properties open the possibility that novel modes of cAMP‐mediated signal transduction exist in the pathogen.

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 Vmax 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 noncanonical 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.