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

Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: Modes of ligand binding and dimerization

In several species, GAF domains, which are widely expressed small-molecule-binding domains that regulate enzyme activity, are known to bind cyclic nucleotides. However, the molecular mechanism by which cyclic nucleotide binding affects enzyme activity is not known for any GAF domain. In the cyanobacterium, Anabaena, the cyaB1 and cyaB2 genes encode adenylyl cyclases that are stimulated by binding of cAMP to their N-terminal GAF domains. Replacement of the tandem GAF-A/B domains in cyaB1 with the mammalian phosphodiesterase 2A GAF-A/B tandem domains allows regulation of the chimeric protein by cGMP, suggesting a highly conserved mechanism of activation. Here, we describe the 1.9-Å crystal structure of the tandem GAF-A/B domains of cyaB2 with bound cAMP and compare it to the previously reported structure of the PDE2A GAF-A/B. Unexpectedly, the cyaB2 GAF-A/B dimer is antiparallel, unlike the parallel dimer of PDE2A. Moreover, there is clear electron density for cAMP in both GAF-A and -B, whereas in PDE2A, cGMP is found only in GAF-B. Phosphate and ribose group contacts are similar to those in PDE2A. However, the purine-binding pockets appear very different from that in PDE2A GAF-B. Differences in the β2–β3 loop suggest that this loop confers much of the ligand specificity in this and perhaps in many other GAF domains. Finally, a conserved asparagine appears to be a new addition to the signature NKFDE motif, and a mechanism for this motif to stabilize the cNMP-binding pocket is proposed

Transnational Dimensions of German Anti-Modern Modernism: Ernst May in Breslau

The internationally acclaimed architect and urban designer Ernst May (1886-1970) is generally considered an exemplary modernist, yet from 1919 to1925 he practiced an anti-moderm modernism in Silesia that calls into question conventional classifications of early modern architecture and has local, national and transnational implications