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 original KSP scales were revised, shortened, modernized and psychometrically evaluated. The psychometric properties and the usefulness of the test battery were found to be substantially improved.

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Class III adenylyl cyclases contain catalytic and regulatory domains, yet structural insight into their interactions is missing. We show that the mycobacterial adenylyl cyclase Rv1264 is rendered a pH sensor by its N-terminal domain. In the structure of the inhibited state, catalytic and regulatory domains share a large interface involving catalytic residues. In the structure of the active state, the two catalytic domains rotate by 55 degrees to form two catalytic sites at their interface. Two alpha helices serve as molecular switches. Mutagenesis is consistent with a regulatory role of the structural transition, and we suggest that the transition is regulated by pH.

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.

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-A͞B domains in cyaB1 with the mammalian phosphodiesterase 2A GAF-A͞B 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-A͞B domains of cyaB2 with bound cAMP and compare it to the previously reported structure of the PDE2A GAF-A͞B. Unexpectedly, the cyaB2 GAF-A͞B 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 ␤2-␤3 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.cAMP ͉ phosphodiesterase

Class III adenylyl cyclases are the most abundant type of cyclic AMP-producing enzymes. The adjustment of the cellular levels of this second messenger is achieved by a variety of regulatory mechanisms which couple signals to adenylyl cyclase activity. Because of the divergent nature of stimuli which impinge on these enzymes, highly individualized class III adenylyl cyclases have evolved in metazoans, eukaryotic unicells and bacteria. Regulation usually exploits the dimeric structure of the catalyst, whose active centres form at the dimer interface. The fold of the catalytic domains and the basic catalytic mechanisms are similar in all class III adenylyl cyclases, and substrate binding generally closes the active site by an induced-fit mechanism. Regulatory inputs can result in dramatic rearrangements of the catalytic domains within the dimer, which often are based on rotational movements.

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 mol of cAMP⅐mg ؊1 ⅐min ؊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 Grampositive bacteria. The purified full-length enzyme was 300-fold less active than the catalytic domain alone. (2), and in lower organisms, particularly in bacteria, class III CHDs seem to be linked with different protein domains that most likely impart peculiar regulatory features. However, so far only a few studies addressed bacterial class III ACs.In the completed genome of Mycobacterium tuberculosis, 15 open reading frames were identified that contain a CHD (2). The availability of this information enables us to study each mycobacterial AC isoform individually in vitro with the perspective to determine its contribution to the cAMP regulatory system during tuberculosis disease development in vivo. Two of the 15 AC open reading frames (Rv1625c and Rv2435c) belong to the mammalian-type ACs, and recent work concentrated on the membrane-bound mammalian-type AC present in Mycobacterium, Rv1625c (3, 4). Four predicted mycobacterial ACs (Rv1318c, Rv1319c, Rv1320c, and Rv3645) contain CHDs that are part of a subclass consisting of ACs from, among others, Anabaena, Stigmatella, Rhizobium, and Treponema (2). The remaining nine mycobacterial CHDs (Rv1264, Rv1647, Rv2212, Rv0386, Rv1358, Rv1359, Rv2488c, Rv0891c, and LipJ) are most similar to CHDs detected in other Gram-positive bacteria.Here we investigated the gene product of Rv1264. Earlier, AC genes of this subtype were identified by complementation cloning in Brevibacterium liquefaciens and Streptomyces (5, 6). Those genes code for modular proteins with the CHD located C-terminally. The dismal expression of these ACs in E. coli precluded detailed biochemical studies (5, 6). Thus, Rv1264 was used in an attempt to characterize this AC subtype. In addition, the biochemical characterization of the Rv1264 catalyst might constitute a starting point for future studies on the remaining eight related AC genes present in M. tuberculosis.We were able to express reasonable amounts of the Rv1264 AC catalytic domain in E. coli with high AC activity. The catalytic site is the result of homodimerization, and catalysis depends on the same amino acids previously identified as crucial in mammalian ACs. The holoenzyme was much less active than the catalytic domain alone, suggesting an autoinhibitory function of this unique N-terminal domain, which contains no similarity to any other known prot...