Solution structure of the cGMP binding GAF domain from phosphodiesterase 5: insights into nucleotide specificity, dimerization, and cGMP-dependent conformational change

Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular nuclear Overhauser effects. Comparison with GAF domains from PDE2A and adenylyl cyclase cyaB2 reveals a conserved overall domain fold of a six-stranded beta-sheet and four alpha-helices that form a well defined cGMP binding pocket. However, the nucleotide coordination is distinct with a series of altered binding contacts. The structure suggests that nucleotide binding specificity is provided by Asp-196, which is positioned to form two hydrogen bonds to the guanine ring of cGMP. An alanine mutation of Asp-196 disrupts cGMP binding and increases cAMP affinity in constructs containing only GAF A causing an altered cAMP-bound structural conformation. NMR studies on the tandem GAF domains reveal a flexible GAF A domain in the absence of cGMP, and indicate a large conformational change upon ligand binding. Furthermore, we identify a region of approximately 20 residues directly N-terminal of GAF A as critical for tight dimerization of the tandem GAF domains. The features of the PDE5 regulatory domain revealed here provide an initial structural basis for future investigations of the regulatory mechanism of PDE5 and the design of GAF-specific regulators of PDE5 function.     

Interactions of the cbbII promoter-operator region with CbbR and RegA (PrrA) regulators indicate distinct mechanisms to control expression of the two cbb operons of Rhodobacter sphaeroides

In a previous study (Dubbs, J. M., Bird, T. H., Bauer, C. E., and Tabita, F. R. (2000) J. Biol. Chem. 275, 19224-19230), it was demonstrated that the regulators CbbR and RegA (PrrA) interacted with both promoter proximal and promoter distal regions of the form I (cbb(I)) promoter operon specifying genes of the Calvin-Benson-Bassham cycle of Rhodobacter sphaeroides. To determine how these regulators interact with the form II (cbb(II)) promoter, three cbbF(II)::lacZ translational fusion plasmids were constructed containing various lengths of sequence 5' to the cbb(II) operon of R. sphaeroides CAC. Expression of beta-galactosidase was monitored under a variety of growth conditions in both the parental strain and knock-out strains that contain mutations that affect synthesis of CbbR and RegA. The binding sites for both CbbR and RegA were determined by DNase I footprinting. A region of the cbb(II) promoter from +38 to -227 bp contained a CbbR binding site and conferred low level regulated cbb(II) expression. The region from -227 to -1025 bp contained six RegA binding sites and conferred enhanced cbb(II) expression under all growth conditions. Unlike the cbb(I) operon, the region between -227 and -545 bp that contains one RegA binding site, was responsible for the majority of the observed enhancement. Both RegA and CbbR were required for maximal cbb(II) expression. Two potentially novel and specific cbb(II) promoter-binding proteins that did not interact with the cbb(I) promoter region were detected in crude extracts of R. sphaeroides. These results, combined with the observation that chemoautotrophic expression of the cbb(I) operon is RegA independent, indicated that the mechanisms controlling cbb(I) and cbb(II) operon expression during chemoautotrophic growth are quite different.     

NMR structure of AbhN and comparison with AbrBN: FIRST insights into the DNA binding promiscuity and specificity of AbrB-like transition state regulator proteins

Understanding the molecular mechanisms of transition state regulator proteins is critical, since they play a pivotal role in the ability of bacteria to cope with changing environments. Although much effort has focused on their genetic characterization, little is known about their structural and functional conservation. Here we present the high resolution NMR solution structure of the N-terminal domain of the Bacillus subtilis transition state regulator Abh (AbhN), only the second such structure to date. We then compare AbhN to the N-terminal DNA-binding domain of B. subtilis AbrB (AbrBN). This is the first such comparison between two AbrB-like transition state regulators. AbhN and AbrBN are very similar, suggesting a common structural basis for their DNA binding. However, we also note subtle variances between the AbhN and AbrBN structures, which may play important roles in DNA target specificity. The results of accompanying in vitro DNA-binding studies serve to highlight binding differences between the two proteins.     

The binding domain on horse cytochrome c and Rhodobacter sphaeroides cytochrome c2 for the Rhodobacter sphaeroides cytochrome bc1 complex

The interaction of the Rhodobacter sphaeroides cytochrome bc1 complex with Rb. sphaeroides cytochrome c2 and horse cytochrome c was studied by using specific lysine modification and ionic strength dependence methods. The rate of the reactions with both cytochrome c and cytochrome c2 decreased rapidly with increasing ionic strength above 0.2 M NaCl. The ionic strength dependence suggested that electrostatic interactions were equally important to the reactions of the two cytochromes, even though they have opposite net charges at pH 7.0. In order to define the interaction domain on horse cytochrome c, the reaction rates of derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This result indicates that lysines surrounding the heme crevice of horse cytochrome c are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. In order to define the reaction domain on cytochrome c2, a fraction consisting of a mixture of singly labeled 4-carboxy-2,6-dinitrophenylcytochrome c2 derivatives modified at lysine-35, -88, -95, -97, and -105 and several unidentified lysines was prepared. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The rate of reaction of this fraction was significantly smaller than that of native cytochrome c2, suggesting that the binding domain on cytochrome c2 is also located at the heme crevice.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR assignment of the Rhodobacter sphaeroides fasciclin-1 domain protein (Fdp)

We report the almost complete assignment of ¹H, ¹³C and ¹⁵N nuclei in the 137-residue his-tagged fasciclin domain protein (Fdp) from Rhodobacter sphaeroides. Fdp is homologous to fasciclin I domains, including Drosophila FAS1 and M. tuberculosis MPB70 and plays a role in cell adhesion.     

Solution structure of the DNA binding domain of AraC protein

We report the solution structure of the DNA binding domain of the Escherichia coli regulatory protein AraC determined in the absence of DNA. The 20 lowest energy structures, determined on the basis of 1507 unambiguous nuclear Overhauser restraints and 180 angle restraints, are well resolved with a pair wise backbone root mean square deviation of 0.7 Å. The protein, free of DNA, is well folded in solution and contains seven helices arranged in two semi‐independent sub domains, each containing one helix‐turn‐helix DNA binding motif, joined by a 19 residue central helix. This solution structure is discussed in the context of extensive biochemical and physiological data on AraC and with respect to the DNA‐bound structures of the MarA and Rob homologs. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Subunit IV (Mr = 14,384) of the cytochrome b-c1 complex from Rhodobacter sphaeroides. Cloning, DNA sequencing, and ubiquinone binding domain.

The Rhodobacter sphaeroides gene encoding subunit IV of the cytochrome b-c1 complex (fbcQ) was cloned and sequenced. The fbcQ cistron is 372 base pairs long and encodes 124 amino acid residues. The molecular mass of subunit IV, deduced from the nucleotide sequence, is 14,384 Da. A hydropathy plot of the predicted amino acid sequence revealed only one transmembrane helix; it is near the C-terminal end. The 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone ([3H]azido-Q)-labeled subunit IV was isolated from the [3H]-azido-Q-treated cytochrome b-c1 complex. A ubiquinone-binding peptide was obtained by digesting the labeled subunit IV with V8 protease followed by high performance liquid chromatography separation. Amino acid analysis and partial N-terminal sequencing of this ubiquinone-binding peptide revealed that it corresponded to residues 77-124 of subunit IV. Based on the hydropathy profile and predicted tendency to form alpha-helices and beta-sheets, we propose a structural model for subunit IV. In this model the ubiquinone-binding domain is located near the surface of the membrane.