In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH- Chemoreception in insects is mediated via sensing of a variety of small, volatile organic compounds. Chemoreception plays a critical role not only in the regulation of the most fundamental chemosensory behaviors in insects but also for intraspecies communication. Pheromone is a chemical signal that triggers a natural behavioral response in another member within the same species. In lepidopteran insects, sex pheromones produced by females are detected by males of the same species with extreme sensitivity and selectivity. These hydrophobic compounds are transported to the membrane-bound receptors (ion channels) (1, 2) across the aqueous sensillar lymph by the pheromone-binding proteins (PBPs).2 PBPs are small, acidic proteins, highly soluble in water, with a molecular mass of 14 -16 kDa. The first PBP to be identified, cloned, and expressed in the bacterial system was that from the giant silk moth Antheraea polyphemus (3, 4). Since then, PBPs have been isolated from at least eight moth species that share about 50% sequence identity, with six conserved cysteine residues forming three disulfide bridges that are important for the formation of the hydrophobic binding pocket (5).Moth PBPs are known to undergo a dramatic conformational switch with a change in pH, which has been proposed to be necessary for the release of ligand at lower pH near the membrane-bound receptors (ion channels) (6 -11). The current view on insect PBPs is that the unliganded protein exists in two conformations as follows: form A (PBP A ), the acidic or "ligand releasing" conformation that exists at low pH, and form B (PBP B ), the basic or "ligand binding" conformation that exists at high pH (7). The low and high pH conformations have been determined by solution NMR for PBPs from two representative moths, Bombyx mori (BmorPBP) and Antheraea polyphemus (ApolPBP1) (8 -11), which are believed to represent the "free" or "unliganded" form of the two proteins. High pH conformations of both "ligand-bound" and free forms of BmorPBP have also been determined by x-ray crystallography (12, 13).The conformation of BmorPBP B in solution at pH 6.5 (9) cons...

Agrobacterium tumefaciens virulence genes are induced by plant signals through the VirA-VirG twocomponent regulatory system. The VirA protein is a membrane-spanning sensor molecule that possesses an autophosphorylating activity, and the VirG protein is a sequence-specific DNA-binding protein. In this report, we demonstrate that the VirG protein is phosphorylated by the VirA protein and that the phosphate is directly transferred from the phosphorylated VirA molecule (phosphohistidine) to the VirG protein. The chemical stability of the phospho-VirG bond suggested that the VirG protein was phosphorylated at the aspartate and/or glutamate residue. The phosphorylated VirG protein was reduced with tritiated sodium borohydride and subjected to proteolytic digestion with the Achromobater protease I enzyme. The resulting peptide fragments were separated by Cs reversed-phase high-pressure liquid chromatography, and the tritium-labeled peptide was sequenced. Amino acid sequence data showed that the aspartate residue at position 52 was the only site phosphorylated. Changing this aspartate into asparagine resulted in a nonphosphorylatable and biologically nonfunctional gene product. As a control, a randomly chosen aspartate was changed into an asparagine (position 72), and no effect on its phosphorylation or biological activity was observed. Unlike its homologs, including CheA-CheY, EnvZ-OmpR, and NtrB-NtrC, the phospho-VirG molecule was very stable in vitro. The possible implications of these observations and the function of VirG phosphorylation in vir gene activation are discussed.

Retinal rod and cone phosphodiesterases are oligomeric enzymes that consist of a dimeric catalytic core (␣ 2 in cones and ␣␤ in rods) with inhibitory subunits (␥) that regulate their activity. In addition, a 17-kDa protein referred to as the ␦ subunit co-purifies with the rod soluble phosphodiesterase and the cone phosphodiesterase. We report here partial protein sequencing of the rod ␦ subunit and isolation of a cDNA clone encoding it. The predicted amino acid sequence is unrelated to any other known protein. Of eight bovine tissue mRNA preparations examined by Northern analysis, the strongest ␦ subunit-specific signal was present in the retina. A less intense signal was seen in the brain and adrenal mRNA. In bovine retinal sections, rod ␦ subunit anti-peptide antibodies label rod but not cone outer segments. ␦ subunit, added back to washed outer segment membranes, solubilizes a large fraction of the membrane-bound phosphodiesterase, indicating that this subunit binds to the classical membrane associated phosphodiesterase. The subunit forms a tight complex with native, but not trypsin-released phosphodiesterase, suggesting that the isoprenylated carboxyl termini of the catalytic subunits may be involved in binding of the ␦ subunit to the phosphodiesterase holoenzyme.Phototransduction in the retina involves a cascade of regulated biochemical processes (for recent reviews, see Refs. 1 and 2). Key components in this light-activated biochemical cascade are the retinal specific phosphodiesterases (PDE6s).1 These phosphodiesterases catalyze the conversion of cGMP to 5Ј-GMP when activated by the G protein, transducin. The retinal phosphodiesterases are multimeric proteins composed of catalytic and inhibitory subunits. The large subunits of the rod and cone phosphodiesterases (the ␣ and ␤ subunits in rods and the ␣Ј subunit in cones) dimerize to form the catalytic core of the isozymes. The smaller 11-and 13-kDa subunits (rod ␥ and cone ␥) serve as inhibitors of the rod and cone phosphodiesterases, respectively (3-5). Hurwitz et al. (6) identified a 17-kDa protein that immunoprecipitated with bovine retinal phosphodiesterases. Gillespie and Beavo (7) and Gillespie et al. (8) later demonstrated that a protein of this size co-purified with the isotonically soluble rod and cone phosphodiesterases, but not with the membrane-bound rod phosphodiesterase. A function for this 17-kDa protein subunit (also referred to as the ␦ or 15-kDa subunit) has not been described.The rod membrane-bound phosphodiesterase is loosely associated with the membrane at least in part due to C-terminal isoprenyl and carboxymethyl post-translational modifications. Several investigators (9, 10) have demonstrated that the ␣ subunit is modified by a farnesyl (C-15) group and the ␤ subunit is modified by a geranylgeranyl (C-20) group on their carboxyl termini. In addition both COOH termini are methylated as reported by Swanson and Applebury (11) and others (12). Mutation of the conserved cysteine residue to serine in the CAAX isoprenylation motif in the ...

Partial amino acid sequence has been determined for the cone, a' subunit of the bovine photoreceptor cyclic nucleotide phosphodiesterase (PDE) and deduced from nucleotide sequences of a partial cDNA clone. These sequences identify the at' subunit as the product of a gene that is distinct from those encoding the a or .3 subunits of the membraneassociated rod photoreceptor PDE. Comparisons between the recently determined cGMP-stimulated-PDE sequence and those of the a and a' photoreceptor PDE subunits reveal an unexpected sequence similarity. In addition to the catalytic domain conserved in eukaryotic PDEs, all three PDEs possess a second conserved segment of -340 residues that contains two internally homologous repeats. Limited proteolysis and direct photolabeling studies indicate that the noncatalytic, cGMPbinding site(s) in the cGMP-stimulated PDE is located within this conserved domain, suggesting that it also may serve this function in the photoreceptor PDEs. Moreover, other PDEs that do not bind cGMP at noncatalytic sites do not contain this conserved domain. The function of the conserved segment in the photoreceptor PDEs is not known, but the homology to allosteric sites of the cGMP-stimulated PDE suggests a role in cGMP binding and modulation of enzyme activity.whereas the cone PDE (5) has two a' subunits (93.5 kDa) and three small subunits (11, 13, and 15 kDa).The cGS-PDE is a homodimer of two identical (105 kDa) subunits and can hydrolyze both cAMP and cGMP with nearly equal efficiency (7,8). cGMP is a positive allosteric regulator of this enzyme, and micromolar concentrations produce a 10-fold stimulation of cAMP hydrolysis (7-10). A cGMP-specific, allosteric site has been demonstrated in several types of cyclic nucleotide binding experiments (7,(9)(10)(11).In this report, we describe the partial amino acid sequence of the cone a' subunit and nucleotide sequences from a partial cDNA clone encoding it. § The protein sequence data confirms the identity of the full-length cDNA clone described in the companion paper by Li et al. (14) and shows that the a, a', and p subunits of photoreceptor PDEs are distinctive gene products. The a and a' subunits and the cGS-PDE have extensive sequence similarity within single segments that are not part of their conserved, catalytic domains (15, 16). Each segment displays internal homology and appears to comprise a regulatory, cGMP-binding domain.Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are found in many eukaryotic species; in some cases, distinct isozymes exist within a single cell type (1, 2). Within several PDE families, additional diversity is created by apparent alternative mRNA splicing (3, 4). Tissue and celltype-specific expression of the isozymes has been demonstrated for several of the isozymes and is suspected in others (1-6). As part of an effort to analyze structure-function relationships among the PDE isozymes, we are determining the sequence of the bovine cone photoreceptor PDE (5) and the cGMP-stimulated (cGS) PDE (7,8). Bot...

In Agrobacterium tumefaciens, a cis-active 24-base-pair sequence adjacent to the right border of the T-DNA, called overdrive, stimulates tumor formation by increasing the level of T-DNA processing. Recent results from our laboratory have suggested that the virC operon which enhances T-DNA processing probably does so because the VirC1 protein interacts with overdrive (N. Toro, A. Datta, M. Yanofsky, and E. W. Nester, Proc. Natl. Acad. Sci. USA 85:8558-8562, 1988). We report here the purification of the VirC1 protein from cells of Escherichia coli harboring a plasmid containing the coding sequences of the virC locus of the octopine Ti plasmid. By gel mobility shift and DNase I footprinting assays, we showed that this purified virC1 gene product binds to overdrive but not to the right border of T-DNA.