Phytochromes are a collection of bilin-containing photoreceptors that regulate numerous photoresponses in plants and microorganisms through their ability to photointerconvert between a red light-absorbing, ground state Pr and a far-red light-absorbing, photoactivated state Pfr1,2. While the structures of several phytochromes as Pr have been determined3-7, little is known about the structure of Pfr and how it initiates signaling. Here, we describe the three-dimensional solution structure of the bilin-binding domain as Pfr using the cyanobacterial phytochrome from Synechococcus OSB’. Contrary to predictions, light-induced rotation of the A but not the D pyrrole ring is the primary motion of the chromophore during photoconversion. Subsequent rearrangements within the protein then affect intra- and interdomain contact sites within the phytochrome dimer. From our models, we propose that phytochromes act by propagating reversible light-driven conformational changes in the bilin to altered contacts between the adjacent output domains, which in most phytochromes direct differential phosphotransfer.

Phytochromes are a collection of bilin-containing photoreceptors that regulate a diverse array of processes in microorganisms and plants through photoconversion between two stable states, a red light-absorbing Pr form, and a far red light-absorbing Pfr form. Recently, a novel set of phytochrome-like chromoproteins was discovered in cyanobacteria, designated here as cyanochromes, that instead photoconvert between stable blue and green light-absorbing forms Pb and Pg, respectively. Here, we show that the distinctive absorption properties of cyanochromes are facilitated through the binding of phycocyanobilin via two stable cysteine-based thioether linkages within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. Absorption, resonance Raman and infrared spectroscopy, and molecular modeling of the Te-PixJ GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domain assembled with phycocyanobilin are consistent with attachments to the C3 1 carbon of the ethylidene side chain and the C4 or C5 carbons in the A-B methine bridge to generate a double thioether-linked phycoviolobilin-type chromophore. These spectroscopic methods combined with NMR data show that the bilin is fully protonated in the Pb and Pg states and that numerous conformation changes occur during Pb 3 Pg photoconversion. Also identified were a number of photochromically inactive mutants with strong yellow or red fluorescence that may be useful for fluorescence-based cell biological assays. Phylogenetic analyses detected cyanochromes capable of different signaling outputs in a wide range of cyanobacterial species. One unusual case is the Synechocystis cyanochrome Etr1 that also binds ethylene, suggesting that it works as a hybrid receptor to simultaneously integrate light and hormone signals. Phytochromes (Phys)3 comprise a large and diverse superfamily of photoreceptors that regulate a wide range of physiological responses in plants, fungi, bacteria, and cyanobacteria (1-3). They are unique among photoreceptors by being able to photoconvert between two stable states, a red light-absorbing Pr form that is typically the dark-adapted and biologically inactive conformer and a far-red light-absorbing Pfr form that requires light for its production and is typically the biologically active conformer. By interconverting between Pr and Pfr, Phys act as light-regulated switches in controlling processes ranging from phototaxis and pigmentation in bacteria to seed germination, photomorphogenesis, and flowering time in higher plants.Light absorption by Phys is directed by a bilin (or linear tetrapyrrole) chromophore produced by the oxidative cleavage of heme. Although bacterial and fungal Phys use the immediate cleavage product biliverdin (BV), cyanobacterial and higher plant Phys use phycocyanobilin (PCB) and phytochromobilin, respectively, produced by enzymatic reduction of BV (1, 2). The bilin is then covalently bound autocatalytically to the photosensory unit of the apoprotein, which typically contains a sequence of Per/Arndt/Sim (PAS), cGMP phosphodiesterase/aden...

Background: Phytochromes are photochromic bili-proteins vital to microbial and plant photoperception. Results: NMR spectroscopy generated three-dimensional structures of the photosensing module from a cyanobacterial variant in the dark and photoactivated states. Conclusion: Photoconversion involves thioether bond rupture, bilin isomerization and sliding, and increased protein disorder. Significance: Combined with crystallographic models, these paired NMR structures provide an unprecedented view into photoconversion of a phytochrome-type photoreceptor.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (1) is a classical example of a signal transduction pathway whereby the transfer of a phosphoryl group through a series of bimolecular protein-protein complexes is coupled with the transport of sugars across the membrane (2-4). The initial events of the cascade involve a common pathway: enzyme I, which is autophosphorylated by phosphoenolpyruvate at His-189 (in Escherichia coli), transfers the phosphoryl group to His-15 (in E. coli) of the histidine-containing phosphocarrier protein (HPr).1 Subsequently, the phosphoryl group on HPr is transferred to a variety of sugar-specific carbohydrate transporters, known as enzymes II (5). The enzymes II are organized into several domains, some of which are covalently linked (5). There are two cytoplasmic domains, IIA and IIB; IIA accepts the phosphoryl group from HPr and then transfers it to IIB. The transmembrane domain IIC (and in some cases IID as well) catalyzes the translocation and phosphoryl transfer from IIB to the incoming sugar. There are five classes of enzymes II as follows: glucose-sucrose, mannitol-fructose, mannose-sorbose, lactose-cellobiose, and glucitol (3, 5). The membranebound IIC domains comprise a variable number of transmembrane helices (5). The IIA and IIB domains for the different sugar classes bear no sequence or structural similarity to one another (6 -15).The bacterial phosphoenolpyruvate:sugar phosphotransferase system provides a paradigm for understanding proteinprotein interactions and the factors governing their specificity. Thus, for example, HPr recognizes enzyme I and the various sugar-specific IIA domains, although all these target proteins are structurally dissimilar. We have recently solved the structures of the complexes between the N-terminal phosphoryl transfer domain of EI (EIN) and HPr (16) and between IIA Glucose (IIA Glc ) and HPr (17). In the present paper, we extend these studies to the solution structure determination of the complex between IIA Mannitol (IIA Mtl ) and HPr. EXPERIMENTAL PROCEDURES Expression Vector for IIAMtl -E. coli chromosomal DNA was used as a template to amplify by PCR the region corresponding to the A domain of the mannitol permease. The forward PCR primer 5Ј-GACAGCTTT-GACGATCATATGGCTAACCTGTTCAAG-3Ј contained an engineered NdeI restriction site (underlined), and the reverse primer 5Ј-TTAAC-CCCACCTTCTCCATGTCGACAGGGTGGGATTGG-3Ј contained an engineered SalI site (underlined). The NdeI-and SalI-cut PCR product was purified and cloned into the corresponding sites of the vector pRE1 * This work was supported in part by the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health (to G. M. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The

Staphylococcus aureus is a major human pathogen that uses quorum sensing (QS) to control virulence. Its QS system is regulated by macrocyclic peptide signals (or autoinducing peptides (AIPs)) and their cognate transmembrane receptors (AgrCs). Four different specificity groups of S. aureus have been identified to date (groups I–IV), each of which uses a different AIP:AgrC pair. Non-native ligands capable of intercepting AIP:AgrC binding, and thereby QS, in S. aureus have attracted considerable interest as chemical tools to study QS pathways and as possible anti-virulence strategies for the treatment of infection. We recently reported a set of analogs of the group-III AIP that are capable of strongly modulating the activity of all four AgrC receptors. Critical to the further development of such ligands is a detailed understanding of the structural features of both native AIPs and non-native analogs that are essential for activity. Herein, we report the first three-dimensional structural analysis of the known native AIP signals (AIPs-I–IV) and several AIP-III analogs with varied biological activities using NMR spectroscopy. Integration of these NMR studies with the known agonism and antagonism profiles of these peptides in AgrC-III revealed two key structural elements that control AIP-III (and non-native peptide) activity: (1) a tri-residue hydrophobic “knob” essential for both activation and inhibition, and (2) a fourth anchor point on the exocyclic tail needed for receptor activation. These results provide strong structural support for a mechanism of AIP-mediated AgrC activation and inhibition in S. aureus, and should facilitate the design of new AgrC ligands with enhanced activities (as agonists or antagonists) and simplified chemical structures.

We report the three-dimensional structure of a late embryogenesis abundant (LEA) protein from Arabidopsis thaliana gene At1g01470.1. This protein is a member of Pfam cluster PF03168, and has been classified as a LEA14 protein. LEA proteins are expressed under conditions of cellular stress, such as desiccation, cold, osmotic stress, and heat. The structure, which was determined by NMR spectroscopy, revealed that the At1g01470.1 protein has an ab-fold consisting of one a-helix and seven b-strands that form two antiparallel b-sheets. The closest structural homologs were discovered to be fibronectin Type III domains, which have <7% sequence identity. Because fibronectins from animal cells have been shown to be involved in cell adhesion, cell motility, wound healing, and maintenance of cell shape, it is interesting to note that in plants wounding or stress results in the overexpression of a protein with fibronectin Type III structural features.

Summary The unique photochromic absorption behavior of phytochromes (Phys) depends on numerous reversible interactions between the bilin chromophore and the associated polypeptide. To help define these dynamic interactions, we determined by NMR spectroscopy the first solution structure of the chromophore-binding cGMP phosphodiesterase/adenylcyclase/FhlA (GAF) domain from a cyanobacterial Phy assembled with phycocyanobilin (PCB). The three dimensional NMR structure of Synechococcus OS-B′ Cph1 in its red light-absorbing Pr state revealed that PCB is bound to Cys-138 of the GAF domain via the A-ring ethylidene side chain and is buried within the GAF domain in a ZZZsyn,syn,anti configuration. The D ring of the chromophore sits within a hydrophobic pocket and is tilted approximately 80° relative to the B/C rings by contacts with Lys-52 and His-169. The solution structure revealed remarkable flexibility for PCB and several adjacent amino acids, indicating that the Pr chromophore has more freedom in the binding pocket than anticipated. The propionic acid side chains of rings B and C and Arg-101 and Arg-133 nearby are especially mobile and can assume several distinct and energetically favorable conformations. Mutagenic studies on these arginines, which are conserved within the Phy superfamily, revealed that they have opposing roles with Arg-101 and Arg-133 helping stabilize and destabilize Pfr, respectively. Given the fact that the Synechococcus OS-B′ GAF domain can by itself complete the Pr→Pfr photocycle, it should now be possible to determine the solution structure of the far-red light-absorbing Pfr chromophore and surrounding pocket using this Pr structure as a framework.

We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell-based and cell-free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N-labeled proteins to be analyzed by 1H-15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell-based and two involved wheat germ cell-free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H-15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC+/- (protein partially disordered or not in a single stable conformational state), HSQC- (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X- for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell-free platform offers the advantage of greater genome coverage for NMR-based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X-ray structure determination.