Richard B. Cooley

Genetically Encoded Tetrazine Amino Acid Directs Rapid Site-Specific In Vivo Bioorthogonal Ligation with trans-Cyclooctenes

Bioorthogonal ligation methods with improved reaction rates and less obtrusive components are needed for site-specifically labeling proteins without catalysts. Currently no general method exists for in vivo site-specific labeling of proteins that combines fast reaction rate with stable, nontoxic, and chemoselective reagents. To overcome these limitations, we have developed a tetrazine-containing amino acid, 1, that is stable inside living cells. We have site-specifically genetically encoded this unique amino acid in response to an amber codon allowing a single 1 to be placed at any location in a protein. We have demonstrated that protein containing 1 can be ligated to a conformationally strained trans-cyclooctene in vitro and in vivo with reaction rates significantly faster than most commonly used labeling methods.

Mechanistic insight into the conserved allosteric regulation of periplasmic proteolysis by the signaling molecule cyclic-di-GMP

Stable surface adhesion of cells is one of the early pivotal steps in bacterial biofilm formation, a prevalent adaptation strategy in response to changing environments. In Pseudomonas fluorescens, this process is regulated by the Lap system and the second messenger cyclic-di-GMP. High cytoplasmic levels of cyclic-di-GMP activate the transmembrane receptor LapD that in turn recruits the periplasmic protease LapG, preventing it from cleaving a cell surface-bound adhesin, thereby promoting cell adhesion. In this study, we elucidate the molecular basis of LapG regulation by LapD and reveal a remarkably sensitive switching mechanism that is controlled by LapDs HAMP domain. LapD appears to act as a coincidence detector, whereby a weak interaction of LapG with LapD transmits a transient outside-in signal that is reinforced only when cyclic-di-GMP levels increase. Given the conservation of key elements of this receptor system in many bacterial species, the results are broadly relevant for cyclic-di-GMP- and HAMP domain-regulated transmembrane signaling. DOI: http://dx.doi.org/10.7554/eLife.03650.001

Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly 'Pseudomonas butanovora'.

Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the C(2)-C(9) alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for C(1)-C(5) alkanes and product inhibition profiles. sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by k(cat) : K(m) ratios). Relative rates of oxidation for C(1)-C(5) alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate K(m) values. The low micromolar K(m) for linear C(2)-C(5) alkanes and the millimolar K(m) for methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similar K(m) and k(cat) values to those of methane. This inability to kinetically discriminate between the C(1) alkane and C(1) alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length C(2)-C(5) do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of C(3)-C(5) alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes > or =C(2,) despite its high sequence homology with sMMO. These results represent, to the best of our knowledge, the first kinetic in vitro characterization of the closest known homologue of sMMO.

Gleaning Unexpected Fruits from Hard-Won Synthetases: Probing Principles of Permissivity in Non-canonical Amino Acid–tRNA Synthetases

The site‐specific incorporation of non‐canonical amino acids (ncAAs) into proteins is an important tool for understanding biological function. Traditionally, each new ncAA targeted for incorporation requires a resource‐consuming process of generating new ncAA aminoacyl tRNA synthetase/tRNACUA pairs. However, the discovery that some tRNA synthetases are “permissive”, in that they can incorporate multiple ncAAs, means that it is no longer always necessary to develop a new synthetase for each newly desired ncAA. Developing a better understanding of what factors make ncAA synthetases more permissive would increase the utility of this new approach. Here, we characterized two synthetases selected for the same ncAA that have markedly different “permissivity profiles.” Remarkably, the more permissive synthetase incorporated an ncAA for which we had not been able to generate a synthetase through de novo selection methods. Crystal structures revealed that the two synthetases recognize their parent ncAA through a conserved core of interactions, with the more permissive synthetase displaying a greater degree of flexibility in its interaction geometries. We also observed that intraprotein interactions not directly involved in ncAA binding can play a crucial role in synthetase permissivity and suggest that optimization of such interactions might provide an avenue to engineering synthetases with enhanced permissivity.

Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate

UNLABELLED We previously identified a second-messenger-regulated signaling system in the environmental bacterium Pseudomonas fluorescens which controls biofilm formation in response to levels of environmental inorganic phosphate. This system contains the transmembrane cyclic di-GMP (c-di-GMP) receptor LapD and the periplasmic protease LapG. LapD regulates LapG and controls the ability of this protease to process a large cell surface adhesin protein, LapA. While LapDG orthologs can be identified in diverse bacteria, predictions of LapG substrates are sparse. Notably, the opportunistic pathogen Pseudomonas aeruginosa harbors LapDG orthologs, but neither the substrate of LapG nor any associated secretion machinery has been identified to date. Here, we identified P. aeruginosa CdrA, a protein known to mediate cell-cell aggregation and biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen. IMPORTANCE Here, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogen Pseudomonas aeruginosa is revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition to its established transcriptional regulation via the same second messenger.

A Diiron Protein Autogenerates a Valine-Phenylalanine Cross-Link

An enzyme creates its own cofactor by linking two nonfunctionalized amino acid side chains. All known internal covalent cross-links in proteins involve functionalized groups having oxygen, nitrogen, or sulfur atoms present to facilitate their formation. Here, we report a carbon-carbon cross-link between two unfunctionalized side chains. This valine-phenyalanine cross-link, produced in an oxygen-dependent reaction, is generated by its own carboxylate-bridged diiron center and serves to stabilize the metallocenter. This finding opens the door to new types of posttranslational modifications, and it demonstrates new catalytic potential of diiron centers.

Symerythrin structures at atomic resolution and the origins of rubrerythrins and the ferritin-like superfamily.

Rubrerythrins are diiron-containing peroxidases that belong to the ferritin-like superfamily (FLSF). Here, we describe the structures of symerythrin, a novel rubrerythrin variant from the oxygenic phototroph Cyanophora paradoxa, at 1.20-1.40 Å resolution in three different states: diferric, azide-bound diferric and chemically reduced. The symerythrin metallocenter has a unique eighth ligating residue compared to rubrerythrin-an additional glutamate inserted into helix A of the four-helix bundle that resides on a π-helical segment. Otherwise, the diferric metallocenter structure is highly similar to that of characterized rubrerythrins. Azide binds the diferric center in a μ-1,1 orientation similar to how peroxide binds to diferric rubrerythrin. The structure of the diferrous metallocenter shows heterogeneity that we ascribe to the acidic pH of the crystals. In what we consider the neutral pH conformation, reduction causes a 2.0-Å shift in Fe1 and the toggling of a Glu to a His ligand, as seen with rubrerythrins. The function of symerythrin remains unknown, but preliminary tests showing oxidase and peroxidase activities and the similarities of its metallocenter to other rubrerythrins suggest similar functionalities between the two despite the additional ligating glutamate in symerythrin. Of particular interest is the high internal symmetry of symerythrin, which supports the notion that its core four-helix bundle was formed by the gene duplication and fusion of a two-helix peptide. Sequence comparisons with another family in the FLSF that also has notable internal symmetry provide compelling evidence that, contrary to previous assumptions, there have been multiple gene fusion events that have generated the single-chain FLSF fold.

Coincidence detection and bi-directional transmembrane signaling control a bacterial second messenger receptor

The second messenger c-di-GMP (or cyclic diguanylate) regulates biofilm formation, a physiological adaptation process in bacteria, via a widely conserved signaling node comprising a prototypical transmembrane receptor for c-di-GMP, LapD, and a cognate periplasmic protease, LapG. Previously, we reported a structure-function study of a soluble LapD•LapG complex, which established conformational changes in the receptor that lead to c-di-GMP-dependent protease recruitment (Chatterjee et al., 2014). This work also revealed a basal affinity of c-di-GMP-unbound receptor for LapG, the relevance of which remained enigmatic. Here, we elucidate the structural basis of coincidence detection that relies on both c-di-GMP and LapG binding to LapD for receptor activation. The data indicate that high-affinity for LapG relies on the formation of a receptor dimer-of-dimers, rather than a simple conformational change within dimeric LapD. The proposed mechanism provides a rationale of how external proteins can regulate receptor function and may also apply to c-di-GMP-metabolizing enzymes that are akin to LapD. DOI: http://dx.doi.org/10.7554/eLife.21848.001

Structure-Based Insights into the Role of the Cys-Tyr Crosslink and Inhibitor Recognition by Mammalian Cysteine Dioxygenase.

In mammals, the non-heme iron enzyme cysteine dioxygenase (CDO) helps regulate Cys levels through converting Cys to Cys sulfinic acid. Its activity is in part modulated by the formation of a Cys93-Tyr157 crosslink that increases its catalytic efficiency over 10-fold. Here, 21 high-resolution mammalian CDO structures are used to gain insight into how the Cys-Tyr crosslink promotes activity and how select competitive inhibitors bind. Crystal structures of crosslink-deficient C93A and Y157F variants reveal similar ~1.0-Å shifts in the side chain of residue 157, and both variant structures have a new chloride ion coordinating the active site iron. Cys binding is also different from wild-type CDO, and no Cys-persulfenate forms in the C93A or Y157F active sites at pH6.2 or 8.0. We conclude that the crosslink enhances activity by positioning the Tyr157 hydroxyl to enable proper Cys binding, proper oxygen binding, and optimal chemistry. In addition, structures are presented for homocysteine (Hcy), D-Cys, thiosulfate, and azide bound as competitive inhibitors. The observed binding modes of Hcy and D-Cys clarify why they are not substrates, and the binding of azide shows that in contrast to what has been proposed, it does not bind in these crystals as a superoxide mimic.

Growth of a non-methanotroph on natural gas: ignoring the obvious to focus on the obscure.

Methanotrophs are well known for their ability to grow on methane in natural gas environments; however, these environments also contain low concentrations of longer-chain-length gaseous alkanes. This mixture of alkanes poses a problem for organisms that might otherwise grow on alkanes ≥ C2 because methane could inhibit oxidation of growth substrates and lead to an accumulation of toxic C1 metabolites. Here, we have characterized the growth of a C2 -C9 alkane-utilizing bacterium, Thauera butanivorans, in conditions containing high concentrations of methane and small amounts (< 3% of total alkane) of C2 -C4 . During such growth, methanol accumulates transiently before being consumed in an O2 -dependent process that leads to the formation of a proton gradient and subsequent ATP generation. In contrast, formaldehyde-dependent O2 consumption is insensitive to uncouplers and does not lead to significant ATP production. This efficient C1 oxidation process that regains much of the energy loss inflicted by oxidizing methane, coupled with an alkane monooxygenase effective at limiting methane oxidation, allows T. butanivorans to grow uninhibited in natural gas environments. Although longer-chain-length gaseous alkane-utilizing organisms have been previously identified to grow in natural gas seepages, the data presented here represent the first detailed characterization of the physiological effects associated with inadvertent methane oxidation by a non-methanotroph, and suggest the presence of a well-evolved series of biochemical processes that allow them to grow in natural gas deposits without the need for developing the unique metabolic machinery characteristic of methanotrophs.