Microorganisms can switch from a planktonic, free-swimming life style to a sessile, colonial state, called a biofilm, conferring resistance to environmental stress. Conversion between the motile and biofilm life style has been attributed to increased levels of the prokaryotic second messenger cyclic di-guanosine monophosphate (c-di-GMP), yet the signaling mechanisms mediating such a global switch are poorly understood. Here we show that the transcriptional regulator VpsT from Vibrio cholerae directly senses c-di-GMP to inversely control extracellular matrix production and motility, identifying VpsT as a master regulator for biofilm formation. Rather than being regulated by phosphorylation, VpsT undergoes a change in oligomerization upon c-di-GMP binding.

X-ray crystallographic structural analyses of the bacterial transmembrane receptor LapD identify conserved molecular mechanisms that control biofilm formation in response to changes in the intracellular levels of the second messenger c-di-GMP.

endocytosis ͉ membrane trafficking ͉ protein structure

Summary Bacterial pathogenesis involves social behavior including biofilm formation and swarming, processes that are regulated by the bacterially unique second messenger cyclic di-GMP (c-di-GMP). Diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL domains have been identified as the enzymes controlling cellular c-di-GMP levels, yet less is known regarding signal transmission and the targets of c-di-GMP. FimX, a protein from Pseudomonas aeruginosa that governs twitching motility, belongs to a large subfamily containing both GGDEF and EAL domains. Biochemical and structural analyses reveals its function as a high-affinity receptor for c-di-GMP. A model for full-length FimX was generated combining solution scattering data and crystal structures of the degenerate GGDEF and EAL domains. While FimX forms a dimer in solution via the N-terminal domains, a crystallographic EAL domain dimer suggests modes for the regulation of FimX by c-di-GMP binding. The results provide the structural basis for c-di-GMP sensing via degenerate phosphodiesterases.

Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ 54 -interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ's AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP-complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs.enhancer binding protein | flagella | structure | gene expression B acterial adaptations to diverse environments, including human hosts, involve collaborative group behaviors, such as quorum sensing, swarming, and biofilm formation (1-5). In general, quorum-sensing during host tissue colonization is associated with virulence gene expression and acute-phase infections, whereas biofilm formation facilitates the development of chronic infections, evasion of host immune response, and increased tolerance to treatments (6). It is now well appreciated that these social behaviors result from highly regulated signal transduction processes, which in many bacteria are choreographed by the nucleotide-based second messenger c-di-GMP (7-9). Synthesized by GGDEF domain-containing diguanylate cyclases and hydrolyzed by EAL or HD-GYP domain-containing phosphodiesterases, c-di-GMP is sensed by a variety of protein-and RNA-based effectors to exert control at transcriptional, translational, and posttranslational levels (10, 11).In Pseudomonas aeruginosa, an opportunistic pathogen that causes severe chronic infections in cystic fibrosis patients, burn victims, and other immunocompromised individuals, the transcription factor FleQ acts as a master regulator of flagellar...

The bacterial second messenger c-di-GMP controls secretion, cell adhesion and motility leading to biofilm formation and increased cytotoxicity. Diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL or HD-GYP domains have been identified as the enzymes controlling cellular c-di-GMP levels, yet less is known regarding the molecular mechanisms governing regulation and signaling specificity. We recently determined a product-inhibition pathway for the diguanylate cyclase response regulator WspR from Pseudomonas, a potent molecular switch that controls biofilm formation. In WspR, catalytic activity is modulated by a helical stalk motif that connects its phospho-receiver (REC) and GGDEF domains. The stalks facilitate the formation of distinct oligomeric states that contribute to both activation and autoinhibition. Here, we provide novel insights into the regulation of diguanylate cyclase activity in WspR based on the crystal structures of full-length WspR, the isolated GGDEF domain, and an artificially dimerized catalytic domain. The structures highlight that inhibition is achieved by restricting the mobility of rigid GGDEF domains, mediated by c-di-GMP binding to an inhibitory site at the GGDEF domain. Kinetic measurements and biochemical characterization corroborate a model in which the activation of WspR requires the formation of a tetrameric species. Tetramerization occurs spontaneously at high protein concentration or upon addition of the phosphomimetic compound beryllium fluoride. Our analyses elucidate common and WspR-specific mechanisms for the fine-tuning of diguanylate cyclase activity.

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.Initially described as a multisubunit RNase complex required for maturation of 5.8 S rRNA in yeast (1), the 3Ј 3 5Ј exoribonuclease complex exosome was subsequently shown to play a central role on numerous pathways related to RNA processing and degradation, both in the nucleus and in the cytoplasm (reviewed in Ref. 2). These include 3Ј end processing of rRNAs, small nuclear RNAs, and small nucleolar RNAs (3-5), degradation of aberrant pre-mRNAs, pre-tRNAs, and pre-rRNAs (6 -9), normal turnover of cytosolic mRNAs (10), and degradation of RNA fragments produced during RNA interference processes (11, 12).Increasing structural and genetic studies with archaeal and eukaryotic exosomes have unveiled the evolutionary conservation of its molecular architecture (1, 13-18). The archaeal exosome consists of two RNase PH subunits (Rrp41 and Rrp42) and two proteins containing the S1/KH (Rrp4) or S1/zinc ribbon (Csl4) RNA-binding domains (17)(18)(19). Determination of the three-dimensional structure of the Sulfolobus solfataricus and Archaeoglobus fulgidus exosomes revealed a PNPase-like fold (20), composed by alternating RNase PH subunits assembled into a hexameric ring capped by a trimer of RNA-binding proteins, which can be formed by either Rrp4 or Csl4 or possibly by a mixture of both (19,21,22). Such an architecture encloses the exoribonucleolytic active sites at the bottom of the RNase PH ring catalytic chamber and restricts the entry to only unstructured RNA through the S1 pore, formed by the RNAbinding subunits of the exosome cap placed at the top of the ring (19,(21)(22)(23). Both Rrp41 and Rrp42 subunits possess the same RNase PH-fold and are involved in substrate binding, but the amino acid residues contributing to the interactions with the phosphate nucle...

Summary. Background: Missense mutations causing conformational alterations in serpins can be responsible for protein deficiency associated with human diseases. However, there are few data about conformational consequences of mutations affecting antithrombin, the main hemostatic serpin. Objectives: To investigate the conformational and clinical effect of mutations affecting the shutter region of antithrombin. Patients and methods: We identified two families with significant reduction of circulating antithrombin displaying early and severe venous thrombosis, frequently associated with pregnancy or infection. Mutations were determined by standard molecular methods. Biochemical studies were performed on plasma samples. One variant (P80S) was purified by heparinaffinity chromatography and gel filtration, and evaluated by proteomic analysis. Finally, we modelled the structure of the mutant dimer. Results: We identified two missense mutations affecting the shutter region of antithrombin: P80S and G424R. Carriers of both mutations presented traces of a similar abnormal antithrombin, supporting inefficiently expressed rather than non-expressed variants. The abnormal antithrombin purified from P80S carriers is an inactive disulfide-linked dimer of mutant antithrombin whose properties are consistent with head-to-head insertion of the reactive loop. Conclusions: Our data support the conclusion that missense mutations affecting the shutter region of serpins have specific conformational effects resulting in the formation of mutant oligomers. The consequent inefficiency of secretion explains the accompanying deficiency and loss of function, but the severity of thrombosis associated with these mutations suggests that the oligomers also have new and undefined pathological properties that could be exacerbated by pregnancy or infection.