GTP-dependent membrane fusion.

Shape changes and topological remodeling of membranes are essential for the identity of organelles and membrane trafficking. Although all cellular membranes have common features, membranes of different organelles create unique environments that support specialized biological functions. The endoplasmic reticulum (ER) is a prime example of this specialization, as its lipid bilayer forms an interconnected system of cisternae, vesicles, and tubules, providing a highly compartmentalized structure for a multitude of biochemical processes. A variety of peripheral and integral membrane proteins that facilitate membrane curvature generation, fission, and/or fusion have been identified over the past two decades. Among these, the dynamin-related proteins (DRPs) have emerged as key players. Here, we review recent advances in our functional and molecular understanding of fusion DRPs, exemplified by atlastin, an ER-resident DRP that controls ER structure, function, and signaling.

Reconstitution of a biofilm adhesin system from a sulfate-reducing bacterium in Pseudomonas fluorescens.

Biofilms of sulfate-reducing bacterium (SRB) like Desulfovibrio vulgaris Hildenborough (DvH) can facilitate metal corrosion in various industrial and environmental settings leading to substantial economic losses. Although the mechanisms of biofilm formation by DvH are not yet well understood, recent studies indicate the large adhesin, DvhA, is a key determinant of biofilm formation. The dvhA gene neighborhood resembles the biofilm-regulating Lap system of Pseudomonas fluorescens but is curiously missing the c-di-GMP-binding regulator LapD. Instead, DvH encodes an evolutionarily unrelated c-di-GMP-binding protein (DVU1020) that we hypothesized is functionally analogous to LapD. To study this unusual Lap system and overcome experimental limitations with the slow-growing anaerobe DvH, we reconstituted its predicted SRB Lap system in a P. fluorescens strain lacking its native Lap regulatory components (ΔlapGΔlapD). Our data support the model that DvhA is a cell surface-associated LapA-like adhesin with a N-terminal "retention module" and that DvhA is released from the cell surface upon cleavage by the LapG-like protease DvhG. Further, we demonstrate DVU1020 (named here DvhD) represents a distinct class of c-di-GMP-binding, biofilm-regulating proteins that regulates DvhG activity in response to intracellular levels of this second messenger. This study provides insight into the key players responsible for biofilm formation by DvH, thereby expanding our understanding of Lap-like systems.

From Input to Output: The Lap/c-di-GMP Biofilm Regulatory Circuit.

Biofilms are the dominant bacterial lifestyle. The regulation of the formation and dispersal of bacterial biofilms has been the subject of study in many organisms. Over the last two decades, the mechanisms of Pseudomonas fluorescens biofilm formation and regulation have emerged as among the best understood of any bacterial biofilm system. Biofilm formation by P. fluorescens occurs through the localization of an adhesin, LapA, to the outer membrane via a variant of the classical type I secretion system. The decision between biofilm formation and dispersal is mediated by LapD, a c-di-GMP receptor, and LapG, a periplasmic protease, which together control whether LapA is retained or released from the cell surface. LapA localization is also controlled by a complex network of c-di-GMP-metabolizing enzymes. This review describes the current understanding of LapA-mediated biofilm formation by P. fluorescens and discusses several emerging models for the regulation and function of this adhesin.

Bacillus subtilis NrnB is expressed during sporulation and acts as a unique 3'-5' exonuclease.

All cells employ a combination of endo- and exoribonucleases to degrade long RNA polymers to fragments 2-5 nucleotides in length. These short RNA fragments are processed to monoribonucleotides by nanoRNases. Genetic depletion of nanoRNases has been shown to increase abundance of short RNAs. This deleteriously affects viability, virulence, and fitness, indicating that short RNAs are a metabolic burden. Previously, we provided evidence that NrnA is the housekeeping nanoRNase for Bacillus subtilis. Herein, we investigate the biological and biochemical functions of the evolutionarily related protein, B. subtilis NrnB (NrnBBs). These experiments show that NrnB is surprisingly different from NrnA. While NrnA acts at the 5' terminus of RNA substrates, NrnB acts at the 3' terminus. Additionally, NrnA is expressed constitutively under standard growth conditions, yet NrnB is selectively expressed during endospore formation. Furthermore, NrnA processes only short RNAs, while NrnB unexpectedly processes both short RNAs and longer RNAs. Indeed, inducible expression of NrnB can even complement the loss of the known global 3'-5' exoribonucleases, indicating that it acts as a general exonuclease. Together, these data demonstrate that NrnB proteins, which are widely found in Firmicutes, Epsilonproteobacteria and Archaea, are fundamentally different than NrnA proteins and may be used for specialized purposes.

MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis.

Lipids are important nutrients for Mycobacterium tuberculosis (Mtb) to support bacterial survival in mammalian tissues and host cells. Fatty acids and cholesterol are imported across the Mtb cell wall via the dedicated Mce1 and Mce4 transporters, respectively. It is thought that the Mce1 and Mce4 transporters are comprised of subunits that confer substrate specificity and proteins that couple lipid transport to ATP hydrolysis, similar to other bacterial ABC transporters. However, unlike canonical bacterial ABC transporters, Mce1 and Mce4 appear to share a single ATPase, MceG. Previously, it was established that Mce1 and Mce4 are destabilized when key transporter subunits are rendered nonfunctional; therefore, we investigated here the role of MceG in Mce1 and Mce4 protein stability. We determined that key residues in the Walker B domain of MceG are required for the Mce1- and Mce4-mediated transport of fatty acids and cholesterol. Previously, it has been established that Mce1 and Mce4 are destabilized and/or degraded when key transporter subunits are rendered nonfunctional, thus we investigated a role for MceG in stabilizing Mce1 and Mce4. Using an unbiased quantitative proteomic approach, we demonstrate that Mce1 and Mce4 proteins are specifically degraded in mutants lacking MceG. Furthermore, bacteria expressing Walker B mutant variants of MceG failed to stabilize Mce1 and Mce4, and we show that deleting MceG impacts the fitness of Mtb in the lungs of mice. Thus, we conclude that MceG represents an enzymatic weakness that can be potentially leveraged to disable and destabilize both the Mce1 and Mce4 transporters in Mtb.

NrnA is a 5'-3' exonuclease that processes short RNA substrates in vivo and in vitro.

Bacterial RNases process RNAs until only short oligomers (2-5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2-4 nucleotides in length, all of which were processed from their 5' terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.

A novel insertion mutation in atlastin 1 is associated with spastic quadriplegia, increased membrane tethering, and aberrant conformational switching.

Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of neuropathies affecting upper motor neurons and causing progressive gait disorder. Mutations in the gene SPG3A/atlastin-1 (ATL1), encoding a dynamin superfamily member, which utilizes the energy from GTP hydrolysis for membrane tethering and fusion to promote the formation of a highly branched, smooth endoplasmic reticulum (ER), account for approximately 10% of all HSP cases. The continued discovery and characterization of novel disease mutations are crucial for our understanding of HSP pathogenesis and potential treatments. Here, we report a novel disease-causing, in-frame insertion in the ATL1 gene, leading to inclusion of an additional asparagine residue at position 417 (N417ins). This mutation correlates with complex, early-onset spastic quadriplegia affecting all four extremities, generalized dystonia, and a thinning of the corpus callosum. We show using limited proteolysis and FRET-based studies that this novel insertion affects a region in the protein central to intramolecular interactions and GTPase-driven conformational change, and that this insertion mutation is associated with an aberrant prehydrolysis state. While GTPase activity remains unaffected by the insertion, membrane tethering is increased, indicative of a gain-of-function disease mechanism uncommon for ATL1-associated pathologies. In conclusion, our results identify a novel insertion mutation with altered membrane tethering activity that is associated with spastic quadriplegia, potentially uncovering a broad spectrum of molecular mechanisms that may affect neuronal function.

Functional inactivation of Plasmodium falciparum glycogen synthase kinase GSK3 modulates erythrocyte invasion and blocks gametocyte maturation.

Malaria is responsible for hundreds of thousands of deaths every year. The lack of an effective vaccine and the global spread of multidrug resistant parasites hampers the fight against the disease and underlines the need for new antimalarial drugs. Central to the pathogenesis of malaria is the proliferation of Plasmodium parasites within human erythrocytes. Parasites invade erythrocytes via a coordinated sequence of receptor-ligand interactions between the parasite and the host cell. Posttranslational modifications such as protein phosphorylation are known to be key regulators in this process and are mediated by protein kinases. For several parasite kinases, including the Plasmodium falciparum glycogen synthase kinase 3 (PfGSK3), inhibitors have been shown to block erythrocyte invasion. Here, we provide an assessment of PfGSK3 function by reverse genetics. Using targeted gene disruption, we show the active gene copy, PfGSK3ß, is not essential for asexual blood stage proliferation, although it modulates efficient erythrocyte invasion. We found functional inactivation leads to a 69% decreased growth rate and confirmed this growth defect by rescue experiments with wildtype and catalytically inactive mutants. Functional knockout of PfGSK3ß does not lead to transcriptional upregulation of the second copy of PfGSK3. We further analyze expression, localization, and function of PfGSK3ß during gametocytogenesis using a parasite line allowing conditional induction of sexual commitment. We demonstrate PfGSK3ß-deficient gametocytes show a strikingly malformed morphology leading to the death of parasites in later stages of gametocyte development. Taken together, these findings are important for our understanding and the development of PfGSK3 as an antimalarial target.

A hereditary spastic paraplegia-associated atlastin variant exhibits defective allosteric coupling in the catalytic core.

The dynamin-related GTPase atlastin (ATL) catalyzes membrane fusion of the endoplasmic reticulum and thus establishes a network of branched membrane tubules. When ATL function is compromised, the morphology of the endoplasmic reticulum deteriorates, and these defects can result in neurological disorders such as hereditary spastic paraplegia and hereditary sensory neuropathy. ATLs harness the energy of GTP hydrolysis to initiate a series of conformational changes that enable homodimerization and subsequent membrane fusion. Disease-associated amino acid substitutions cluster in regions adjacent to ATL's catalytic site, but the consequences for the GTPase's molecular mechanism are often poorly understood. Here, we elucidate structural and functional defects of an atypical hereditary spastic paraplegia mutant, ATL1-F151S, that is impaired in its nucleotide-hydrolysis cycle but can still adopt a high-affinity homodimer when bound to a transition-state analog. Crystal structures of mutant proteins yielded models of the monomeric pre- and post-hydrolysis states of ATL. Together, these findings define a mechanism for allosteric coupling in which Phe151 is the central residue in a hydrophobic interaction network connecting the active site to an interdomain interface responsible for nucleotide loading.

Versatile modes of cellular regulation via cyclic dinucleotides.

Since the discovery of c-di-GMP almost three decades ago, cyclic dinucleotides (CDNs) have emerged as widely used signaling molecules in most kingdoms of life. The family of second messengers now includes c-di-AMP and distinct versions of mixed cyclic GMP-AMP (cGAMP) compounds. In addition to these nucleotides, a vast number of proteins for the production and turnover of these molecules have been described, as well as effectors that translate the signals into physiological responses. The latter include, but are not limited to, mechanisms for adaptation and survival in prokaryotes, persistence and virulence of bacterial pathogens, and immune responses to viral and bacterial invasion in eukaryotes. In this review, we will focus on recent discoveries and emerging themes that illustrate the ubiquity and versatility of cyclic dinucleotide function at the transcriptional and post-translational levels and, in particular, on insights gained through mechanistic structure-function analyses.

Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa.

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.

Type 1 Does the Two-Step: Type 1 Secretion Substrates with a Functional Periplasmic Intermediate.

Bacteria have evolved several secretion strategies for polling and responding to environmental flux and insult. Of these, the type 1 secretion system (T1SS) is known to secrete an array of biologically diverse proteins-from small, <10-kDa bacteriocins to gigantic adhesins with a mass >1 MDa. For the last several decades, T1SSs have been characterized as a one-step translocation strategy whereby the secreted substrate is transported directly into the extracellular environment from the cytoplasm with no periplasmic intermediate. Recent phylogenetic, biochemical, and genetic evidences point to a distinct subgroup of T1SS machinery linked with a bacterial transglutaminase-like cysteine proteinase (BTLCP), which uses a two-step secretion mechanism. BTLCP-linked T1SSs transport a class of repeats-in-toxin (RTX) adhesins that are critical for biofilm formation. The prototype of this RTX adhesin group, LapA of Pseudomonas fluorescens Pf0-1, uses a novel N-terminal retention module to anchor the adhesin at the cell surface as a secretion intermediate threaded through the outer membrane-localized TolC-like protein LapE. This secretion intermediate is posttranslationally cleaved by the BTLCP family LapG protein to release LapA from its cognate T1SS pore. Thus, the secretion of LapA and related RTX adhesins into the extracellular environment appears to be a T1SS-mediated two-step process that involves a periplasmic intermediate. In this review, we contrast the T1SS machinery and substrates of the BLTCP-linked two-step secretion process with those of the classical one-step T1SS to better understand the newly recognized and expanded role of this secretion machinery.

An N-Terminal Retention Module Anchors the Giant Adhesin LapA of Pseudomonas fluorescens at the Cell Surface: a Novel Subfamily of Type I Secretion Systems.

LapA of Pseudomonas fluorescens Pf0-1 belongs to a diverse family of cell surface-associated bacterial adhesins that are secreted via the type I secretion system (T1SS). We previously reported that the periplasmic protease LapG cleaves the N terminus of LapA at a canonical dialanine motif to release the adhesin from the cell surface under conditions unfavorable to biofilm formation, thus decreasing biofilm formation. Here, we characterize LapA as the first type I secreted substrate that does not follow the "one-step" rule of T1SS. Rather, a novel N-terminal element, called the retention module (RM), localizes LapA at the cell surface as a secretion intermediate. Our genetic, biochemical, and molecular modeling analyses support a model wherein LapA is tethered to the cell surface through its T1SS outer membrane TolC-like pore, LapE, until LapG cleaves LapA in the periplasm. We further demonstrate that this unusual retention strategy is likely conserved among LapA-like proteins, and it reveals a new subclass of T1SS ABC transporters involved in transporting this group of surface-associated LapA-like adhesins. These studies demonstrate a novel cell surface retention strategy used throughout the Proteobacteria and highlight a previously unappreciated flexibility of function for T1SS.IMPORTANCE Bacteria have evolved multiple secretion strategies to interact with their environment. For many bacteria, the secretion of cell surface-associated adhesins is key for initiating contact with a preferred substratum to facilitate biofilm formation. Our work demonstrates that P. fluorescens uses a previously unrecognized secretion strategy to retain the giant adhesin LapA at its cell surface. Further, we identify likely LapA-like adhesins in various pathogenic and commensal proteobacteria and provide phylogenetic evidence that these adhesins are secreted by a new subclass of T1SS ABC transporters.

Disrupted Hydrogen-Bond Network and Impaired ATPase Activity in an Hsc70 Cysteine Mutant.

The ATPase domain of members of the 70 kDa heat shock protein (Hsp70) family shows a high degree of sequence, structural, and functional homology across species. A broadly conserved residue within the Hsp70 ATPase domain that captured our attention is an unpaired cysteine, positioned proximal to the site of nucleotide binding. Prior studies of several Hsp70 family members show this cysteine is not required for Hsp70 ATPase activity, yet select amino acid replacements of the cysteine can dramatically alter ATP hydrolysis. Moreover, post-translational modification of the cysteine has been reported to limit ATP hydrolysis for several Hsp70s. To better understand the underlying mechanism for how perturbation of this noncatalytic residue modulates Hsp70 function, we determined the structure for a cysteine-to-tryptophan mutation in the constitutively expressed, mammalian Hsp70 family member Hsc70. Our work reveals that the steric hindrance produced by a cysteine-to-tryptophan mutation disrupts the hydrogen-bond network within the active site, resulting in a loss of proper catalytic magnesium coordination. We propose that a similarly altered active site is likely observed upon post-translational oxidation. We speculate that the subtle changes we detect in the hydrogen-bonding network may relate to the previously reported observation that cysteine oxidation can influence Hsp70 interdomain communication.

Structural basis for conformational switching and GTP loading of the large G protein atlastin.

Atlastin, a member of the dynamin superfamily, is known to catalyse homotypic membrane fusion in the smooth endoplasmic reticulum (ER). Recent studies of atlastin have elucidated key features about its structure and function; however, several mechanistic details, including the catalytic mechanism and GTP hydrolysis-driven conformational changes, are yet to be determined. Here, we present the crystal structures of atlastin-1 bound to GDP·AlF(4)(-) and GppNHp, uncovering an intramolecular arginine finger that stimulates GTP hydrolysis when correctly oriented through rearrangements within the G domain. Utilizing Förster Resonance Energy Transfer, we describe nucleotide binding and hydrolysis-driven conformational changes in atlastin and their sequence. Furthermore, we discovered a nucleotide exchange mechanism that is intrinsic to atlastin's N-terminal domains. Our results indicate that the cytoplasmic domain of atlastin acts as a tether and homotypic interactions are timed by GTP binding and hydrolysis. Perturbation of these mechanisms may be implicated in a group of atlastin-associated hereditary neurodegenerative diseases.

The Inhibitory Site of a Diguanylate Cyclase Is a Necessary Element for Interaction and Signaling with an Effector Protein.

UNLABELLED: Many bacteria contain large cyclic diguanylate (c-di-GMP) signaling networks made of diguanylate cyclases (DGCs) and phosphodiesterases that can direct cellular activities sensitive to c-di-GMP levels. While DGCs synthesize c-di-GMP, many DGCs also contain an autoinhibitory site (I-site) that binds c-di-GMP to halt excess production of this small molecule, thus controlling the amount of c-di-GMP available to bind to target proteins in the cell. Many DGCs studied to date have also been found to signal for a specific c-di-GMP-related process, and although recent studies have suggested that physical interaction between DGCs and target proteins may provide this signaling fidelity, the importance of the I-site has not yet been incorporated into this model. Our results from Pseudomonas fluorescens indicate that mutation of residues at the I-site of a DGC disrupts the interaction with its target receptor. By creating various substitutions to a DGC's I-site, we show that signaling between a DGC (GcbC) and its target protein (LapD) is a combined function of the I-site-dependent protein-protein interaction and the level of c-di-GMP production. The dual role of the I-site in modulating DGC activity as well as participating in protein-protein interactions suggests caution in interpreting the function of the I-site as only a means to negatively regulate a cyclase. These results implicate the I-site as an important positive and negative regulatory element of DGCs that may contribute to signaling specificity. IMPORTANCE: Some bacteria contain several dozen diguanylate cyclases (DGCs), nearly all of which signal to specific receptors using the same small molecule, c-di-GMP. Signaling specificity in these networks may be partially driven by physical interactions between DGCs and their receptors, in addition to the autoinhibitory site of DGCs preventing the overproduction of c-di-GMP. In this study, we show that disruption of the autoinhibitory site of a DGC in Pseudomonas fluorescens can result in the loss of interactions with its target receptor and reduced biofilm formation, despite increased production of c-di-GMP. Our findings implicate the autoinhibitory site as both an important feature for signaling specificity through the regulation of c-di-GMP production and a necessary element for the physical interaction between a diguanylate cyclase and its receptor.

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 c-di-GMP effector system controls cell adhesion by inside-out signaling and surface protein cleavage.

In Pseudomonas fluorescens Pf0-1 the availability of inorganic phosphate (Pi) is an environmental signal that controls biofilm formation through a cyclic dimeric GMP (c-di-GMP) signaling pathway. In low Pi conditions, a c-di-GMP phosphodiesterase (PDE) RapA is expressed, depleting cellular c-di-GMP and causing the loss of a critical outer-membrane adhesin LapA from the cell surface. This response involves an inner membrane protein LapD, which binds c-di-GMP in the cytoplasm and exerts a periplasmic output promoting LapA maintenance on the cell surface. Here we report how LapD differentially controls maintenance and release of LapA: c-di-GMP binding to LapD promotes interaction with and inhibition of the periplasmic protease LapG, which targets the N-terminus of LapA. We identify conserved amino acids in LapA required for cleavage by LapG. Mutating these residues in chromosomal lapA inhibits LapG activity in vivo, leading to retention of the adhesin on the cell surface. Mutations with defined effects on LapD's ability to control LapA localization in vivo show concomitant effects on c-di-GMP-dependent LapG inhibition in vitro. To establish the physiological importance of the LapD-LapG effector system, we track cell attachment and LapA protein localization during Pi starvation. Under this condition, the LapA adhesin is released from the surface of cells and biofilms detach from the substratum. This response requires c-di-GMP depletion by RapA, signaling through LapD, and proteolytic cleavage of LapA by LapG. These data, in combination with the companion study by Navarro et al. presenting a structural analysis of LapD's signaling mechanism, give a detailed description of a complete c-di-GMP control circuit--from environmental signal to molecular output. They describe a novel paradigm in bacterial signal transduction: regulation of a periplasmic enzyme by an inner membrane signaling protein that binds a cytoplasmic second messenger.

Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis.

The bacterial second messenger bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a central regulator for biofilm formation. Increased cellular c-di-GMP levels lead to stable cell attachment, which in Pseudomonas fluorescens requires the transmembrane receptor LapD. LapD exhibits a conserved and widely used modular architecture containing a HAMP domain and degenerate diguanylate cyclase and phosphodiesterase domains. c-di-GMP binding to the LapD degenerate phosphodiesterase domain is communicated via the HAMP relay to the periplasmic domain, triggering sequestration of the protease LapG, thus preventing cleavage of the surface adhesin LapA. Here, we elucidate the molecular mechanism of autoinhibition and activation of LapD based on structure-function analyses and crystal structures of the entire periplasmic domain and the intracellular signaling unit in two different states. In the absence of c-di-GMP, the intracellular module assumes an inactive conformation. Binding of c-di-GMP to the phosphodiesterase domain disrupts the inactive state, permitting the formation of a trans-subunit dimer interface between adjacent phosphodiesterase domains via interactions conserved in c-di-GMP-degrading enzymes. Efficient mechanical coupling of the conformational changes across the membrane is realized through an extensively domain-swapped, unique periplasmic fold. Our structural and functional analyses identified a conserved system for the regulation of periplasmic proteases in a wide variety of bacteria, including many free-living and pathogenic species.

Structural basis for the nucleotide-dependent dimerization of the large G protein atlastin-1/SPG3A.

The large GTPase atlastin belongs to the dynamin superfamily that has been widely implicated in facilitating membrane tubulation, fission, and in select cases, fusion. Mutations spread across atlastin isoform 1 (atlastin-1) have been identified in patients suffering from hereditary spastic paraplegia (HSP), a neurodegenerative disorder affecting motor neuron function in the lower extremities. On a molecular level, atlastin-1 associates with high membrane curvature and fusion events at the endoplasmic reticulum and cis-Golgi. Here we report crystal structures of atlastin-1 comprising the G and middle domains in two different conformations. Although the orientation of the middle domain relative to the G domain is different in the two structures, both reveal dimeric assemblies with a common, GDP-bound G domain dimer. In contrast, dimer formation in solution is observed only in the presence of GTP and transition state analogs, similar to other G proteins that are activated by nucleotide-dependent dimerization. Analyses of solution scattering data suggest that upon nucleotide binding, the protein adopts a somewhat extended, dimeric conformation that is reminiscent of one of the two crystal structures. These structural studies suggest a model for nucleotide-dependent regulation of atlastin with implications for membrane fusion. This mechanism is affected in several mutants associated with HSP, providing insights into disease pathogenesis.