Neuronal expression in Drosophila of an evolutionarily conserved metallophosphodiesterase reveals pleiotropic roles in longevity and odorant response.

Evolutionarily conserved genes often play critical roles in organismal physiology. Here, we describe multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. We observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism.

The evolutionary divergence of receptor guanylyl cyclase C has implications for preclinical models for receptor-directed therapeutics.

Mutations in receptor guanylyl cyclase C (GC-C) cause severe gastrointestinal disease, including meconium ileus, early onset acute diarrhea, and pediatric inflammatory bowel disease that continues into adulthood. Agonists of GC-C are US Food and Drug Administration-approved drugs for the treatment of constipation and irritable bowel syndrome. Therapeutic strategies targeting GC-C are tested in preclinical mouse models, assuming that murine GC-C mimics human GC-C in its biochemical properties and downstream signaling events. Here, we reveal important differences in ligand-binding affinity and GC activity between mouse GC-C and human GC-C. We generated a series of chimeric constructs of various domains of human and mouse GC-C to show that the extracellular domain of mouse GC-C contributed to log-orders lower affinity of mouse GC-C for ligands than human GC-C. Further, the Vmax of the murine GC domain was lower than that of human GC-C, and allosteric regulation of the receptor by ATP binding to the intracellular kinase-homology domain also differed. These altered properties are reflected in the high concentrations of ligands required to elicit signaling responses in the mouse gut in preclinical models and the specificity of a GC inhibitor towards human GC-C. Therefore, our studies identify considerations in using the murine model to test molecules for therapeutic purposes that work as either agonists or antagonists of GC-C, and vaccines for the bacterial heat-stable enterotoxin that causes watery diarrhea in humans.

Cyclic AMP binding to a universal stress protein in Mycobacterium tuberculosis is essential for viability.

Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways these bacteria utilize cAMP. We identified universal stress proteins, Rv1636 and MSMEG_3811 in Mycobacterium tuberculosis and Mycobacterium smegmatis, respectively, as abundantly expressed, novel cAMP-binding proteins. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. In slow-growing mycobacteria, intrabacterial concentrations of Rv1636 were equivalent to the concentrations of cAMP present in the cell. In contrast, levels of intrabacterial MSMEG_3811 in M. smegmatis were lower than that of cAMP and therefore, overexpression of Rv1636 increased levels of "bound" cAMP. While msmeg_3811 could be readily deleted from the genome of M. smegmatis, we found that the rv1636 gene is essential for the viability of M. tuberculosis and is dependent on the cAMP-binding ability of Rv1636. Therefore, Rv1636 may function to regulate cAMP signaling by direct sequestration of the second messenger. This is the first evidence of a "sponge" for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intrabacterial "free" pool of cAMP.

Mechanistic Insights into Pore Formation by an α-Pore Forming Toxin: Protein and Lipid Bilayer Interactions of Cytolysin A.

Pore forming toxins (PFTs) are the largest class of bacterial toxins playing a central role in bacterial pathogenesis. They are proteins specifically designed to form nanochannels in the membranes of target cells, ultimately resulting in cell death and establishing infection. PFTs are broadly classified as α- and ß-PFTs, depending on secondary structures that form the transmembrane channel. A unique feature about this class of proteins is the drastic conformational changes and complex oligomerization pathways that occur upon exposure to the plasma membrane. A molecular understanding of pore formation has implications in designing novel intervention strategies to combat rising antimicrobial resistance, targeted-cancer therapy, as well as designing nanopores for specialized technologies. Central to unraveling the pore formation pathway is the availability of high resolution crystal structures. In this regard, ß-toxins are better understood, when compared with α-toxins whose pore forming mechanisms are complicated by an incomplete knowledge of the driving forces for amphiphatic membrane-inserted helices to organize into functional pores. With the publication of the first crystal structure for an α-toxin, cytolysin A (ClyA), in 2009 we embarked on an extensive multiscale study to unravel its pore forming mechanism. This Account represents the collective mechanistic knowledge gained in our laboratories using a variety of experimental and theoretical techniques which include large scale molecular dynamics (MD) simulations, kinetic modeling studies, single-molecule fluorescence imaging, and super-resolution spectroscopy. We reported MD simulations of the ClyA protomer, oligomeric intermediates, and full pore complex in a lipid bilayer and mapped the conformational transitions that accompany membrane binding. Using single-molecule fluorescence imaging, the conformational transition was experimentally verified by analysis of various diffusion states of membrane bound ClyA. Importantly, we have uncovered a hitherto unknown putative cholesterol binding motif in the membrane-inserted helix of ClyA. Distinct binding pockets for cholesterol formed by adjacent membrane-inserted helices are revealed in MD simulations. Cholesterol appears to play a dual role by stabilizing both the membrane-inserted protomer as well as oligomeric intermediates. Molecular dynamics simulations and kinetic modeling studies suggest that the membrane-inserted arcs oligomerize reversibly to form the predominant transmembrane oligomeric intermediates during pore formation. We posit that this mechanistic understanding of the complex action of α-PFTs has implications in unraveling pore assembly across the wider family of bacterial toxins. With emerging antimicrobial resistance, alternate therapies may rely on disrupting pore functionality or oligomerization of these pathogenic determinants utilized by bacteria, and our study includes assessing the potential for dendrimers as pore blockers.

Strategies to Promote Vascularization in 3D Printed Tissue Scaffolds: Trends and Challenges.

Three-dimensional (3D) printing techniques for scaffold fabrication have shown promising advancements in recent years owing to the ability of the latest high-performance printers to mimic the native tissue down to submicron scales. Nevertheless, host integration and performance of scaffolds in vivo have been severely limited owing to the lack of robust strategies to promote vascularization in 3D printed scaffolds. As a result, researchers over the past decade have been exploring strategies that can promote vascularization in 3D printed scaffolds toward enhancing scaffold functionality and ensuring host integration. Various emerging strategies to enhance vascularization in 3D printed scaffolds are discussed. These approaches include simple strategies such as the enhancement of vascular in-growth from the host upon implantation by scaffold modifications to complex approaches wherein scaffolds are fabricated with their own vasculature that can be directly anastomosed or microsurgically connected to the host vasculature, thereby ensuring optimal integration. The key differences among the techniques, their pros and cons, and the future opportunities for utilizing each technique are highlighted here. The Review concludes with the current limitations and future directions that can help 3D printing emerge as an effective biofabrication technique to realize tissues with physiologically relevant vasculatures to ultimately accelerate clinical translation.

Mycobacterial STAND adenylyl cyclases: The HTH domain binds DNA to form biocrystallized nucleoids.

Mycobacteria harbor a unique class of adenylyl cyclases with a complex domain organization consisting of an N-terminal putative adenylyl cyclase domain fused to a nucleotide-binding adaptor shared by apoptotic protease-activating factor-1, plant resistance proteins, and CED-4 (NB-ARC) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal helix-turn-helix (HTH) domain. The products of the rv0891c-rv0890c genes represent a split gene pair, where Rv0891c has sequence similarity to adenylyl cyclases, and Rv0890c harbors the NB-ARC-TPR-HTH domains. Rv0891c had very low adenylyl cyclase activity so it could represent a pseudoenzyme. By analyzing the genomic locus, we could express and purify Rv0890c and find that the NB-ARC domain binds ATP and ADP, but does not hydrolyze these nucleotides. Using systematic evolution of ligands by exponential enrichment (SELEX), we identified DNA sequences that bound to the HTH domain of Rv0890c. Uniquely, the HTH domain could also bind RNA. Atomic force microscopy revealed that binding of Rv0890c to DNA was sequence independent, and binding of adenine nucleotides to the protein induced the formation of higher order structures that may represent biocrystalline nucleoids. This represents the first characterization of this group of proteins and their unusual biochemical properties warrant further studies into their physiological roles in future.

A universal stress protein in Mycobacterium smegmatis sequesters the cAMP-regulated lysine acyltransferase and is essential for biofilm formation.

Universal stress proteins (USPs) are present in many bacteria, and their expression is enhanced under various environmental stresses. We have previously identified a USP in Mycobacterium smegmatis that is a product of the msmeg_4207 gene and is a substrate for a cAMP-regulated protein lysine acyltransferase (KATms; MSMEG_5458). Here, we explored the role of this USP (USP4207) in M. smegmatis and found that its gene is present in an operon that also contains genes predicted to encode a putative tripartite tricarboxylate transporter (TTT). Transcription of the TTT-usp4207 operon was induced in the presence of citrate and tartrate, perhaps by the activity of a divergent histidine kinase-response regulator gene pair. A usp4207-deleted strain had rough colony morphology and reduced biofilm formation compared with the WT strain; however, both normal colony morphology and biofilm formation were restored in a Δusp4207Δkatms strain. We identified several proteins whose acetylation was lost in the Δkatms strain, and whose transcript levels increased in M. smegmatis biofilms along with that of USP4207, suggesting that USP4207 insulates KATms from its other substrates in the cell. We propose that USP4207 sequesters KATms from diverse substrates whose activities are down-regulated by acylation but are required for biofilm formation, thus providing a defined role for this USP in mycobacterial physiology and stress responses.

Cholesterol promotes Cytolysin A activity by stabilizing the intermediates during pore formation.

Pore-forming toxins (PFTs) form nanoscale pores across target membranes causing cell death. Cytolysin A (ClyA) from Escherichia coli is a prototypical α-helical toxin that contributes to cytolytic phenotype of several pathogenic strains. It is produced as a monomer and, upon membrane exposure, undergoes conformational changes and finally oligomerizes to form a dodecameric pore, thereby causing ion imbalance and finally cell death. However, our current understanding of this assembly process is limited to studies in detergents, which do not capture the physicochemical properties of biological membranes. Here, using single-molecule imaging and molecular dynamics simulations, we study the ClyA assembly pathway on phospholipid bilayers. We report that cholesterol stimulates pore formation, not by enhancing initial ClyA binding to the membrane but by selectively stabilizing a protomer-like conformation. This was mediated by specific interactions by cholesterol-interacting residues in the N-terminal helix. Additionally, cholesterol stabilized the oligomeric structure using bridging interactions in the protomer-protomer interfaces, thereby resulting in enhanced ClyA oligomerization. This dual stabilization of distinct intermediates by cholesterol suggests a possible molecular mechanism by which ClyA achieves selective membrane rupture of eukaryotic cell membranes. Topological similarity to eukaryotic membrane proteins suggests evolution of a bacterial α-toxin to adopt eukaryotic motifs for its activation. Broad mechanistic correspondence between pore-forming toxins hints at a wider prevalence of similar protein membrane insertion mechanisms.

Mutational landscape of receptor guanylyl cyclase C: Functional analysis and disease-related mutations.

Guanylyl cyclase C (GC-C) is the receptor for the heat-stable enterotoxin, which causes diarrhea, and the endogenous ligands, guanylin and uroguanylin. GC-C is predominantly expressed in the intestinal epithelium and regulates fluid and ion secretion in the gut. The receptor has a complex domain organization, and in the absence of structural information, mutational analysis provides clues to mechanisms of regulation of this protein. Here, we review the mutational landscape of this receptor that reveals regulatory features critical for its activity. We also summarize the available information on mutations in GC-C that have been reported in humans and contribute to severe gastrointestinal abnormalities. Since GC-C is also expressed in extra-intestinal tissues, it is likely that mutations thus far reported in humans may also affect other organ systems, warranting a close observation of these patients in future.

Absence of Receptor Guanylyl Cyclase C Enhances Ileal Damage and Reduces Cytokine and Antimicrobial Peptide Production during Oral Salmonella enterica Serovar Typhimurium Infection.

Nontyphoidal Salmonella disease contributes toward significant morbidity and mortality across the world. Host factors, including gamma interferon, tumor necrosis factor alpha, and gut microbiota, significantly influence the outcome of Salmonella pathogenesis. However, the entire repertoire of host protective mechanisms contributing to Salmonella pathogenicity is not completely appreciated. Here, we investigated the roles of receptor guanylyl cyclase C (GC-C), which is predominantly expressed in the intestine and regulates intestinal cell proliferation and fluid-ion homeostasis. Mice deficient in GC-C (Gucy2c-/-) displayed accelerated mortality compared with that for wild-type mice following infection via the oral route, even though both groups possessed comparable systemic Salmonella infection burdens. Survival following intraperitoneal infection remained similar in both groups, indicating that GC-C offered protection via a gut-mediated response. The serum cortisol level was higher in Gucy2c-/- mice than wild-type (Gucy2c+/+) mice, and an increase in infection-induced thymic atrophy with a loss of immature CD4+ CD8+ double-positive thymocytes was observed. Accelerated and enhanced damage in the ileum, including submucosal edema, epithelial cell damage, focal tufting, and distortion of the villus architecture, was seen in Gucy2c-/- mice concomitantly with a larger number of ileal tissue-associated bacteria. Transcription of key mediators of Salmonella-induced inflammation (interleukin-22/Reg3ß) was altered in Gucy2c-/- mice in comparison to that in Gucy2c+/+ mice. A reduction in fecal lactobacilli, which are protective against Salmonella infection, was observed in Gucy2c-/- mice. Gucy2c-/- mice cohoused with wild-type mice continued to show reduced amounts of lactobacilli and increased susceptibility to infection. Our study, therefore, suggests that the receptor GC-C confers a survival advantage during gut-mediated Salmonella enterica serovar Typhimurium pathogenesis, presumably by regulating Salmonella effector mechanisms and maintaining a beneficial microbiome.

The regulatory role of the kinase-homology domain in receptor guanylyl cyclases: nothing 'pseudo' about it!

The availability of genome sequence information and a large number of protein structures has allowed the cataloging of genes into various families, based on their function and predicted biochemical activity. Intriguingly, a number of proteins harbor changes in the amino acid sequence at residues, that from structural elucidation, are critical for catalytic activity. Such proteins have been categorized as 'pseudoenzymes'. Here, we review the role of the pseudokinase (or kinase-homology) domain in receptor guanylyl cyclases. These are multidomain single-pass, transmembrane proteins harboring an extracellular ligand-binding domain, and an intracellular domain composed of a kinase-homology domain that regulates the activity of the associated guanylyl cyclase domain. Mutations that lie in the kinase-homology domain of these receptors are associated with human disease, and either abolish or enhance cGMP production by these receptors to alter downstream signaling events. This raises the interesting possibility that one could identify molecules that bind to the pseudokinase domain and regulate the activities of these receptors, in order to alleviate symptoms in patients harboring these mutations.

Mycobacterial phenolic glycolipid synthesis is regulated by cAMP-dependent lysine acylation of FadD22.

The mycobacterial cell envelope is unique in its chemical composition, and has an important role to play in pathogenesis. Phthiocerol dimycocerosates (PDIMs) and glycosylated phenolphthiocerol dimycocerosates, also known as phenolic glycolipids (PGLs), contribute significantly to the virulence of Mycobacterium tuberculosis. FadD22 is essential for PGL biosynthesis. We have recently shown in vitro that FadD22 is a substrate for lysine acylation by a unique cAMP-dependent, protein lysine acyltransferase found only in mycobacteria. The lysine residue that is acylated is at the active site of FadD22. Therefore, acylation is likely to inhibit FadD22 activity and reduce PGL biosynthesis. Here, we show accumulation of PGLs in a strain of M. bovis BCG deleted for the gene encoding the cAMP-dependent acyltransferase, katbcg, with no change seen in PDIM synthesis. Complementation using KATbcg mutants that are deficient in cAMP-binding or acyltransferase activity shows that PGL accumulation is regulated by cAMP-dependent protein acylation in vivo. Expression of FadD22 and KATbcg mutants in Mycobacterium smegmatis confirmed that FadD22 is a substrate for lysine acylation by KATbcg. We have therefore described a mechanism by which cAMP can regulate mycobacterial virulence as a result of the ability of this second messenger to modulate critical cell wall components that affect the host immune response.

Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C.

OBJECTIVE: Congenital sodium diarrhoea (CSD) refers to a form of secretory diarrhoea with intrauterine onset and high faecal losses of sodium without congenital malformations. The molecular basis for CSD remains unknown. We clinically characterised a cohort of infants with CSD and set out to identify disease-causing mutations by genome-wide genetic testing. DESIGN: We performed whole-exome sequencing and chromosomal microarray analyses in 4 unrelated patients, followed by confirmatory Sanger sequencing of the likely disease-causing mutations in patients and in their family members, followed by functional studies. RESULTS: We identified novel de novo missense mutations in GUCY2C, the gene encoding receptor guanylate cyclase C (GC-C) in 4 patients with CSD. One patient developed severe, early-onset IBD and chronic arthritis at 4 years of age. GC-C is an intestinal brush border membrane-bound guanylate cyclase, which functions as receptor for guanylin, uroguanylin and Escherichia coli heat-stable enterotoxin. Mutations in GUCY2C were present in different intracellular domains of GC-C, and were activating mutations that enhanced intracellular cyclic guanosine monophosphate accumulation in a ligand-independent and ligand-stimulated manner, following heterologous expression in HEK293T cells. CONCLUSIONS: Dominant gain-of-function GUCY2C mutations lead to elevated intracellular cyclic guanosine monophosphate levels and could explain the chronic diarrhoea as a result of decreased intestinal sodium and water absorption and increased chloride secretion. Thus, mutations in GUCY2C indicate a role for this receptor in the pathogenesis of sporadic CSD.

The Solvent-Exposed C-Terminus of the Cytolysin A Pore-Forming Toxin Directs Pore Formation and Channel Function in Membranes.

Pore-forming toxins (PFTs) bind to cell membranes and form nanoscale pores that allow leakage of cellular components, resulting in cell death. The water-soluble, monomeric form of these toxins shows a dramatic conformational change during pore formation, as exemplified by crystal structures of the monomer and functional pore of cytolysin A (ClyA). The solvent-exposed, C-terminal residues of the protein are essential for activity, but the mechanism by which this region regulates pore formation remains unknown. We show here that deletion of the C-terminus of ClyA did not alter its ability to bind to the membrane or oligomerize in detergent. However, the truncated toxin lysed erythrocytes poorly, was more susceptible to proteolysis and thermal unfolding, and showed low calcein leakage from small unilamellar vesicles. Using fully atomistic molecular dynamics (MD) simulations, we find that deletion of C-terminal residues from the ClyA monomer significantly altered stability and unfolding trajectories in the transmembrane N-terminal helix, a region that is pivotal in maintaining the structural integrity of the helical bundle. MD simulations of pores with or without the C-terminus showed minor differences, implying that if oligomerization could be induced prior to the addition to vesicles, then an active pore could be generated. Via generation of oligomers in a detergent prior to the addition to vesicles, the truncated toxin could induce calcein leakage from vesicles, albeit to a lower extent. Therefore, regions of pore-forming toxins, not directly involved in the pore structure, are not passive players but have important roles in undergoing the transition through intermediary steps leading to successful pore formation in a membrane environment.

A universal stress protein (USP) in mycobacteria binds cAMP.

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as "sinks" for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell.

Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase.

Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins has not been studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to the identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein-tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys-224. This residue is situated in a lid region that covers the enzyme's active site. Consequently, acetylation and succinylation negatively regulate the activity of PtpB.

Super-resolution Stimulated Emission Depletion-Fluorescence Correlation Spectroscopy Reveals Nanoscale Membrane Reorganization Induced by Pore-Forming Proteins.

Membrane-protein interactions play a central role in membrane mediated cellular processes ranging from signaling, budding, and fusion, to transport across the cell membrane. Of particular significance is the process of efficient protein olgomerization and transmembrane pore formation on the membrane surface; the primary virulent pathway for the action of antimicrobial peptides and pore forming toxins (PFTs). The suggested nanoscopic length scales and dynamic nature of such membrane lipid-protein interactions makes their detection extremely challenging. Using a combination of super-resolution stimulated emission depletion nanoscopy with fluorescence correlation spectroscopy (STED-FCS) we unravel the emergence of nanoscale lateral heterogeneity in supported bilayer membranes made up of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol upon interaction with the PFT, listeriolysin O (LLO). A distinct length scale-dependent dynamical crossover (<200 nm) from a Brownian diffusive regime is observed at 33 and 50% cholesterol compositions, indicating the partitioning of lipids into domains with variable cholesterol content. At 25% cholesterol content, this dyamical crossover is observed only in bilayers incubated with LLO providing evidence for the existence of sub ∼100 nm dynamical lipid nanodomains bound to LLO pore assemblies. By introducing asymmetry in cholesterol composition across the bilayer leaflets we infer that this domain formation is driven largely due to active cholesterol sequestration and transient trapping of lipids to the membrane bound motifs present in the toxins, en route to LLO oligomerization and subsequent pore formation. Bilayers prepared with labeled lipids present in either the proximal or distal leaflet allow us to track the dynamical perturbation in a leaflet-dependent manner upon LLO incubation. From the differences in the extent and intensity of the dynamical crossover as observed with STED-FCS, these experiments reveal that the affinity for cholesterol in the membrane binding motifs of the LLO subdomains induce cholesterol and lipid reorganization to a greater extent in the distal (upper) leaflet when compared with the proximal (lower) leaflet. The observed length scale-dependent membrane reorganization that occurs due to invasion by LLO could be generalized to other cholesterol-dependent cytolysins and emphasizes the significant advantage of using super-resolution STED nanoscopy to unravel complex lipid-protein interactions in membrane and cellular biophysics.

Nanoscale dynamics of phospholipids reveals an optimal assembly mechanism of pore-forming proteins in bilayer membranes.

Cell membranes are believed to be highly complex dynamical systems having compositional heterogeneity involving several types of lipids and proteins as the major constituents. This dynamical and compositional heterogeneity is suggested to be critical to the maintenance of active functionality and response to chemical, mechanical, electrical and thermal stresses. However, delineating the various factors responsible for the spatio-temporal response of actual cell membranes to stresses can be quite challenging. In this work we show how biomimetic phospholipid bilayer membranes with variable lipid fluidity determine the optimal assembly mechanism of the pore-forming protein, listeriolysin O (LLO), belonging to the class of cholesterol dependent cytolysins (CDCs). By combining atomic force microscopy (AFM) and super-resolution stimulated emission depletion (STED) microscopy imaging on model membranes, we show that pores formed by LLO in supported lipid bilayers can have variable conformation and morphology depending on the fluidity of the bilayer. At a fixed cholesterol concentration, pores formed in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes were larger, flexible and more prone to coalescence when compared with the smaller and more compact pores formed in the lower fluidity 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. In contrast, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes did not show any evidence of pore formation. Fluorescence correlation spectroscopy (FCS) in STED mode revealed the appearance of a length scale, ξ, below which lipid dynamics, under the influence of LLO protein binding and assembly, becomes anomalous. Interestingly, the magnitude of ξ is found to correlate with both lipid fluidity and pore dimensions (and flexibility) in DOPC and POPC bilayers. However this length scale dependent crossover, signalling the onset of anomalous diffusion, was not observed in DMPC bilayers. Our study highlights the subtle interplay of lipid membrane mediated protein assembly and lipid fluidity in determining proteo-lipidic complexes formed in biomembranes and the significant insight that STED microscopy provides in unraveling critical aspects of nanoscale membrane biophysics.

Metallophosphoesterases: structural fidelity with functional promiscuity.

Calcineurin-like metallophosphoesterases (MPEs) form a large superfamily of binuclear metal-ion-centre-containing enzymes that hydrolyse phosphomono-, phosphodi- or phosphotri-esters in a metal-dependent manner. The MPE domain is found in Mre11/SbcD DNA-repair enzymes, mammalian phosphoprotein phosphatases, acid sphingomyelinases, purple acid phosphatases, nucleotidases and bacterial cyclic nucleotide phosphodiesterases. Despite this functional diversity, MPEs show a remarkably similar structural fold and active-site architecture. In the present review, we summarize the available structural, biochemical and functional information on these proteins. We also describe how diversification and specialization of the core MPE fold in various MPEs is achieved by amino acid substitution in their active sites, metal ions and regulatory effects of accessory domains. Finally, we discuss emerging roles of these proteins as non-catalytic protein-interaction scaffolds. Thus we view the MPE superfamily as a set of proteins with a highly conserved structural core that allows embellishment to result in dramatic and niche-specific diversification of function.

Genomic mapping of cAMP receptor protein (CRP Mt) in Mycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMt as a transcription factor.

Chromatin immunoprecipitation identified 191 binding sites of Mycobacterium tuberculosis cAMP receptor protein (CRP(Mt)) at endogenous expression levels using a specific α-CRP(Mt) antibody. Under these native conditions an equal distribution between intragenic and intergenic locations was observed. CRP(Mt) binding overlapped a palindromic consensus sequence. Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRP(Mt) during exponential growth, and in response to nutrient starvation. Differential expression of genes with a CRP(Mt)-binding site represented only a minor portion of this transcriptional reprogramming with ∼ 19% of those representing transcriptional regulators potentially controlled by CRP(Mt). The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation. CRP(Mt) can function as a classical transcription factor in M. tuberculosis, though this occurs at only a subset of CRP(Mt)-binding sites.