Bacterial Homolog Research Articles

Allosteric modulation of the GTPase activity of a bacterial LRRK2 homolog by conformation-specific Nanobodies

Mutations in the Parkinson's disease (PD)-associated protein leucine-rich repeat kinase 2 (LRRK2) commonly lead to a reduction of GTPase activity and increase in kinase activity. Therefore, strategies for drug development have mainly been focusing on the design of LRRK2 kinase inhibitors. We recently showed that the central RocCOR domains (Roc: Ras of complex proteins; COR: C-terminal of Roc) of a bacterial LRRK2 homolog cycle between a dimeric and monomeric form concomitant with GTP binding and hydrolysis. PD-associated mutations can slow down GTP hydrolysis by stabilizing the protein in its dimeric form. Here, we report the identification of two Nanobodies (NbRoco1 and NbRoco2) that bind the bacterial Roco protein (CtRoco) in a conformation-specific way, with a preference for the GTP-bound state. NbRoco1 considerably increases the GTP turnover rate of CtRoco and reverts the decrease in GTPase activity caused by a PD-analogous mutation. We show that NbRoco1 exerts its effect by allosterically interfering with the CtRoco dimer–monomer cycle through the destabilization of the dimeric form. Hence, we provide the first proof of principle that allosteric modulation of the RocCOR dimer–monomer cycle can alter its GTPase activity, which might present a potential novel strategy to overcome the effect of LRRK2 PD mutations.

Functional Characterization of Position 271 in NTCP, a Predicted Rheostat Location

BackgroundHuman Na+/taurocholate cotransporting polypeptide (NTCP) is a sodium dependent bile acid transporter expressed at the basolateral membrane of human hepatocytes. It is important for the enterohepatic circulation of bile acids, such as taurocholate (TCA), but it also transports other compounds including estrone‐3‐sulfate (E3S) and rosuvastatin. We previously identified a rheostat position at a naturally occurring polymorphism, S267F. That is, when other amino acids were substituted into this position, progressive changes in function were observed.AimThe purpose of the current study is to determine whether position N271, a predicted rheostat, is indeed another rheostat position in NTCP.MethodsThe crystal structures of two bacterial NTCP homologs were used to model the structure of human NTCP in the inward‐open and outward‐open conformations. Mutations were then introduced into the structural models and the energetic effect on protein stability was evaluated using the Rosetta software. Based on these calculations we predicted that N271 is a rheostat. Mutants for position N271 were generated using site‐directed mutagenesis and the resulting proteins were transiently expressed in HEK293 cells. The functions of the mutants were characterized by measuring uptake of radiolabeled model substrates: [3H]‐taurocholate, [3H]‐estrone‐3‐sulfate, and [3H]‐rosuvastatin. Plasma membrane expression levels were determined by surface biotinylation assays.ResultsInitial transport studies using nanomolar concentrations of TCA, E3S, and rosuvastatin indicated a variety of intermediate functional outcomes for the mutants. Furthermore, this alteration in function was substrate dependent, which is consistent with our findings for S267. Some mutants, such as N271A, transport substrates similar to wild‐type NTCP, while others, like N271K, show variation depending on the substrate. N271P consistently transports all substrates minimally. In addition, there is no correlation between the initial uptake results and the Rosetta energies.Summary and ConclusionsInitial transport studies demonstrate that position N271 is a rheostat position, as predicted by the alteration in Rosetta energy scores. However, there was no correlation between the calculated energy scores and the functional results. This suggests that functional outcomes of specific amino acid replacements at position N271 may arise from more complex conformational changes than were captured by the two modeled states. Additional experiments should help to determine to what extent the observed change in function is due to variations in the substrate affinity, the turnover number or the transport capacity of the mutants.Support or Funding InformationP30 GM118247, GM077336, and W. M. Keck Foundation

Probing the Role of Chloride in Facilitating System xc− Function

System xc− is a heterodimeric transporter that functions as a Na+‐independent antiporter exchanging extracellular cystine for intracellular glutamate. The transporter functions as an obligate heterodimer and is comprised of two proteins, xCT, which functions specifically in amino acid exchange, and 4F2HC, which appears to play a role in transporter stability. System xc− belongs to the SLC7 family of transporters, which is further subdivided into the cationic amino acid transporters (CAT) and the L‐type amino acid transporters (LAT), the latter of which is the subfamily which includes System xc−. In recent years, there have been advances in our understanding of the structure of the SLC7 transporters as crystal structures of bacterial homologs of CAT and LAT transporters have been published. As a result, we can now begin to ask important questions about the mechanism by which System xc− exchanges cystine and glutamate across the membrane. Previous studies in the Chase lab have demonstrated that System xc− is Cl−‐dependent. More recently we have shown that transport of one molecule of cystine requires more than one Cl− ion, and that Cl− binding affects the affinity of the transporter for cystine. We have also shown that chloride dependence of cystine transport is pH dependent. The K0.5 of chloride increases as the pH increases, suggesting that the Cl− binding affinity decreases at higher pH. Using this collective information, we examined the structure of xCT to identify the most likely binding site for cystine and glutamate. Specifically, we used Chimera 1.13.1 and Pyrx to dock cystine and glutamate into its most energetically favorable positions on xCT and identified several ionizable side chains within the putative permeation pathway associated with transmembrane domains 3 and 6 that appear to interact with substrate. These residues include R135, T138, Y246 and Y251. We are currently assessing this model in the presence of Cl− to identify putative residues which may be important for the docking of Cl− near the substrate binding site and using site‐directed mutagenesis to determine whether these residues are responsible for Cl−‐dependent cystine transport.Support or Funding InformationThis research is supported by the Campbell Foundation and Schaap Endowed Funds for Undergraduate Research.

Bacterial alginate regulators and phage homologs repress CRISPR-Cas immunity.

CRISPR-Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection1. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases2. While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR-Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB/AlgB), previously characterized in regulating Pseudomonas aeruginosa alginate biosynthesis3,4, as a regulator of the expression and activity of the P. aeruginosa Type I-F CRISPR-Cas system. Downstream of KinB/AlgB, activators of alginate production AlgU (a σE orthologue) and AlgR, repress CRISPR-Cas activity during planktonic and surface-associated growth5. AmrZ, another alginate regulator6, is triggered to repress CRISPR-Cas immunity during surface-association. Pseudomonas phages and plasmids have taken advantage of this regulatory scheme, and carry hijacked homologs of AmrZ that repress CRISPR-Cas expression and activity. This suggests that while CRISPR-Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation.

Nitric oxide disrupts bacterial cytokinesis by poisoning purine metabolism.

Cytostasis is the most salient manifestation of the potent antimicrobial activity of nitric oxide (NO), yet the mechanism by which NO disrupts bacterial cell division is unknown. Here, we show that in respiring Escherichia coli, Salmonella, and Bacillus subtilis, NO arrests the first step in division, namely, the GTP-dependent assembly of the bacterial tubulin homolog FtsZ into a cytokinetic ring. FtsZ assembly fails in respiring cells because NO inactivates inosine 5'-monophosphate dehydrogenase in de novo purine nucleotide biosynthesis and quinol oxidases in the electron transport chain, leading to drastic depletion of nucleoside triphosphates, including the GTP needed for the polymerization of FtsZ. Despite inhibiting respiration and dissipating proton motive force, NO does not destroy Z ring formation and only modestly decreases nucleoside triphosphates in glycolytic cells, which obtain much of their ATP by substrate-level phosphorylation and overexpress inosine 5'-monophosphate dehydrogenase. Purine metabolism dictates the susceptibility of early morphogenic steps in cytokinesis to NO toxicity.

Insights into the Structure, Function, and Dynamics of the Bacterial Cytokinetic FtsZ-Ring

The FtsZ protein is a highly conserved bacterial tubulin homolog. In vivo, the functional form of FtsZ is the polymeric, ring-like structure (Z-ring) assembled at the future division site during cell division. While it is clear that the Z-ring plays an essential role in orchestrating cytokinesis, precisely what its functions are and how these functions are achieved remain elusive. In this article, we review what we have learned during the past decade about the Z-ring's structure, function, and dynamics, with a particular focus on insights generated by recent high-resolution imaging and single-molecule analyses. We suggest that the major function of the Z-ring is to govern nascent cell pole morphogenesis by directing the spatiotemporal distribution of septal cell wall remodeling enzymes through the Z-ring's GTP hydrolysis-dependent treadmilling dynamics. In this role, FtsZ functions in cell division as the counterpart of the cell shape-determining actin homolog MreB in cell elongation.

Conformational analysis of the riboflavin-responsive ETF:QO-p.Pro456Leu variant associated with mild multiple acyl-CoA dehydrogenase deficiency

Multiple-CoA dehydrogenase deficiency (MADD) is an inborn disorder of fatty acid and amino acid metabolism caused by mutations in the genes encoding for human electron transfer flavoprotein (ETF) and its partner electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). Albeit a rare disease, extensive newborn screening programs contributed to a wider coverage of MADD genotypes. However, the impact of non-lethal mutations on ETF:QO function remains scarcely understood from a structural perspective. To this end, we here revisit the relatively common MADD mutation ETF:QO-p.Pro456Leu, in order to clarify how it affects enzyme structure and folding. Given the limitation in recombinant expression of human ETF:QO, we resort to its bacterial homologue from Rhodobacter sphaeroides (Rs), in which the corresponding mutation (p.Pro389Leu) was inserted. The in vitro biochemical and biophysical investigations of the Rs ETF:QO-p.Pro389Leu variant showed that, while the mutation does not significantly affect the protein α/β fold, it introduces some plasticity on the tertiary structure and within flavin interactions. Indeed, in the p.Pro389Leu variant, FAD exhibits a higher thermolability during thermal denaturation and a faster rate of release in temperature-induced dissociation experiments, in comparison to the wild type. Therefore, although this clinical mutation occurs in the ubiquinone domain, its effect likely propagates to the nearby FAD binding domain, probably influencing electron transfer and redox potentials. Overall, our results provide a molecular rational for the decreased enzyme activity observed in patients and suggest that compromised FAD interactions in ETF:QO might account for the known riboflavin responsiveness of this mutation.

Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences

ABSTRACTRNA helicases are fundamental players in RNA metabolism: they remodel RNA secondary structures and arrange ribonucleoprotein complexes. While DExH-box RNA helicases function in ribosome biogenesis and splicing in eukaryotes, information is scarce about bacterial homologs. HrpB is the only bacterial DExH-box protein whose structure is solved. Besides the catalytic core, HrpB possesses three accessory domains, conserved in all DExH-box helicases, plus a unique C-terminal extension (CTE). The function of these auxiliary domains remains unknown. Here, we characterize genetically and biochemically Pseudomonas aeruginosa HrpB homolog. We reveal that the auxiliary domains shape HrpB RNA preferences, affecting RNA species recognition and catalytic activity. We show that, among several types of RNAs, the single-stranded poly(A) and the highly structured MS2 RNA strongly stimulate HrpB ATPase activity. In addition, deleting the CTE affects only stimulation by structured RNAs like MS2 and rRNAs, while deletion of accessory domains results in gain of poly(U)-dependent activity. Finally, using hydrogen-deuterium exchange, we dissect the molecular details of HrpB interaction with poly(A) and MS2 RNAs. The catalytic core interacts with both RNAs, triggering a conformational change that reorients HrpB. Regions within the accessory domains and CTE are, instead, specifically responsive to MS2. Altogether, we demonstrate that in bacteria, like in eukaryotes, DExH-box helicase auxiliary domains are indispensable for RNA handling.

Targeting Bacterial Cell Division: A Binding Site-Centered Approach to the Most Promising Inhibitors of the Essential Protein FtsZ.

Binary fission is the most common mode of bacterial cell division and is mediated by a multiprotein complex denominated the divisome. The constriction of the Z-ring splits the mother bacterial cell into two daughter cells of the same size. The Z-ring is formed by the polymerization of FtsZ, a bacterial protein homologue of eukaryotic tubulin, and it represents the first step of bacterial cytokinesis. The high grade of conservation of FtsZ in most prokaryotic organisms and its relevance in orchestrating the whole division system make this protein a fascinating target in antibiotic research. Indeed, FtsZ inhibition results in the complete blockage of the division system and, consequently, in a bacteriostatic or a bactericidal effect. Since many papers and reviews already discussed the physiology of FtsZ and its auxiliary proteins, as well as the molecular mechanisms in which they are involved, here, we focus on the discussion of the most compelling FtsZ inhibitors, classified by their main protein binding sites and following a medicinal chemistry approach.

Mapping pH-Dependent State Transitions of a Pentameric Ligand-gated Ion Channel through Markov State Modeling

Pentameric ligand-gated ion channels are of great importance as intercellular communicators that mediate electrochemical signaling in neurons and other excitable cells. Recently, representative structures in this protein family have been determined through X-ray crystallography or cryo-electron microscopy, confirming the conservation of several topological features. However, details of the gating transition between open and closed states remain unclear, which is hampering efforts towards further mechanistic modeling and drug design. The bacterial homolog, GLIC from Gloeobacter violaceus, is gated by changes in pH and has the advantage of being associated with structural and functional data covering multiple functional states. Here, we present Markov state models of the gating conformational transitions of GLIC by using hundreds of microsecond-long molecular dynamics simulations. These have been started from different initial conditions along a transition path and at different pH values, thus allowing sampling of the whole conformational landscape between open and resting states. From these models, we could confirm the experimental result that the channels prefer a closed conformation at neutral pH, while a small fraction of channels open at low pH. In contrast, simulations of the proton-sensitized mutant, I233T, yielded a clear shift of the free energy profile towards the open state, evident at both pH values. This observation agrees with our electrophysiology recordings of both wild-type and mutant GLIC, demonstrating that simulations are able to predict non-trivial shifts in activation profiles. Furthermore, these results offer insight into the fraction of channels opened upon activation, and provide a basis for mechanistic modeling of intermediate states along the gating pathway.

Characterizing the Interplay between Dynamics and Regulation in the Trypsinogen/Trypsin Protease System

Trypsin (Tn) is a digestive serine protease found in eukaryotes and prokaryotes. Tn is manufactured by the vertebrate pancreas as its proenzyme trypsinogen (Tgn) which includes an N-terminal six amino acid zymogen region. The zymogen form, Tgn, is disabled against cleavage of substrate until Tgn reaches the small intestine. Within the small intestine, enterokinase cleaves the inhibiting peptide from folded Tgn to release active Tn protease. Fluorescence experiments with bovine Tn demonstrate remarkably slow global unfolding, with a kinetic barrier to unfolding as large as kinetically stabilized bacterial homologues. Whereas bacterial homologues have evolved to resist proteolysis, thereby prolonging extracellular activity, mammalian Tgn/Tn is tightly regulated through a series of auto-cleavage events. We propose that Tgn/Tn experiences subglobal dynamics, and that sampling of these semi-unfolded states under physiological conditions causes this rapid autolysis. To explore this, we will perform native-state hydrogen exchange mass spectrometry of a purified inactive mutant. Exploring the Tgn/Tn subglobal dynamics which expose the native-state to autolysis will grant understanding of the link between Tn dynamics and the regulation of its function through its specific sequence of auto-cleavage events. By comprehensively characterizing the sampling of subglobally and globally unfolded states of Tgn/Tn, more can be inferred about the occurrence of non-native state protein aggregation and susceptibility to proteolytic attack. Studying Tgn/Tn from the inactive Tgn/Tn mutant allows direct assessment of the role of subglobal protein dynamics and the regulation of these dynamics in Tn folding disorders without the complication of autoproteolysis. The Tgn/Tn system will allow investigation of diseases characterized by abnormal protein aggregation and vulnerability to proteolytic attack, in addition to diseases directly related to Tn function such as pancreatitis.

Comparison of the Dynamic Properties of the Bacterial Tubulin Homolog FtsZ, from Bacteria to Chloroplast

The tubulin homolog FtsZ is the major cytoskeletal protein in the bacterial cell division machinery, conserved in almost all bacteria, archaea, and chloroplasts. Bacterial FtsZ assembles spontaneously into dynamic protofilaments in vitro and forms a dynamic contractile ring or Z-ring in vivo. FtsZ from different species can have different dynamic properties. Here, we compare the dynamic properties of FtsZ from Escherichia coli (EcFtsZ), from the chloroplast of red alga Galdieria sulphuraria (GsFtsZA and GsFtsZB), and from the cyanobacterium Synechocystis sp. PCC 6803 (SyFtsZ). EcFtsZ assembles in vitro into mostly single protofilaments with an average length of 200 nm, which is highly dynamic, turning over with a half time of 5-8 s both in vitro and in vivo. The mechanism of dynamics is now established to be treadmilling, which involves adding subunits at one end and disassembling at the other. Chloroplasts arose in eukaryotes as endosymbionts of a cyanobacterium. The ancestral ftsZ gene was duplicated early in chloroplast evolution, and most express two FtsZ proteins for its division. Our studies on the GsFtsZA and GsFtsZB showed that both FtsZs assembled into large bundles of protofilaments. Random co-assembly of GsFtsZs enhanced their disassembly kinetics, suggesting GsFtsZB mostly as a regulator. In contrast to the two FtsZs in chloroplasts, there is only one FtsZ in cyanobacteria, similar to other bacteria. Our studies showed SyFtsZ assembled into thick bundles either straight or curved as toroids. This bundling is similar to that of chloroplast FtsZ, consistent with its origin in cyanobacteria. The assembly of SyFtsZ occurred in two stages. The first stage consisted of the assembly of single-stranded protofilaments and opened circles; the second stage formed bundles and toroids. The bundling might benefit the cell division of large cells.

The Bacterial Tubulin Homolog FtsZ Forms 2D-sheets that Sustain Electrical Oscillations

FtsZ, a major cytoskeletal protein present in all bacteria and archaea forms a cytokinetic ring, the Z ring, at the site of septation, which directs cytokinesis. Multiple sequence alignment and secondary structure predictions indicate that bacterial FtsZ is a homolog of the eukaryotic tubulins which, as their mammalian counterpart, binds guanine nucleotides, has GTPase activity, and is able to assemble into protofilamental structures in a GTP-dependent manner. Previous studies from our laboratory demonstrated that microtubule (MTs) structures isolated from murine, bovine and apian brains spontaneously generate electrical oscillations and bursts of electrical activity similar to action potentials. Although the electrical behavior of brain MTs may have important implication in eukaryotic cell function, no information is available as to whether FtsZ may share similar properties. Therefore, the aim of the present study was to explore whether FtsZ protofilamental sheets may sustain electrical activity. FtsZ protein from E. coli ATCC 25922 was purified by ammonium sulfate precipitation, and assembled into protofilamental sheets by incubation with a polymerization buffer. 2D FtsZ sheets were voltage-clamped, and gigaseals obtained in most experiments (n = 7). Subsequently, electrical recordings from output currents were obtained, showing a complex oscillatory behavior with several peak frequencies between 7 and 110 Hz in the power spectra. As a result, it was observed that FtsZ 2D sheets had a much wider range of frequencies compared to MTs sheets from eukaryotic origin. The findings demonstrate that FtsZ protofilamental sheets are capable of generating electrical signals and behave as electrical oscillators that may be relevant to the cell division process and/or yet unknown signaling mechanisms.

Defining Substrate Binding Site in Sodium-Dependent Bile Acid Transporters

Bile acids are stored in the gallbladder and excreted into the digestive tract where they assist absorption of lipids and vitamins. Bile acids are synthesized from cholesterol in the liver before they are stored and entered into the small intestine. In the small intestine, more than 95% of the bile acids are absorbed and sent back to the liver through a process known as enterohepatic circulation. In humans, there are two Na+-dependent transporters that play an essential role in enterohepatic circulation—Na+-taurocholate co-transporting polypeptide (NTCP) and apical sodium-dependent bile acid transporter (ASBT). NTCP and ASBT share 35% sequence identity and both belong to the solute carrier 10 (Slc10A) family of secondary transporters. NTCP, in addition to its role in bile acid transport, is required for infection of hepatitis B virus because it serves as a co-receptor for human hepatitis B and D virus. ASBT is crucial to enterohepatic circulation as it has been correlated to multiple digestive illnesses, and has become a potential drug target for hypercholesterolaemia, type 2 diabetes, and constipation. Crystals structures of two bacterial homologs of human ASBT, ASBTNm and ASBTYf, have been reported, however, the two reports differ in their definition of the bile acid binding site. We will better define the bile acid binding site and its selectivity to various bile acids by making mutations to solvent exposed residues on the ASBTYf transporter and measure binding and transport of bile acids. These studies will give a better understanding of the substrate binding site of ASBT.

Bile Acid Transport by the Symporter ASBTNM: Substrate Binding and Conformational Change

The apical sodium-dependent bile acid transporter (ASBT) allows reabsorption of bile acids from the intestine by coupling bile acid movement to the sodium gradient. Such transporters are attractive targets for drug delivery and in the treatment for hypercholesterolaemia. Several structures of bacterial homologues in both inward and outward facing conformations have been obtained experimentally, including a substrate-bound inward facing structure of the homologue from Neisseria meningitidis (ASBTNM). However, many details surrounding the binding of substrates and the conformational transition remain unclear. We have used experimental and computational methods to explore these details at an atomistic scale using the homologue ASBTNM and the bile acid taurocholate. Models of apo and substrate-bound ASBTNM in the outward-facing conformation were generated. Biased molecular dynamics simulations and free energy calculations were used to explore conformational changes and possible movement of taurocholate during the transition by an elevator mechanism. Probability distributions and the residues important for binding of sodium and taurocholate in wild type and mutant ASBTNM were investigated with unbiased simulations, revealing alternative sodium binding sites. These results further our understanding of the important molecular details of ASBT function.

The functional role of the C-terminal tail of the human ribosomal protein uS19

The eukaryotic ribosomal protein uS19 has a C-terminal tail that is absent in its bacterial homologue. This tail has been shown to be involved in the formation of the decoding site of human ribosomes. We studied here the previously unexplored functional significance of the 15 C-terminal amino acid residues of human uS19 for the assembly of ribosomes and translation using HEK293-based cell cultures capable of producing FLAG-labeled uS19 (uS19FLAG) or its mutant form deprived of the mentioned amino acid ones. The examination of polysome profiles of cytoplasmic extracts from the respective cells revealed that the deletion of the above uS19 amino acid residues barely affected the assembly and maturation of 40S subunits and the initiation of translation, but completely prevented the formation of polysomes. This implied the crucial importance of the uS19 tail in the elongation process. Analysis of tRNAs associated with 40S subunits and 80S ribosomes containing wild type uS19FLAG or its truncated form showed that the deletion of the C-terminal pentadecapeptide fragment of uS19 did not interfere with the binding of aminoacyl-tRNA (aa-tRNA) at the ribosomal A site. The results led to the conclusion that the transpeptidation, which occurs on the large ribosomal subunit after decoding the A site codon by the incoming aa-tRNA, is the most likely elongation stage, where this uS19 fragment can play a critical role. Our findings suggest that the uS19 tail is a keystone player in the accommodation of aa-tRNA at the A site, which is a pre-requisite for the peptide transfer.

Study on a Novel Cold-Active and Halotolerant Monoacylglycerol Lipase Widespread in Marine Bacteria Reveals a New Group of Bacterial Monoacylglycerol Lipases Containing Unusual C(A/S)HSMG Catalytic Motifs

Monoacylglycerol lipases (MGLs) are present in all domains of life. However, reports on bacterial MGLs are still limited. Until now, reported bacterial MGLs are all thermophilic/mesophilic enzymes from warm terrestrial environments or deep-sea hydrothermal vent, and none of them originates from marine environments vastly subject to low temperature, high salts, and oligotrophy. Here, we characterized a novel MGL, GnMgl, from the marine cold-adapted and halophilic bacterium Glaciecola nitratireducens FR1064T. GnMgl shares quite low sequence similarities with characterized MGLs (lower than 31%). GnMgl and most of its bacterial homologs harbor a catalytic Ser residue located in the conserved C(A/S)HSMG motif rather than in the typical GxSxG motif reported on other MGLs, suggesting that GnMgl-like enzymes might be different from reported MGLs in catalysis. Phylogenetic analysis suggested that GnMgl and its bacterial homologs are clustered as a separate group in the monoglyceridelipase_lysophospholipase family of the Hydrolase_4 superfamily. Recombinant GnMgl has no lysophospholipase activity but could hydrolyze saturated (C12:0-C16:0) and unsaturated (C18:1 and C18:2) MGs and short-chain triacylglycerols, displaying distinct substrate selectivity from those of reported bacterial MGLs. The substrate preference of GnMgl, predicted to be a membrane protein, correlates to the most abundant fatty acids within the strain FR1064T, suggesting the role of GnMgl in the lipid catabolism in this marine bacterium. In addition, different from known bacterial MGLs that are all thermostable enzymes, GnMgl is a cold-adapted enzyme, with the maximum activity at 30°C and retaining 30% activity at 0°C. GnMgl is also a halotolerant enzyme with full activity in 3.5M NaCl. The cold-adapted and salt-tolerant characteristics of GnMgl may help its source strain FR1064T adapt to the cold and saline marine environment. Moreover, homologs to GnMgl are found to be abundant in various marine bacteria, implying their important physiological role in these marine bacteria. Our results on GnMgl shed light on marine MGLs.

Wheat protein disulfide isomerase improves bread properties via different mechanisms

Gluten network formation by the oxidation of glutenin sulfhydryl group majorly impacts the subsequent dough and bread properties, and an evolutionary list of chemical oxidants has been used as improvers in bread making. A systematic comparison between azodicarbonamide (ADA), Vc, wheat protein disulfide isomerase (wPDI) and disulfide bond formation protein C (DsbC) of their effects on the alveographic characters of dough and texture properties of subsequent bread was performed. Results show that wPDI improves dough alveographic characters and bread texture properties better in most aspects than other reagents. Free sulfhydryl analysis finds that addition of wPDI increased the free sulfhydryl content in both dough and bread. Compare with inorganic reagents and its bacterial homologue, improving the dough and bread properties with less oxidation of sulfhydryl lead to the proposal that wPDI acts by catalyzing the formation of rheologically active disulfide and reduction of inactive ones in a substrate specific manner.

Cryo-EM structures of a lipid-sensitive pentameric ligand-gated ion channel embedded in a phosphatidylcholine-only bilayer

The lipid dependence of the nicotinic acetylcholine receptor from the Torpedo electric organ has long been recognized, and one of the most consistent experimental observations is that, when reconstituted in membranes formed by zwitterionic phospholipids alone, exposure to agonist fails to elicit ion-flux activity. More recently, it has been suggested that the bacterial homolog ELIC (Erwinia chrysanthemi ligand-gated ion channel) has a similar lipid sensitivity. As a first step toward the elucidation of the structural basis of this phenomenon, we solved the structures of ELIC embedded in palmitoyl-oleoyl-phosphatidylcholine- (POPC-) only nanodiscs in both the unliganded (4.1-Å resolution) and agonist-bound (3.3 Å) states using single-particle cryoelectron microscopy. Comparison of the two structural models revealed that the largest differences occur at the level of loop C-at the agonist-binding sites-and the loops at the interface between the extracellular and transmembrane domains (ECD and TMD, respectively). On the other hand, the transmembrane pore is occluded in a remarkably similar manner in both structures. A straightforward interpretation of these findings is that POPC-only membranes frustrate the ECD-TMD coupling in such a way that the "conformational wave" of liganded-receptor gating takes place in the ECD and the interfacial M2-M3 linker but fails to penetrate the membrane and propagate into the TMD. Furthermore, analysis of the structural models and molecular simulations suggested that the higher affinity for agonists characteristic of the open- and desensitized-channel conformations results, at least in part, from the tighter confinement of the ligand to its binding site; this limits the ligand's fluctuations, and thus delays its escape into bulk solvent.

Hemerythrins enhance aerobic respiration in Methylomicrobium alcaliphilum 20ZR, a methane-consuming bacterium.

Numerous hemerythrins, di-iron proteins, have been identified in prokaryote genomes, but in most cases their function remains elusive. Bacterial hemerythrin homologs (bacteriohemerythrins, Bhrs) may contribute to various cellular functions, including oxygen sensing, metal binding and antibiotic resistance. It has been proposed that methanotrophic Bhrs support methane oxidation by supplying oxygen to a core enzyme, particulate methane monooxygenase. In this study, the consequences of the overexpression or deletion of the Bhr gene (bhr) in Methylomicrobiam alcaliphillum 20ZR were investigated. We found that the bhrknockout (20ZRΔbhr) displays growth kinetics and methane consumption rates similar to wild type. However, the 20ZRΔbhr accumulates elevated concentrations of acetate at aerobic conditions, indicating slowed respiration. The methanotrophic strain overproducing Bhr shows increased oxygen consumption and reduced carbon-conversion efficiency, while its methane consumption rates remain unchanged. These results suggest that the methanotrophic Bhr proteins specifically contribute to oxygen-dependent respiration, while they have minimal, if any, input of oxygen for the methane oxidation machinery.