Structural Basis for the Interactions of the Colibactin Resistance Gene Product ClbS with DNA.

The natural product colibactin, along with its associated biosynthetic gene cluster, is an example system for the role microbially derived small molecules play in the human microbiome. This is particularly relevant in the human gut, where host microbiota is involved in various disorders, including colorectal cancer pathogenesis. Bacteria harboring the colibactin gene cluster induce alkylation of nucleobases in host DNA, forming interstrand cross-links both in vivo and in vitro. These lesions can lead to deleterious double-strand breaks and have been identified as the primary mechanism of colibactin-induced cytotoxicity. The gene product ClbS is one of several mechanisms utilized by the producing bacteria to maintain genome integrity. ClbS catalyzes hydrolytic inactivation of colibactin and has been shown to bind DNA, incurring self-resistance. Presented is the molecular basis for ClbS bound to a DNA oligonucleotide. The structure shows the interaction of the protein with the ends of a DNA duplex with terminal nucleotides flipped to the enzyme active site. The structure suggests an additional function for ClbS, the binding to damaged DNA followed by repair. Additionally, our study provides general insight into the function of the widely distributed and largely uncharacterized DUF1706 protein family.

The role of Escherichia coli FhlA transcriptional activator in generation of proton motive force and FO F1 -ATPase activity at pH 7.5.

Escherichia coli is able to utilize the mixture of carbon sources and produce molecular hydrogen (H2 ) via formate hydrogen lyase (FHL) complexes. In current work role of transcriptional activator of formate regulon FhlA in generation of fermentation end products and proton motive force, N'N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity at 20 and 72 hr growth during utilization of mixture of glucose, glycerol, and formate were investigated. It was shown that in fhlA mutant specific growth rate was ~1.5 fold lower compared to wt, while addition of DCCD abolished the growth in fhlA but not in wt. Formate was not utilized in fhlA mutant but wt cells simultaneously utilized formate with glucose. Glycerol utilization started earlier (from 2 hr) in fhlA than in wt. The DCCD-sensitive ATPase activity in wt cells membrane vesicles increased ~2 fold at 72 hr and was decreased 70% in fhlA. Addition of formate in the assays increased proton ATPase activity in wt and mutant strain. FhlA absence mainly affected the ΔpH but not ΔΨ component of Δp in the cells grown at 72 hr but not in 24 hr. The Δp in wt cells decreased from 24 to 72 hr of growth ~40 mV while in fhlA mutant it was stable. Taken together, it is suggested that FhlA regulates the concentration of fermentation end products and via influencing FO F1 -ATPase activity contributes to the proton motive force generation.

Basic mechanism of the autonomous ClpG disaggregase.

Bacterial survival during lethal heat stress relies on the cellular ability to reactivate aggregated proteins. This activity is typically executed by the canonical 70-kDa heat shock protein (Hsp70)-ClpB bichaperone disaggregase, which is most widespread in bacteria. The ClpB disaggregase is a member of the ATPase associated with diverse cellular activities protein family and exhibits an ATP-driven threading activity. Substrate binding and stimulation of ATP hydrolysis depends on the Hsp70 partner, which initiates the disaggregation reaction. Recently elevated heat resistance in gamma-proteobacterial species was shown to be mediated by the ATPase associated with diverse cellular activities protein ClpG as an alternative disaggregase. Pseudomonas aeruginosa ClpG functions autonomously and does not cooperate with Hsp70 for substrate binding, enhanced ATPase activity, and disaggregation. With the underlying molecular basis largely unknown, the fundamental differences in ClpG- and ClpB-dependent disaggregation are reflected by the presence of sequence alterations and additional ClpG-specific domains. By analyzing the effects of mutants lacking ClpG-specific domains and harboring mutations in conserved motifs implicated in ATP hydrolysis and substrate threading, we show that the N-terminal, ClpG-specific N1 domain generally mediates protein aggregate binding as the molecular basis of autonomous disaggregation activity. Peptide substrate binding strongly stimulates ClpG ATPase activity by overriding repression by the N-terminal N1 and N2 domains. High ATPase activity requires two functional nucleotide binding domains and drives substrate threading which ultimately extracts polypeptides from the aggregate. ClpG ATPase and disaggregation activity is thereby directly controlled by substrate availability.

Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx-dependent pyroptosis in intestinal epithelial cells.

Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyroptosis. We reveal a novel mechanism through which infection with an extracellular pathogen leads to pyroptosis in IECs.

ATP-Driven Separation of Liquid Phase Condensates in Bacteria.

Liquid-liquid phase-separated (LLPS) states are key to compartmentalizing components in the absence of membranes; however, it is unclear whether LLPS condensates are actively and specifically organized in the subcellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB), and a motor (ParA). We show that parS and ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favored by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates, and localizes non-canonical LLPS condensates in the subcellular space.

Alkyltransferase-like protein clusters scan DNA rapidly over long distances and recruit NER to alkyl-DNA lesions.

Alkylation of guanine bases in DNA is detrimental to cells due to its high mutagenic and cytotoxic potential and is repaired by the alkyltransferase AGT. Additionally, alkyltransferase-like proteins (ATLs), which are structurally similar to AGTs, have been identified in many organisms. While ATLs are per se catalytically inactive, strong evidence has suggested that ATLs target alkyl lesions to the nucleotide excision repair system (NER). Using a combination of single-molecule and ensemble approaches, we show here recruitment of UvrA, the initiating enzyme of prokaryotic NER, to an alkyl lesion by ATL. We further characterize lesion recognition by ATL and directly visualize DNA lesion search by highly motile ATL and ATL-UvrA complexes on DNA at the molecular level. Based on the high similarity of ATLs and the DNA-interacting domain of AGTs, our results provide important insight in the lesion search mechanism, not only by ATL but also by AGT, thus opening opportunities for controlling the action of AGT for therapeutic benefit during chemotherapy.

ATP serves as a nucleotide switch coupling the genome maturation and packaging motor complexes of a virus assembly machine.

The assembly of double-stranded DNA viruses, from phages to herpesviruses, is strongly conserved. Terminase enzymes processively excise and package monomeric genomes from a concatemeric DNA substrate. The enzymes cycle between a stable maturation complex that introduces site-specific nicks into the duplex and a dynamic motor complex that rapidly translocates DNA into a procapsid shell, fueled by ATP hydrolysis. These tightly coupled reactions are catalyzed by terminase assembled into two functionally distinct nucleoprotein complexes; the maturation complex and the packaging motor complex, respectively. We describe the effects of nucleotides on the assembly of a catalytically competent maturation complex on viral DNA, their effect on maturation complex stability and their requirement for the transition to active packaging motor complex. ATP plays a major role in regulating all of these activities and may serve as a 'nucleotide switch' that mediates transitions between the two complexes during processive genome packaging. These biological processes are recapitulated in all of the dsDNA viruses that package monomeric genomes from concatemeric DNA substrates and the nucleotide switch mechanism may have broad biological implications with respect to virus assembly mechanisms.

The TraK accessory factor activates substrate transfer through the pKM101 type IV secretion system independently of its role in relaxosome assembly.

A large subfamily of the type IV secretion systems (T4SSs), termed the conjugation systems, transmit mobile genetic elements (MGEs) among many bacterial species. In the initiating steps of conjugative transfer, DNA transfer and replication (Dtr) proteins assemble at the origin-of-transfer (oriT) sequence as the relaxosome, which nicks the DNA strand destined for transfer and couples the nicked substrate with the VirD4-like substrate receptor. Here, we defined contributions of the Dtr protein TraK, a predicted member of the Ribbon-Helix-Helix (RHH) family of DNA-binding proteins, to transfer of DNA and protein substrates through the pKM101-encoded T4SS. Using a combination of cross-linking/affinity pull-downs and two-hybrid assays, we determined that TraK self-associates as a probable tetramer and also forms heteromeric contacts with pKM101-encoded TraI relaxase, VirD4-like TraJ receptor, and VirB11-like and VirB4-like ATPases, TraG and TraB, respectively. TraK also promotes stable TraJ-TraB complex formation and stimulates binding of TraI with TraB. Finally, TraK is required for or strongly stimulates the transfer of cognate (pKM101, TraI relaxase) and noncognate (RSF1010, MobA relaxase) substrates. We propose that TraK functions not only to nucleate pKM101 relaxosome assembly, but also to activate the TrapKM101 T4SS via interactions with the ATPase energy center positioned at the channel entrance.

LptB-LptF coupling mediates the closure of the substrate-binding cavity in the LptB2 FGC transporter through a rigid-body mechanism to extract LPS.

Lipopolysaccharides (LPS) are essential envelope components in many Gram-negative bacteria and provide intrinsic resistance to antibiotics. LPS molecules are synthesized in the inner membrane and then transported to the cell surface by the LPS transport (Lpt) machinery. In this system, the ATP-binding cassette (ABC) transporter LptB2 FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge through a poorly understood mechanism. Here, we show that residue E86 of LptB is essential for coupling the function of this ATPase to that of its partners LptFG, specifically at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS-binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge. We also show that defects caused by changing residue E86 are suppressed by mutations altering either LPS structure or transmembrane helices in LptG. Furthermore, these suppressors also fix defects in the coupling helix of LptF, but not of LptG. Together, these results support a transport mechanism in which the ATP-driven movements of LptB and those of the substrate-binding cavity in LptFG are bi-directionally coordinated through the rigid-body coupling, with LptF's coupling helix being important in coordinating cavity collapse with LptB dimerization.

Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

Neutrophil activation by Escherichia coli isolates from human intestine: effects of bacterial hydroperoxidase activity and surface hydrophobicity.

Successful colonization of the intestine requires that bacteria interact with the innate immune system and, in particular, neutrophils. Progression of inflammatory bowel diseases (IBD) is associated with alterations in gut microbiota, and dysbiosis in Crohn's disease (CD) patients is often associated with an expansion of Escherichia coli. Here, we investigated the ability of such E. coli isolates to avoid neutrophil activation and to utilize reactive oxygen species. Neutrophil activation was detected in vitro in normal human blood via luminol chemiluminescence (CL) induced by reactive oxygen and halogen species generated by neutrophils. No significant difference in neutrophil activation in vitro was detected between isolates from inflamed (23 isolates) vs healthy intestines (5 isolates), with 10-fold variation within both groups (2.9-61.2 mV). CL activity of isolates from the same patient differed by 1.5-5 times. Twenty-four isolates from ileal aspirate, biopsy, and feces of seven patients with CD and one patient with no intestine inflammation were tested for extracellular peroxidase and catalase activity and cell surface hydrophobicity. Average values between patients varied from 26 ± 3 to 73 ± 18 µmol·g-1 of air dry weight for peroxidase activity, from 15 ± 2 to 189 ± 56 mmol·g-1 of air dry weight for catalase activity, and from 5 ± 3 to 105 ± 9 a.u. for the hydrophobic probe fluorescence. Extracellular peroxidase activity and hydrophobicity of bacterial cell surface correlated negatively with stimulated neutrophil CL. The ability of some isolates to avoid neutrophil activation and to utilize reactive oxygen species may provide a strategy to survive assault by the innate immune system.

Mechanical Stimuli Affect Escherichia coli Heat-Stable Enterotoxin-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Model.

Modeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a virulence factor of a pathogenic intestinal bacterium, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips) exposed to enterotoxigenic Escherichia coli heat-stable enterotoxin A (ST) and evaluated under conditions of static fluid, apical and basolateral flow, and flow plus repetitive stretch. Application of flow increased epithelial cell height and apical and basolateral secretion of cyclic GMP (cGMP) under baseline, unstimulated conditions. Addition of ST under flow conditions increased apical and basolateral secretion of cGMP relative to the level under static conditions but did not enhance intracellular cGMP accumulation. Cyclic stretch did not have any significant effect beyond that contributed by flow. This study demonstrates that fluid flow application initiates changes in intestinal epithelial cell characteristics relative to those of static culture conditions under both baseline conditions and with exposure to ST enterotoxin and suggests that further investigations of the application of these mechanical forces will provide insights into physiology and pathophysiology that more closely resemble intact intestine than study under static conditions.

Nucleoside Diphosphate Kinase Escalates A-to-C Mutations in MutT-Deficient Strains of Escherichia coli.

The chemical integrity of the nucleotide pool and its homeostasis are crucial for genome stability. Nucleoside diphosphate kinase (NDK) is a crucial enzyme that carries out reversible conversions from nucleoside diphosphate (NDP) to nucleoside triphosphate (NTP) and deoxynucleoside diphosphate (dNDP) to deoxynucleoside triphosphate (dNTP). Guanosine nucleotides (GDP, GTP, dGDP, and dGTP) are highly susceptible to oxidative damage to 8-oxo-GDP (8-O-GDP), 8-O-dGTP, 8-O-GTP, and 8-O-dGTP. MutT proteins in cells hydrolyze 8-O-GTP to 8-O-GMP or 8-O-dGTP to 8-O-dGMP to avoid its incorporation in nucleic acids. In Escherichia coli, 8-O-dGTP is also known to be hydrolyzed by RibA (GTP cyclohydrolase II). In this study, we show that E. coli NDK catalyzes the conversion of 8-O-dGDP to 8-O-dGTP or vice versa. However, the rate of NDK-mediated phosphorylation of 8-O-dGDP to 8-O-dGTP is about thrice as efficient as the rate of dephosphorylation of 8-O-dGTP to 8-O-dGDP, suggesting an additive role of NDK in net production of 8-O-dGTP in cells. Consistent with this observation, the depletion of NDK (Δndk) in E. coli ΔmutT or ΔmutT ΔribA strains results in a decrease of A-to-C mutations. These observations suggest that NDK contributes to the physiological load of MutT in E. coliIMPORTANCE Nucleoside diphosphate kinase (NDK), a ubiquitous enzyme, is known for its critical role in homeostasis of cellular nucleotide pools. However, NDK has now emerged as a molecule with pleiotropic effects in DNA repair, protein phosphorylation, gene expression, tumor metastasis, development, and pathogen virulence and persistence inside the host. In this study, we reveal an unexpected role of NDK in genome instability because of its activity in converting 8-O-dGDP to 8-O-dGTP. This observation has important consequences in escalating A-to-C mutations in Escherichia coli The severity of NDK in enhancing these mutations may be higher in the organisms challenged with high oxidative stress, which promotes 8-O-dGDP/8-O-dGTP production.

Deciphering the interplay between the genotoxic and probiotic activities of Escherichia coli Nissle 1917.

Although Escherichia coli Nissle 1917 (EcN) has been used therapeutically for over a century, the determinants of its probiotic properties remain elusive. EcN produces two siderophore-microcins (Mcc) responsible for an antagonistic activity against other Enterobacteriaceae. EcN also synthesizes the genotoxin colibactin encoded by the pks island. Colibactin is a virulence factor and a putative pro-carcinogenic compound. Therefore, we aimed to decouple the antagonistic activity of EcN from its genotoxic activity. We demonstrated that the pks-encoded ClbP, the peptidase that activates colibactin, is required for the antagonistic activity of EcN. The analysis of a series of ClbP mutants revealed that this activity is linked to the transmembrane helices of ClbP and not the periplasmic peptidase domain, indicating the transmembrane domain is involved in some aspect of Mcc biosynthesis or secretion. A single amino acid substitution in ClbP inactivates the genotoxic activity but maintains the antagonistic activity. In an in vivo salmonellosis model, this point mutant reduced the clinical signs and the fecal shedding of Salmonella similarly to the wild type strain, whereas the clbP deletion mutant could neither protect nor outcompete the pathogen. The ClbP-dependent antibacterial effect was also observed in vitro with other E. coli strains that carry both a truncated form of the Mcc gene cluster and the pks island. In such strains, siderophore-Mcc synthesis also required the glucosyltransferase IroB involved in salmochelin production. This interplay between colibactin, salmochelin, and siderophore-Mcc biosynthetic pathways suggests that these genomic islands were co-selected and played a role in the evolution of E. coli from phylogroup B2. This co-evolution observed in EcN illustrates the fine margin between pathogenicity and probiotic activity, and the need to address both the effectiveness and safety of probiotics. Decoupling the antagonistic from the genotoxic activity by specifically inactivating ClbP peptidase domain opens the way to the safe use of EcN.

The E. coli MinCDE system in the regulation of protein patterns and gradients.

Molecular self-organziation, also regarded as pattern formation, is crucial for the correct distribution of cellular content. The processes leading to spatiotemporal patterns often involve a multitude of molecules interacting in complex networks, so that only very few cellular pattern-forming systems can be regarded as well understood. Due to its compositional simplicity, the Escherichia coli MinCDE system has, thus, become a paradigm for protein pattern formation. This biological reaction diffusion system spatiotemporally positions the division machinery in E. coli and is closely related to ParA-type ATPases involved in most aspects of spatiotemporal organization in bacteria. The ATPase MinD and the ATPase-activating protein MinE self-organize on the membrane as a reaction matrix. In vivo, these two proteins typically oscillate from pole-to-pole, while in vitro they can form a variety of distinct patterns. MinC is a passenger protein supposedly operating as a downstream cue of the system, coupling it to the division machinery. The MinCDE system has helped to extract not only the principles underlying intracellular patterns, but also how they are shaped by cellular boundaries. Moreover, it serves as a model to investigate how patterns can confer information through specific and non-specific interactions with other molecules. Here, we review how the three Min proteins self-organize to form patterns, their response to geometric boundaries, and how these patterns can in turn induce patterns of other molecules, focusing primarily on experimental approaches and developments.

An Active, Ligand-Responsive Pulling Geometry Reports on Internal Signaling between Subdomains of the DnaK Nucleotide-Binding Domain in Single-Molecule Mechanical Experiments.

Single-molecule mechanical experiments have proven to be ideal tools for probing the energetics and mechanics of large proteins and domains. In this paper, we investigate the nucleotide-dependent unfolding mechanics of the nucleotide-binding domain (NBD) of the Hsp70 chaperone DnaK. The NBD binds ADP or ATP in the binding cleft formed by lobe I and lobe II, which consists of two subdomains each. When force is applied to the termini of the NBD, the observed unfolding forces are independent of the nucleotide state. In contrast, when force is applied across the nucleotide-binding pocket, the unfolding forces report specifically on the nucleotide-phosphate state. In this active, ligand-responsive pulling geometry, we observed a bifurcation of the unfolding pathway; the pathway proceeds either through a cooperative "coupled pathway" or through a noncooperative "uncoupled pathway". The partitioning between individual unfolding pathways can be effectively tuned by mutation or by the nucleotide exchange factor GrpE, i.e., by the factors affecting the strength of the lobe I-lobe II interactions within the native NBD. These experiments provide important insight into the molecular origin of the internal signaling between the subdomains of the nucleotide-binding domain of Hsp70 proteins and how signals are efficiently transferred inside the protein molecule.

Microbial Siderophore Enterobactin Promotes Mitochondrial Iron Uptake and Development of the Host via Interaction with ATP Synthase.

Elucidating the benefits of individual microbiota-derived molecules in host animals is important for understanding the symbiosis between humans and their microbiota. The bacteria-secreted enterobactin (Ent) is an iron scavenging siderophore with presumed negative effects on hosts. However, the high prevalence of Ent-producing commensal bacteria in the human gut raises the intriguing question regarding a potential host mechanism to beneficially use Ent. We discovered an unexpected and striking role of Ent in supporting growth and the labile iron pool in C. elegans. We show that Ent promotes mitochondrial iron uptake and does so, surprisingly, by binding to the ATP synthase α subunit, which acts inside of mitochondria and independently of ATP synthase. We also demonstrated the conservation of this mechanism in mammalian cells. This study reveals a distinct paradigm for the "iron tug of war" between commensal bacteria and their hosts and an important mechanism for mitochondrial iron uptake and homeostasis.

Motor-like DNA motion due to an ATP-hydrolyzing protein under nanoconfinement.

We report that long double-stranded DNA confined to quasi-1D nanochannels undergoes superdiffusive motion under the action of the enzyme T4 DNA ligase in the presence of necessary co-factors. Inside the confined environment of the nanochannel, double-stranded DNA molecules stretch out due to self-avoiding interactions. In absence of a catalytically active enzyme, we see classical diffusion of the center of mass. However, cooperative interactions of proteins with the DNA can lead to directed motion of DNA molecules inside the nanochannel. Here we show directed motion in this configuration for three different proteins (T4 DNA ligase, MutS, E. coli DNA ligase) in the presence of their energetic co-factors (ATP, NAD+).

MinE conformational switching confers robustness on self-organized Min protein patterns.

Protein patterning is vital for many fundamental cellular processes. This raises two intriguing questions: Can such intrinsically complex processes be reduced to certain core principles and, if so, what roles do the molecular details play in individual systems? A prototypical example for protein patterning is the bacterial Min system, in which self-organized pole-to-pole oscillations of MinCDE proteins guide the cell division machinery to midcell. These oscillations are based on cycling of the ATPase MinD and its activating protein MinE between the membrane and the cytoplasm. Recent biochemical evidence suggests that MinE undergoes a reversible, MinD-dependent conformational switch from a latent to a reactive state. However, the functional relevance of this switch for the Min network and pattern formation remains unclear. By combining mathematical modeling and in vitro reconstitution of mutant proteins, we dissect the two aspects of MinE's switch, persistent membrane binding and a change in MinE's affinity for MinD. Our study shows that the MinD-dependent change in MinE's binding affinity for MinD is essential for patterns to emerge over a broad and physiological range of protein concentrations. Mechanistically, our results suggest that conformational switching of an ATPase-activating protein can lead to the spatial separation of its distinct functional states and thereby confer robustness on an intracellular protein network with vital roles in bacterial cell division.

Structural basis for the alternating access mechanism of the cation diffusion facilitator YiiP.

YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn2+ across bacterial membranes. Previous work defined the atomic structure of an outward-facing conformation, the location of several Zn2+ binding sites, and hydrophobic residues that appear to control access to the transport sites from the cytoplasm. A low-resolution cryo-EM structure revealed changes within the membrane domain that were associated with the alternating access mechanism for transport. In the current work, the resolution of this cryo-EM structure has been extended to 4.1 Å. Comparison with the X-ray structure defines the differences between inward-facing and outward-facing conformations at an atomic level. These differences include rocking and twisting of a four-helix bundle that harbors the Zn2+ transport site and controls its accessibility within each monomer. As previously noted, membrane domains are closely associated in the dimeric structure from cryo-EM but dramatically splayed apart in the X-ray structure. Cysteine crosslinking was used to constrain these membrane domains and to show that this large-scale splaying was not necessary for transport activity. Furthermore, dimer stability was not compromised by mutagenesis of elements in the cytoplasmic domain, suggesting that the extensive interface between membrane domains is a strong determinant of dimerization. As with other secondary transporters, this interface could provide a stable scaffold for movements of the four-helix bundle that confers alternating access of these ions to opposite sides of the membrane.