The copper-linked Escherichia coli AZY operon: Structure, metal binding, and a possible physiological role in copper delivery.

The Escherichia coli yobA-yebZ-yebY (AZY) operon encodes the proteins YobA, YebZ, and YebY. YobA and YebZ are homologs of the CopC periplasmic copper-binding protein and the CopD putative copper importer, respectively, whereas YebY belongs to the uncharacterized Domain of Unknown Function 2511 family. Despite numerous studies of E. coli copper homeostasis and the existence of the AZY operon in a range of bacteria, the operon's proteins and their functional roles have not been explored. In this study, we present the first biochemical and functional studies of the AZY proteins. Biochemical characterization and structural modeling indicate that YobA binds a single Cu2+ ion with high affinity. Bioinformatics analysis shows that YebY is widespread and encoded either in AZY operons or in other genetic contexts unrelated to copper homeostasis. We also determined the 1.8 Å resolution crystal structure of E. coli YebY, which closely resembles that of the lantibiotic self-resistance protein MlbQ. Two strictly conserved cysteine residues form a disulfide bond, consistent with the observed periplasmic localization of YebY. Upon treatment with reductants, YebY binds Cu+ and Cu2+ with low affinity, as demonstrated by metal-binding analysis and tryptophan fluorescence. Finally, genetic manipulations show that the AZY operon is not involved in copper tolerance or antioxidant defense. Instead, YebY and YobA are required for the activity of the copper-related NADH dehydrogenase II. These results are consistent with a potential role of the AZY operon in copper delivery to membrane proteins.

Titratable transmembrane residues and a hydrophobic plug are essential for manganese import via the Bacillus anthracis ABC transporter MntBC-A.

All extant life forms require trace transition metals (e.g., Fe2/3+, Cu1/2+, and Mn2+) to survive. However, as these are environmentally scarce, organisms have evolved sophisticated metal uptake machineries. In bacteria, high-affinity import of transition metals is predominantly mediated by ABC transporters. During bacterial infection, sequestration of metal by the host further limits the availability of these ions, and accordingly, bacterial ABC transporters (importers) of metals are key virulence determinants. However, the structure-function relationships of these metal transporters have not been fully elucidated. Here, we used metal-sensitivity assays, advanced structural modeling, and enzymatic assays to study the ABC transporter MntBC-A, a virulence determinant of the bacterial human pathogen Bacillus anthracis. We find that despite its broad metal-recognition profile, MntBC-A imports only manganese, whereas zinc can function as a high-affinity inhibitor of MntBC-A. Computational analysis shows that the transmembrane metal permeation pathway is lined with six titratable residues that can coordinate the positively charged metal, and mutagenesis studies show that they are essential for manganese transport. Modeling suggests that access to these titratable residues is blocked by a ladder of hydrophobic residues, and ATP-driven conformational changes open and close this hydrophobic seal to permit metal binding and release. The conservation of this arrangement of titratable and hydrophobic residues among ABC transporters of transition metals suggests a common mechanism. These findings advance our understanding of transmembrane metal recognition and permeation and may aid the design and development of novel antibacterial agents.

Substrate recognition and ATPase activity of the E. coli cysteine/cystine ABC transporter YecSC-FliY.

Sulfur is essential for biological processes such as amino acid biogenesis, iron-sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of their transport mechanism remain poorly characterized. Here we provide results from a biochemical analysis of the purified Escherichia coli YecSC-FliY cysteine/cystine import system. We found that the substrate-binding protein FliY binds l-cystine, l-cysteine, and d-cysteine with micromolar affinities. However, binding of the l- and d-enantiomers induced different conformational changes of FliY, where the l- enantiomer-substrate-binding protein complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo FliY, more strongly by d-cysteine-bound FliY, and maximally by l-cysteine- or l-cystine-bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independent of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities but equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family.

Identification of conserved slow codons that are important for protein expression and function.

ABSTRASTDue to the redundancy of the genetic code most amino acids are encoded by several 'synonymous' codons. These codons are used unevenly, and each organism demonstrates its own unique codon usage bias, where the 'preferred' codons are associated with tRNAs that are found in high concentrations. Therefore, for decades, the prevailing view had been that preferred and non-preferred codons are linked to high or slow translation rates, respectively.However, this simplified view is contrasted by the frequent failures of codon-optimization efforts and by evidence of non-preferred (i.e. 'slow') codons having specific roles important for efficient production of functional proteins. One such specific role of slower codons is the regulation of co-translational protein folding, a complex biophysical process that is very challenging to model or to measure.Here, we combined a genome-wide approach with experiments to investigate the role of slow codons in protein production and co-translational folding. We analysed homologous gene groups from divergent bacteria and identified positions of inter-species conservation of bias towards slow codons. We then generated mutants where the conserved slow codons are substituted with 'fast' ones, and experimentally studied the effects of these codon substitutions. Using cellular and biochemical approaches we find that at certain locations, slow-to-fast codon substitutions reduce protein expression, increase protein aggregation, and impair protein function.This report provides an approach for identifying functionally relevant regions with slower codons and demonstrates that such codons are important for protein expression and function.

ABC transporters: the power to change.

ATP-binding cassette (ABC) transporters constitute a ubiquitous superfamily of integral membrane proteins that are responsible for the ATP-powered translocation of many substrates across membranes. The highly conserved ABC domains of ABC transporters provide the nucleotide-dependent engine that drives transport. By contrast, the transmembrane domains that create the translocation pathway are more variable. Recent structural advances with prokaryotic ABC transporters have provided a qualitative molecular framework for deciphering the transport cycle. An important goal is to develop quantitative models that detail the kinetic and molecular mechanisms by which ABC transporters couple the binding and hydrolysis of ATP to substrate translocation.

Single-molecule probing of the conformational homogeneity of the ABC transporter BtuCD.

ATP-binding cassette (ABC) transporters use the energy of ATP hydrolysis to move molecules through cellular membranes. They are directly linked to human diseases, cancer multidrug resistance, and bacterial virulence. Very little is known of the conformational dynamics of ABC transporters, especially at the single-molecule level. Here, we combine single-molecule spectroscopy and a novel molecular simulation approach to investigate the conformational dynamics of the ABC transporter BtuCD. We observe a single dominant population of molecules in each step of the transport cycle and tight coupling between conformational transitions and ligand binding. We uncover transient conformational changes that allow substrate to enter the transporter. This is followed by a 'squeezing' motion propagating from the extracellular to the intracellular side of the translocation cavity. This coordinated sequence of events provides a mechanism for the unidirectional transport of vitamin B12 by BtuCD.

The size of the proteasomal substrate determines whether its degradation will be mediated by mono- or polyubiquitylation.

A polyubiquitin chain anchored to the substrate has been the hallmark of proteasomal recognition. However, the degradation signal appears to be more complex and to contain also a substrate's unstructured region. Recent reports have shown that the proteasome can degrade also monoubiquitylated proteins, which adds an additional layer of complexity to the signal. Here, we demonstrate that the size of the substrate is an important determinant in its extent of ubiquitylation: a single ubiquitin moiety fused to a tail of up to ∼150 residues derived from either short artificial repeats or from naturally occurring proteins, is sufficient to target them for proteasomal degradation. Importantly, chemically synthesized adducts, where ubiquitin is attached to the substrate via a naturally occurring isopeptide bond, display similar characteristics. Taken together, these findings suggest that the ubiquitin proteasomal signal is adaptive, and is not always made of a long polyubiquitin chain.

L-glutamine Induces Expression of Listeria monocytogenes Virulence Genes.

The high environmental adaptability of bacteria is contingent upon their ability to sense changes in their surroundings. Bacterial pathogen entry into host poses an abrupt and dramatic environmental change, during which successful pathogens gauge multiple parameters that signal host localization. The facultative human pathogen Listeria monocytogenes flourishes in soil, water and food, and in ~50 different animals, and serves as a model for intracellular infection. L. monocytogenes identifies host entry by sensing both physical (e.g., temperature) and chemical (e.g., metabolite concentrations) factors. We report here that L-glutamine, an abundant nitrogen source in host serum and cells, serves as an environmental indicator and inducer of virulence gene expression. In contrast, ammonia, which is the most abundant nitrogen source in soil and water, fully supports growth, but fails to activate virulence gene transcription. We demonstrate that induction of virulence genes only occurs when the Listerial intracellular concentration of L-glutamine crosses a certain threshold, acting as an on/off switch: off when L-glutamine concentrations are below the threshold, and fully on when the threshold is crossed. To turn on the switch, L-glutamine must be present, and the L-glutamine high affinity ABC transporter, GlnPQ, must be active. Inactivation of GlnPQ led to complete arrest of L-glutamine uptake, reduced type I interferon response in infected macrophages, dramatic reduction in expression of virulence genes, and attenuated virulence in a mouse infection model. These results may explain observations made with other pathogens correlating nitrogen metabolism and virulence, and suggest that gauging of L-glutamine as a means of ascertaining host localization may be a general mechanism.

ATP binding and hydrolysis disrupt the high-affinity interaction between the heme ABC transporter HmuUV and its cognate substrate-binding protein.

Using the energy of ATP hydrolysis, ABC transporters catalyze the trans-membrane transport of molecules. In bacteria, these transporters partner with a high-affinity substrate-binding protein (SBP) to import essential micronutrients. ATP binding by Type I ABC transporters (importers of amino acids, sugars, peptides, and small ions) stabilizes the interaction between the transporter and the SBP, thus allowing transfer of the substrate from the latter to the former. In Type II ABC transporters (importers of trace elements, e.g. vitamin B12, heme, and iron-siderophores) the role of ATP remains debatable. Here we studied the interaction between the Yersinia pestis ABC heme importer (HmuUV) and its partner substrate-binding protein (HmuT). Using real-time surface plasmon resonance experiments and interaction studies in membrane vesicles, we find that in the absence of ATP the transporter and the SBP tightly bind. Substrate in excess inhibits this interaction, and ATP binding by the transporter completely abolishes it. To release the stable docked SBP from the transporter hydrolysis of ATP is required. Based on these results we propose a mechanism for heme acquisition by HmuUV-T where the substrate-loaded SBP docks to the nucleotide-free outward-facing conformation of the transporter. ATP binding leads to formation of an occluded state with the substrate trapped in the trans-membrane translocation cavity. Subsequent ATP hydrolysis leads to substrate delivery to the cytoplasm, release of the SBP, and resetting of the system. We propose that other Type II ABC transporters likely share the fundamentals of this mechanism.

A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin.

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7-/- mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.

Two molybdate/tungstate ABC transporters that interact very differently with their substrate binding proteins.

In all kingdoms of life, ATP Binding Cassette (ABC) transporters participate in many physiological and pathological processes. Despite the diversity of their functions, they have been considered to operate by a largely conserved mechanism. One deviant is the vitamin B12 transporter BtuCD that has been shown to operate by a distinct mechanism. However, it is unknown if this deviation is an exotic example, perhaps arising from the nature of the transported moiety. Here we compared two ABC importers of identical substrate specificity (molybdate/tungstate), and find that their interactions with their substrate binding proteins are utterly different. One system forms a high-affinity, slow-dissociating complex that is destabilized by nucleotide and substrate binding. The other forms a low-affinity, transient complex that is stabilized by ligands. The results highlight significant mechanistic divergence among ABC transporters, even when they share the same substrate specificity. We propose that these differences are correlated with the different folds of the transmembrane domains of ABC transporters.

A single intact ATPase site of the ABC transporter BtuCD drives 5% transport activity yet supports full in vivo vitamin B12 utilization.

In all kingdoms of life, ATP binding cassette (ABC) transporters are essential to many cellular functions. In this large superfamily of proteins, two catalytic sites hydrolyze ATP to power uphill substrate translocation. A central question in the field concerns the relationship between the two ATPase catalytic sites: Are the sites independent of one another? Are both needed for function? Do they function cooperatively? These issues have been resolved for type I ABC transporters but never for a type II ABC transporter. The many mechanistic differences between type I and type II ABC transporters raise the question whether in respect to ATP hydrolysis the two subtypes are similar or different. We have addressed this question by studying the Escherichia coli vitamin B12 type II ABC transporter BtuCD. We have constructed and purified a series of BtuCD variants where both, one, or none of the ATPase sites were rendered inactive by mutation. We find that, in a membrane environment, the ATPase sites of BtuCD are highly cooperative with a Hill coefficient of 2. We also find that, when one of the ATPase sites is inactive, ATP hydrolysis and vitamin B12 transport by BtuCD is reduced by 95%. These exact features are also shared by the archetypical type I maltose ABC transporter. Remarkably, mutants that have lost 95% of their ATPase and transport capabilities still retain the ability to fully use vitamin B12 in vivo. The results demonstrate that, despite the many differences between type I and type II ABC transporters, the fundamental mechanism of ATP hydrolysis remains conserved.

Identification of functionally important conserved trans-membrane residues of bacterial PIB -type ATPases.

Powered by ATP hydrolysis, P(IB) -ATPases drive the energetically uphill transport of transition metals. These high affinity pumps are essential for heavy metal detoxification and delivery of metal cofactors to specific cellular compartments. Amino acid sequence alignment of the trans-membrane (TM) helices of P(IB)-ATPases reveals a high degree of conservation, with ∼ 60-70 fully conserved positions. Of these conserved positions, 6-7 were previously identified to be important for transport. However, the functional importance of the majority of the conserved TM residues remains unclear. To investigate the role of conserved TM residues of P(IB)-ATPases we conducted an extensive mutagenesis study of a Zn(2+)/Cd(2+) P(IB)-ATPase from Rhizobium radiobacter (rrZntA) and seven other P(IB)-ATPases. Of the 38 conserved positions tested, 24 had small effects on metal tolerance. Fourteen mutations compromised in vivo metal tolerance and in vitro metal-stimulated ATPase activity. Based on structural modelling, the functionally important residues line a constricted 'channel', tightly surrounded by the residues that were found to be inconsequential for function. We tentatively propose that the distribution of the mutable and immutable residues marks a possible trans-membrane metal translocation pathway. In addition, by substituting six trans-membrane amino acids of rrZntA we changed the in vivo metal specificity of this pump from Zn(2+)/Cd(2+) to Ag(+).

Computational analysis of long-range allosteric communications in CFTR.

Malfunction of the CFTR protein results in cystic fibrosis, one of the most common hereditary diseases. CFTR functions as an anion channel, the gating of which is controlled by long-range allosteric communications. Allostery also has direct bearings on CF treatment: the most effective CFTR drugs modulate its activity allosterically. Herein, we integrated Gaussian network model, transfer entropy, and anisotropic normal mode-Langevin dynamics and investigated the allosteric communications network of CFTR. The results are in remarkable agreement with experimental observations and mutational analysis and provide extensive novel insight. We identified residues that serve as pivotal allosteric sources and transducers, many of which correspond to disease-causing mutations. We find that in the ATP-free form, dynamic fluctuations of the residues that comprise the ATP-binding sites facilitate the initial binding of the nucleotide. Subsequent binding of ATP then brings to the fore and focuses on dynamic fluctuations that were present in a latent and diffuse form in the absence of ATP. We demonstrate that drugs that potentiate CFTR's conductance do so not by directly acting on the gating residues, but rather by mimicking the allosteric signal sent by the ATP-binding sites. We have also uncovered a previously undiscovered allosteric 'hotspot' located proximal to the docking site of the phosphorylated regulatory (R) domain, thereby establishing a molecular foundation for its phosphorylation-dependent excitatory role. This study unveils the molecular underpinnings of allosteric connectivity within CFTR and highlights a novel allosteric 'hotspot' that could serve as a promising target for the development of novel therapeutic interventions.

Initiation of fibronectin fibrillogenesis is an enzyme-dependent process.

Fibronectin fibrillogenesis and mechanosensing both depend on integrin-mediated force transmission to the extracellular matrix. However, force transmission is in itself dependent on fibrillogenesis, and fibronectin fibrils are found in soft embryos where high forces cannot be applied, suggesting that force cannot be the sole initiator of fibrillogenesis. Here, we identify a nucleation step prior to force transmission, driven by fibronectin oxidation mediated by lysyl oxidase enzyme family members. This oxidation induces fibronectin clustering, which promotes early adhesion, alters cellular response to soft matrices, and enhances force transmission to the matrix. In contrast, absence of fibronectin oxidation abrogates fibrillogenesis, perturbs cell-matrix adhesion, and compromises mechanosensation. Moreover, fibronectin oxidation promotes cancer cell colony formation in soft agar as well as collective and single-cell migration. These results reveal a force-independent enzyme-dependent mechanism that initiates fibronectin fibrillogenesis, establishing a critical step in cell adhesion and mechanosensing.

A P-type ATPase importer that discriminates between essential and toxic transition metals.

Transition metals, although being essential cofactors in many physiological processes, are toxic at elevated concentrations. Among the membrane-embedded transport proteins that maintain appropriate intracellular levels of transition metals are ATP-driven pumps belonging to the P-type ATPase superfamily. These metal transporters may be differentiated according to their substrate specificities, where the majority of pumps can extrude either silver and copper or zinc, cadmium, and lead. In the present report, we have established the substrate specificities of nine previously uncharacterized prokaryotic transition-metal P-type ATPases. We find that all of the newly identified exporters indeed fall into one of the two above-mentioned categories. In addition to these exporters, one importer, Pseudomonas aeruginosa Q9I147, was also identified. This protein, designated HmtA (heavy metal transporter A), exhibited a different substrate recognition profile from the exporters. In vivo metal susceptibility assays, intracellular metal measurements, and transport experiments all suggest that HmtA mediates the uptake of copper and zinc but not of silver, mercury, or cadmium. The substrate selectivity of this importer ensures the high-affinity uptake of essential metals, while avoiding intracellular contamination by their toxic counterparts.

Listeria monocytogenes TcyKLMN Cystine/Cysteine Transporter Facilitates Glutathione Synthesis and Virulence Gene Expression.

Listeria monocytogenes is a saprophyte and a human intracellular pathogen. Upon invasion into mammalian cells, it senses multiple metabolic and environmental signals that collectively trigger its transition to the pathogenic state. One of these signals is the tripeptide glutathione, which acts as an allosteric activator of L. monocytogenes's master virulence regulator, PrfA. While glutathione synthesis by L. monocytogenes was shown to be critical for PrfA activation and virulence gene expression, it remains unclear how this tripeptide is synthesized in changing environments, especially in light of the observation that L. monocytogenes is auxotrophic to one of its precursors, cysteine. Here, we show that the ABC transporter TcyKLMN is a cystine/cysteine importer that supplies cysteine for glutathione synthesis, hence mediating the induction of the virulence genes. Further, we demonstrate that this transporter is negatively regulated by three metabolic regulators, CodY, CymR, and CysK, which sense and respond to changing concentrations of branched-chain amino acids (BCAA) and cysteine. The data indicate that under low concentrations of BCAA, TcyKLMN is upregulated, driving the production of glutathione by supplying cysteine, thereby facilitating PrfA activation. These findings provide molecular insight into the coupling of L. monocytogenes metabolism and virulence, connecting BCAA sensing to cysteine uptake and glutathione biosynthesis as a mechanism that controls virulence gene expression. This study exemplifies how bacterial pathogens sense their intracellular environment and exploit essential metabolites as effectors of virulence. IMPORTANCE Bacterial pathogens sense the repertoire of metabolites in the mammalian niche and use this information to shift into the pathogenic state to accomplish a successful infection. Glutathione is a virulence-activating signal that is synthesized by L. monocytogenes during infection of mammalian cells. In this study, we show that cysteine uptake via TcyKLMN drives glutathione synthesis and virulence gene expression. The data emphasize the intimate cross-regulation between metabolism and virulence in bacterial pathogens.

Structures of ABC transporters: handle with care.

In the past two decades, the ATP-binding cassette (ABC) transporters' field has undergone a structural revolution. The importance of structural biology to the development of the field of ABC transporters cannot be overstated, as the ensemble of structures not only revealed the architecture of ABC transporters but also shaped our mechanistic view of these remarkable molecular machines. Nevertheless, we advocate that the mechanistic interpretation of the structures is not trivial and should be carried out with prudence. Herein, we bring several examples of structures of ABC transporters that merit re-interpretation via careful comparison to experimental data. We propose that it is of the upmost importance to place new structures within the context of the available experimental data.

Distinct Allosteric Networks Underlie Mechanistic Speciation of ABC Transporters.

ABC transporters couple the energy of ATP hydrolysis to the transmembrane transport of biomolecules. Here, we investigated the allosteric networks of three representative ABC transporters using a hybrid molecular simulations approach validated by experiments. Each of the three transporters uses a different allosteric network: in the constitutive B12 importer BtuCD, ATP binding is the main driver of allostery and docking/undocking of the substrate-binding protein (SBP) is the driven event. The allosteric signal originates at the cytoplasmic side of the membrane before propagating to the extracellular side. In the substrate-controlled maltose transporter, the SBP is the main driver of allostery, ATP binding is the driven event, and the allosteric signal propagates from the extracellular to the cytoplasmic side of the membrane. In the lipid flippase PglK, a cyclic crosstalk between ATP and substrate binding underlies allostery. These results demonstrate speciation of biological functions may arise from variations in allosteric connectivity.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.