Structural Determinants of Vibrio cholerae FeoB Nucleotide Promiscuity.

Ferrous iron (Fe2+) is required for the growth and virulence of many pathogenic bacteria, including Vibrio cholerae (Vc), the causative agent of the disease cholera. For this bacterium, Feo is the primary system that transports Fe2+ into the cytosol. FeoB, the main component of this system, is regulated by a soluble cytosolic domain termed NFeoB. Recent reanalysis has shown that NFeoBs can be classified as either GTP-specific or NTP-promiscuous, but the structural and mechanistic bases for these differences were not known. To explore this intriguing property of FeoB, we solved the X-ray crystal structures of VcNFeoB in both the apo and GDP-bound forms. Surprisingly, this promiscuous NTPase displayed a canonical NFeoB G-protein fold like GTP-specific NFeoBs. Using structural bioinformatics, we hypothesized that residues surrounding the nucleobase could be important for both nucleotide affinity and specificity. We then solved the X-ray crystal structures of N150T VcNFeoB in the apo and GDP-bound forms to reveal H-bonding differences surround the guanine nucleobase. Interestingly, isothermal titration calorimetry revealed similar binding thermodynamics of the WT and N150T proteins to guanine nucleotides, while the behavior in the presence of adenine nucleotides was dramatically different. AlphaFold models of VcNFeoB in the presence of ADP and ATP showed important conformational changes that contribute to nucleotide specificity among FeoBs. Combined, these results provide a structural framework for understanding FeoB nucleotide promiscuity, which could be an adaptive measure utilized by pathogens to ensure adequate levels of intracellular iron across multiple metabolic landscapes.

Metal Messengers: Communication in the Bacterial World through Transition-Metal-Sensing Two-Component Systems.

Bacteria survive in highly dynamic and complex environments due, in part, to the presence of systems that allow the rapid control of gene expression in the presence of changing environmental stimuli. The crosstalk between intra- and extracellular bacterial environments is often facilitated by two-component signal transduction systems that are typically composed of a transmembrane histidine kinase and a cytosolic response regulator. Sensor histidine kinases and response regulators work in tandem with their modular domains containing highly conserved structural features to control a diverse array of genes that respond to changing environments. Bacterial two-component systems are widespread and play crucial roles in many important processes, such as motility, virulence, chemotaxis, and even transition metal homeostasis. Transition metals are essential for normal prokaryotic physiological processes, and the presence of these metal ions may also influence pathogenic virulence if their levels are appropriately controlled. To do so, bacteria use transition-metal-sensing two-component systems that bind and respond to rapid fluctuations in extracytosolic concentrations of transition metals. This perspective summarizes the structural and metal-binding features of bacterial transition-metal-sensing two-component systems and places a special emphasis on understanding how these systems are used by pathogens to establish infection in host cells and how these systems may be targeted for future therapeutic developments.

Reconstitution of the Arginyltransferase (ATE1) Iron-Sulfur Cluster.

As global regulators of eukaryotic homeostasis, arginyltransferases (ATE1s) have essential functions within the cell. Thus, the regulation of ATE1 is paramount. It was previously postulated that ATE1 was a hemoprotein and that heme was an operative cofactor responsible for enzymatic regulation and inactivation. However, we have recently shown that ATE1 instead binds an iron-sulfur ([Fe-S]) cluster that appears to function as an oxygen sensor to regulate ATE1 activity. As this cofactor is oxygen-sensitive, purification of ATE1 in the presence of O2 results in cluster decomposition and loss. Here, we describe an anoxic chemical reconstitution protocol to assemble the [Fe-S] cluster cofactor in Saccharomyces cerevisiae ATE1 (ScATE1) and Mus musculus ATE1 isoform 1 (MmATE1-1).

Iron-sulfur clusters are involved in post-translational arginylation.

Eukaryotic arginylation is an essential post-translational modification that modulates protein stability and regulates protein half-life. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite their conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we show that ATE1s bind a previously undiscovered [Fe-S] cluster that is conserved across evolution. We characterize the nature of this [Fe-S] cluster and find that the presence of the [Fe-S] cluster in ATE1 is linked to its arginylation activity, both in vitro and in vivo, and the initiation of the yeast stress response. Importantly, the ATE1 [Fe-S] cluster is oxygen-sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Taken together, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.

The preparation of recombinant arginyltransferase 1 (ATE1) for biophysical characterization.

Arginyltransferases (ATE1s) are eukaryotic enzymes that catalyze the non-ribosomal, post-translational addition of the amino acid arginine to an acceptor protein. While understudied, post-translation arginylation and ATE1 have major impacts on eukaryotic cellular homeostasis through both degradative and non-degradative effects on the intracellular proteome. Consequently, ATE1-catalyzed arginylation impacts major eukaryotic biological processes including the stress response, cellular motility, cardiovascular maturation, and even neurological function. Despite this importance, there is a lack of information on the structural and biophysical characteristics of ATE1, prohibiting a comprehensive understanding of the mechanism of this post-translational modification, and hampering efforts to design ATE1-specific therapeutics. To that end, this chapter details a protocol designed for the expression and the purification of ATE1 from Saccharomyces cerevisiae, although the approaches described herein should be generally applicable to other eukaryotic ATE1s. The detailed procedures afford high amounts of pure, homogeneous, monodisperse ATE1 suitable for downstream biophysical analyses such as X-ray crystallography, small angle X-ray scattering (SAXS), and cryo-EM techniques.

The Structure of Saccharomyces cerevisiae Arginyltransferase 1 (ATE1).

Eukaryotic post-translational arginylation, mediated by the family of enzymes known as the arginyltransferases (ATE1s), is an important post-translational modification that can alter protein function and even dictate cellular protein half-life. Multiple major biological pathways are linked to the fidelity of this process, including neural and cardiovascular developments, cell division, and even the stress response. Despite this significance, the structural, mechanistic, and regulatory mechanisms that govern ATE1 function remain enigmatic. To that end, we have used X-ray crystallography to solve the crystal structure of ATE1 from the model organism Saccharomyces cerevisiae ATE1 (ScATE1) in the apo form. The three-dimensional structure of ScATE1 reveals a bilobed protein containing a GCN5-related N-acetyltransferase (GNAT) fold, and this crystalline behavior is faithfully recapitulated in solution based on size-exclusion chromatography-coupled small angle X-ray scattering (SEC-SAXS) analyses and cryo-EM 2D class averaging. Structural superpositions and electrostatic analyses point to this domain and its domain-domain interface as the location of catalytic activity and tRNA binding, and these comparisons strongly suggest a mechanism for post-translational arginylation. Additionally, our structure reveals that the N-terminal domain, which we have previously shown to bind a regulatory [Fe-S] cluster, is dynamic and disordered in the absence of metal bound in this location, hinting at the regulatory influence of this region. When taken together, these insights bring us closer to answering pressing questions regarding the molecular-level mechanism of eukaryotic post-translational arginylation.

The structure of Vibrio cholerae FeoC reveals conservation of the helix-turn-helix motif but not the cluster-binding domain.

Most pathogenic bacteria require ferrous iron (Fe2+) in order to sustain infection within hosts. The ferrous iron transport (Feo) system is the most highly conserved prokaryotic transporter of Fe2+, but its mechanism remains to be fully characterized. Most Feo systems are composed of two proteins: FeoA, a soluble SH3-like accessory protein, and FeoB, a membrane protein that translocates Fe2+ across a lipid bilayer. Some bacterial feo operons encode FeoC, a third soluble, winged-helix protein that remains enigmatic in function. We previously demonstrated that selected FeoC proteins bind O2-sensitive [4Fe-4S] clusters via Cys residues, leading to the proposal that some FeoCs could sense O2 to regulate Fe2+ transport. However, not all FeoCs conserve these Cys residues, and FeoC from the causative agent of cholera (Vibrio cholerae) notably lacks any Cys residues, precluding cluster binding. In this work, we determined the NMR structure of VcFeoC, which is monomeric and conserves the helix-turn-helix domain seen in other FeoCs. In contrast, however, the structure of VcFeoC reveals a truncated winged ß-sheet in which the cluster-binding domain is notably absent. Using homology modeling, we predicted the structure of VcNFeoB and used docking to identify an interaction site with VcFeoC, which is confirmed by NMR spectroscopy. These findings provide the first atomic-level structure of VcFeoC and contribute to a better understanding of its role vis-à-vis FeoB.

A general protocol for the expression and purification of the intact transmembrane transporter FeoB.

Ferrous iron (Fe2+) transport is an essential process that supports the growth, intracellular survival, and virulence of several drug-resistant pathogens, and the ferrous iron transport (Feo) system is the most important and widespread protein complex that mediates Fe2+ transport in these organisms. The Feo system canonically comprises three proteins (FeoA/B/C). FeoA and FeoC are both small, accessory proteins localized to the cytoplasm, and their roles in the Fe2+ transport process have been of great debate. FeoB is the only wholly-conserved component of the Feo system and serves as the inner membrane-embedded Fe2+ transporter with a soluble G-protein-like N-terminal domain. In vivo studies have underscored the importance of Feo during infection, emphasizing the need to better understand Feo-mediated Fe2+ uptake, although a paucity of research exists on intact FeoB. To surmount this problem, we designed an overproduction and purification system that can be applied generally to a suite of intact FeoBs from several organisms. Importantly, we noted that FeoB is extremely sensitive to excess salt while in the membrane of a recombinant host, and we designed a workflow to circumvent this issue. We also demonstrated effective protein extraction from the lipid bilayer through small-scale solubilization studies. We then applied this approach to the large-scale purifications of Escherichia coli and Pseudomonas aeruginosa FeoBs to high purity and homogeneity. Lastly, we show that our protocol can be generally applied to various FeoB proteins. Thus, this workflow allows for isolation of suitable quantities of FeoB for future biochemical and biophysical characterization.

A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB.

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe2+ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein-protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen-deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein-protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA-NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.

Ins and Outs: Recent Advancements in Membrane Protein-Mediated Prokaryotic Ferrous Iron Transport.

Iron is an essential nutrient for virtually every living organism, especially pathogenic prokaryotes. Despite its importance, however, both the acquisition and the export of this element require dedicated pathways that are dependent on oxidation state. Due to its solubility and kinetic lability, reduced ferrous iron (Fe2+) is useful to bacteria for import, chaperoning, and efflux. Once imported, ferrous iron may be loaded into apo and nascent enzymes and even sequestered into storage proteins under certain conditions. However, excess labile ferrous iron can impart toxicity as it may spuriously catalyze Fenton chemistry, thereby generating reactive oxygen species and leading to cellular damage. In response, it is becoming increasingly evident that bacteria have evolved Fe2+ efflux pumps to deal with conditions of ferrous iron excess and to prevent intracellular oxidative stress. In this work, we highlight recent structural and mechanistic advancements in our understanding of prokaryotic ferrous iron import and export systems, with a focus on the connection of these essential transport systems to pathogenesis. Given the connection of these pathways to the virulence of many increasingly antibiotic resistant bacterial strains, a greater understanding of the mechanistic details of ferrous iron cycling in pathogens could illuminate new pathways for future therapeutic developments.

Non-canonical LexA proteins regulate the SOS response in the Bacteroidetes.

Lesions to DNA compromise chromosome integrity, posing a direct threat to cell survival. The bacterial SOS response is a widespread transcriptional regulatory mechanism to address DNA damage. This response is coordinated by the LexA transcriptional repressor, which controls genes involved in DNA repair, mutagenesis and cell-cycle control. To date, the SOS response has been characterized in most major bacterial groups, with the notable exception of the Bacteroidetes. No LexA homologs had been identified in this large, diverse and ecologically important phylum, suggesting that it lacked an inducible mechanism to address DNA damage. Here, we report the identification of a novel family of transcriptional repressors in the Bacteroidetes that orchestrate a canonical response to DNA damage in this phylum. These proteins belong to the S24 peptidase family, but are structurally different from LexA. Their N-terminal domain is most closely related to CI-type bacteriophage repressors, suggesting that they may have originated from phage lytic phase repressors. Given their role as SOS regulators, however, we propose to designate them as non-canonical LexA proteins. The identification of a new class of repressors orchestrating the SOS response illuminates long-standing questions regarding the origin and plasticity of this transcriptional network.

Ferric iron reductases and their contribution to unicellular ferrous iron uptake.

Iron is a necessary element for nearly all forms of life, and the ability to acquire this trace nutrient has been identified as a key virulence factor for the establishment of infection by unicellular pathogens. In the presence of O2, iron typically exists in the ferric (Fe3+) oxidation state, which is highly unstable in aqueous conditions, necessitating its sequestration into cofactors and/or host proteins to remain soluble. To counter this insolubility, and to compete with host sequestration mechanisms, many unicellular pathogens will secrete low molecular weight, high-affinity Fe3+ chelators known as siderophores. Once acquired, unicellular pathogens must liberate the siderophore-bound Fe3+ in order to assimilate this nutrient into metabolic pathways. While these organisms may hydrolyze the siderophore backbone to release the chelated Fe3+, this approach is energetically costly. Instead, iron may be liberated from the Fe3+-siderophore complex through reduction to Fe2+, which produces a lower-affinity form of iron that is highly soluble. This reduction is performed by a class of enzymes known as ferric reductases. Ferric reductases are broadly-distributed electron-transport proteins that are expressed by numerous infectious organisms and are connected to the virulence of unicellular pathogens. Despite this importance, ferric reductases remain poorly understood. This review provides an overview of our current understanding of unicellular ferric reductases (both soluble and membrane-bound), with an emphasis on the important but underappreciated connection between ferric-reductase mediated Fe3+ reduction and the transport of Fe2+ via ferrous iron transporters.

ATE1-Mediated Post-Translational Arginylation Is an Essential Regulator of Eukaryotic Cellular Homeostasis.

Arginylation is a protein post-translational modification catalyzed by arginyl-tRNA transferases (ATE1s), which are critical enzymes conserved across all eukaryotes. Arginylation is a key step in the Arg N-degron pathway, a hierarchical cellular signaling pathway that links the ubiquitin-dependent degradation of a protein to the identity of its N-terminal amino acid side chain. The fidelity of ATE1-catalyzed arginylation is imperative, as this post-translational modification regulates several essential biological processes such as cardiovascular maturation, chromosomal segregation, and even the stress response. While the process of ATE1-catalyzed arginylation has been studied in detail at the cellular level, much remains unknown about the structure of this important enzyme, its mechanism of action, and its regulation. In this work, we detail the current state of knowledge on ATE1-catalyzed arginylation, and we discuss both ongoing and future directions that will reveal the structural and mechanistic details of this essential eukaryotic cellular regulator.

The FeoC [4Fe-4S] Cluster Is Redox-Active and Rapidly Oxygen-Sensitive.

The acquisition of iron is essential to establishing virulence among most pathogens. Under acidic and/or anaerobic conditions, most bacteria utilize the widely distributed ferrous iron (Fe2+) uptake (Feo) system to import metabolically-required iron. The Feo system is inadequately understood at the atomic, molecular, and mechanistic levels, but we do know it is composed of a main membrane component (FeoB) essential for iron translocation, as well as two small, cytosolic proteins (FeoA and FeoC) hypothesized to function as accessories to this process. FeoC has many hypothetical functions, including that of an iron-responsive transcriptional regulator. Here, we demonstrate for the first time that Escherichia coli FeoC (EcFeoC) binds an [Fe-S] cluster. Using electronic absorption, X-ray absorption, and electron paramagnetic resonance spectroscopies, we extensively characterize the nature of this cluster. Under strictly anaerobic conditions after chemical reconstitution, we demonstrate that EcFeoC binds a redox-active [4Fe-4S]2+/+ cluster that is rapidly oxygen-sensitive and decays to a [2Fe-2S]2+ cluster (t1/2 ≈ 20 s), similar to the [Fe-S] cluster in the fumarate and nitrate reductase (FNR) transcriptional regulator. We further show that this behavior is nearly identical to the homologous K. pneumoniae FeoC, suggesting a redox-active, oxygen-sensitive [4Fe-4S]2+ cofactor is a general phenomenon of cluster-binding FeoCs. Finally, in contrast to FNR, we show that the [4Fe-4S]2+ cluster binding to FeoC is associated with modest conformational changes of the polypeptide, but not protein dimerization. We thus posit a working hypothesis in which the cluster-binding FeoCs may function as oxygen-sensitive iron sensors that fine-tune pathogenic ferrous iron acquisition.

The crystal structure of Klebsiella pneumoniae FeoA reveals a site for protein-protein interactions.

In order to establish infection, pathogenic bacteria must obtain essential nutrients such as iron. Under acidic and/or anaerobic conditions, most bacteria utilize the Feo system in order to acquire ferrous iron (Fe2+ ) from their host environment. The mechanism of this process, including its regulation, remains poorly understood. In this work, we have determined the crystal structure of FeoA from the nosocomial agent Klebsiella pneumoniae (KpFeoA). Our structure reveals an SH3-like domain that mediates interactions between neighboring polypeptides via hydrophobic intercalations into a Leu-rich surface ridge. Using docking of a small peptide corresponding to a postulated FeoB partner binding site, we demonstrate that KpFeoA can assume both "open" and "closed" conformations, controlled by binding at this Leu-rich ridge. We propose a model in which a "C-shaped" clamp along the FeoA surface mediates interactions with its partner protein, FeoB. These findings are the first to demonstrate atomic-level details of FeoA-based protein-protein interactions and provide a framework for testing FeoA-FeoB interactions, which could be exploited for future antibiotic developments.

Toward a mechanistic understanding of Feo-mediated ferrous iron uptake.

Virtually all organisms require iron and have evolved to obtain this element in free or chelated forms. Under anaerobic or low pH conditions commonly encountered by numerous pathogens, iron predominantly exists in the ferrous (Fe2+) form. The ferrous iron transport (Feo) system is the only widespread mechanism dedicated solely to bacterial ferrous iron import, and this system has been linked to pathogenic virulence, bacterial colonization, and microbial survival. The canonical feo operon encodes for three proteins that comprise the Feo system: FeoA, a small cytoplasmic ß-barrel protein; FeoB, a large, polytopic membrane protein with a soluble G-protein domain capable of hydrolyzing GTP; and FeoC, a small, cytoplasmic protein containing a winged-helix motif. While previous studies have revealed insight into soluble and fragmentary domains of the Feo system, the chief membrane-bound component FeoB remains poorly studied. However, recent advances have demonstrated that large quantities of intact FeoB can be overexpressed, purified, and biophysically characterized, revealing glimpses into FeoB function. Two models of full-length FeoB have been published, providing starting points for hypothesis-driven investigations into the mechanism of FeoB-mediated ferrous iron transport. Finally, in vivo studies have begun to shed light on how this system functions as a unique multicomponent complex. In light of these new data, this review will summarize what is known about the Feo system, including recent advancements in FeoB structure and function.

Expression and purification of functionally active ferrous iron transporter FeoB from Klebsiella pneumoniae.

The acquisition of ferrous iron (Fe2+) is an important virulence factor utilized by several hospital-acquired (nosocomial) pathogens such as Klebsiella pneumoniae to establish infection within human hosts. Virtually all bacteria use the ferrous iron transport system (Feo) to acquire ferrous iron from their environments, which are often biological niches that stabilize Fe2+ relative to Fe3+. However, the details of this process remain poorly understood, likely owing to the few expression and purification systems capable of supplying sufficient quantities of the chief component of the Feo system, the integral membrane GTPase FeoB. This bottleneck has undoubtedly hampered efforts to understand this system in order to target it for therapeutic intervention. In this study, we describe the expression, solubilization, and purification of the Fe2+ transporter from K. pneumoniae, KpFeoB. We show that this protein may be heterologously overexpressed in Escherichia coli as the host organism. After testing several different commercially-available detergents, we have developed a solubilization and purification protocol that produces milligram quantities of KpFeoB with sufficient purity for enzymatic and biophysical analyses. Importantly, we demonstrate that KpFeoB displays robust GTP hydrolysis activity (kcatGTP of ∼10-1 s-1) in the absence of any additional stimulatory factors. Our findings suggest that K. pneumoniae may be capable of using its Feo system to drive Fe2+ import in an active manner.

Ethanol Interactions With Dexmethylphenidate and dl-Methylphenidate Spheroidal Oral Drug Absorption Systems in Healthy Volunteers.

BACKGROUND/PURPOSE: Ethanol coadministered with immediate-release dl-methylphenidate (dl-MPH) or dexmethylphenidate (d-MPH) significantly increases the geomean maximum plasma concentration (Cmax) of d-MPH 22% and 15%, respectively, and elevates overall drug exposure and psychostimulant effects. We asked the question: Are these ethanol-MPH interactions based more fundamentally on (1) inhibition of postabsorption d-MPH metabolism or (2) acceleration of MPH formulation gastric dissolution by ethanol in the stomach? This was investigated using the pulsatile, distinctly biphasic, spheroidal oral drug absorption systems of dl-MPH and d-MPH. METHODS: In a randomized, 4-way crossover study, 14 healthy subjects received pulsatile dl-MPH (40 mg) or d-MPH (20 mg), with or without ethanol (0.6 g/kg), dosed 4 hours later. These 4 hours allowed the delayed-release second MPH pulse to reach a more distal region of the gut to preclude gastric biopharmaceutical influences. Plasma was analyzed using a highly sensitive chiral method. Subjective/physiological effects were recorded. FINDINGS/RESULTS: Ethanol increased the second pulse of d-MPH Cmax for dl-MPH by 35% (P < 0.01) and the partial area under the plasma concentration curve from 4 to 8 hours by 25% (P < 0.05). The respective values for enantiopure d-MPH were 27% (P = 0.001) and 20% (P < 0.01). The carboxylesterase 1-mediated transesterification metabolite ethylphenidate served as a biomarker for coexposure. Ethanol significantly potentiated stimulant responses to either formulation. IMPLICATIONS/CONCLUSIONS: These findings support drug dispositional interactions between ethanol and MPH as dominant over potential biopharmaceutical considerations. Understanding the pharmacology underlying the frequent coabuse of MPH-ethanol provides rational guidance in the selection of first-line pharmacotherapy for comorbid attention-deficit/hyperactivity disorder-alcohol use disorder.

Metal Selectivity of a Cd-, Co-, and Zn-Transporting P1B-type ATPase.

The P1B-ATPases, a family of transmembrane metal transporters important for transition metal homeostasis in all organisms, are subdivided into classes based on sequence conservation and metal specificity. The multifunctional P1B-4-ATPase CzcP is part of the cobalt, zinc, and cadmium resistance system from the metal-tolerant, model organism Cupriavidus metallidurans. Previous work revealed the presence of an unusual soluble metal-binding domain (MBD) at the CzcP N-terminus, but the nature, extent, and selectivity of the transmembrane metal-binding site (MBS) of CzcP have not been resolved. Using homology modeling, we show that four wholly conserved amino acids from the transmembrane (TM) domain (Met254, Ser474, Cys476, and His807) are logical candidates for the TM MBS, which may communicate with the MBD via interactions with the first TM helix. Metal-binding analyses indicate that wild-type (WT) CzcP has three MBSs, and data on N-terminally truncated (ΔMBD) CzcP suggest the presence of a single TM MBS. Electronic absorption and electron paramagnetic resonance spectroscopic analyses of ΔMBD CzcP and variant proteins thereof provide insight into the details of Co2+ coordination by the TM MBS. These spectroscopic data, combined with in vitro functional studies of WT and variant CzcP proteins, show that the side chains of Met254, Cys476, and His807 contribute to Cd2+, Co2+, and Zn2+ binding and transport, whereas the side chain of Ser474 appears to play a minimal role. By comparison to other P1B-4-ATPases, we suggest that an evolutionarily adapted flexibility in the TM region likely afforded CzcP the ability to transport Cd2+ and Zn2+ in addition to Co2+.

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity.

The RNA-binding heme protein DiGeorge critical region 8 (DGCR8) and its ribonuclease partner Drosha cleave primary transcripts of microRNA (pri-miRNA) as part of the canonical microRNA (miRNA) processing pathway. Previous studies show that bis-cysteine thiolate-coordinated Fe(III) DGCR8 supports pri-miRNA processing activity, while Fe(II) DGCR8 does not. In this study, we further characterized Fe(II) DGCR8 and tested whether CO or NO might bind and restore pri-miRNA processing activity to the reduced protein. Fe(II) DGCR8 RNA-binding heme domain (Rhed) undergoes a pH-dependent transition from 6-coordinate to 5-coordinate, due to protonation and loss of a lysine ligand; the ligand bound throughout the pH change is a histidine. Fe(II) Rhed binds CO and NO from 6- and 5-coordinate states, forming common CO and NO adducts at all pHs. Fe(II)-CO Rhed is 6-coordinate, low-spin, and pH insensitive with the histidine ligand retained, suggesting that the protonatable lysine ligand has been replaced by CO. Fe(II)-NO Rhed is 5-coordinate and pH insensitive. Fe(II)-NO also forms slowly upon reaction of Fe(III) Rhed with excess NO via a stepwise process. Heme reduction by NO is rate-limiting, and the rate would be negligible at physiological NO concentrations. Importantly, in vitro pri-miRNA processing assays show that both CO- and NO-bound DGCR8 species are inactive. Fe(II), Fe(II)-CO, and Fe(II)-NO Rhed do not bear either of the cysteine ligands found in the Fe(III) state. These data support a model in which the bis-cysteine thiolate ligand environment of Fe(III) DGCR8 is necessary for establishing proper pri-miRNA binding and enabling processing activity.