NiaR is a regulatory protein that represses the expression of proteins involved in the de novo biosynthesis and uptake of nicotinic acid (NA), with NA acting as a co-repressor. The previously published structure of NiaR from Thermotoga maritima (TmNiaR) identified it as a functional homodimer containing a transition metal ion in a suspected NA-binding pocket. Here, we present the crystal structure of NA bound to the iron-metalated form of TmNiaR. Supported by spectroscopic and solution studies, this structure shows that NA binds to a protein-bound ferrous ion via its ring nitrogen. In addition, the carboxylate group on NA interacts with Tyr108 from the dyad-related subunit, repositioning the likely DNA-binding domains of the dimer to promote high-affinity interactions with DNA operators. The specificity of TmNiaR for NA can be explained by the hydrogen bonding scheme within the NA-binding pocket.

It has been challenging to test how proteins acquire specific metals in cells. The speciation of metalation is thought to depend on the preferences of proteins for different metals competing at intracellular metal-availabilities. This implies mis-metalation may occur if proteins become mis-matched to metal-availabilities in heterologous cells. Here we use a cyanobacterial MnII-cupin (MncA) as a metal trap, to test predictions of metalation. By re-folding MncA in buffered competing metals, metal-preferences are determined. Relating metal-preferences to metal-availabilities estimated using cellular metal sensors, predicts mis-metalation of MncA with FeII in E. coli. After expression in E. coli, predominantly FeII-bound MncA is isolated experimentally. It is predicted that in metal-supplemented viable cells metal-MncA speciation should switch. MnII-, CoII-, or NiII-MncA are recovered from the respective metal-supplemented cells. Differences between observed and predicted metal-MncA speciation are used to refine estimated metal availabilities. Values are provided as blueprints to guide engineering biological protein metalation.

AbstractHere we provide the background data supporting an additional web-based calculator to predict in-cell metalation of proteins withinE. coliBW25113 grown in M9 media. Intracellular metal availabilities have been estimated from the calibrated responses of DNA-binding, metal-sensing, transcriptional regulators. We have previously tested and validated metalation calculator predictions. They confirm that when proteins are expressed heterologously inE. coliin LB medium, they can become mismatched to metal availability and be mismetalated. Mg2+GTP-CobW fromRhodobacteris predicted to be correctly metalated with cobalt inE. coligrown in M9 media, although mismetalated with zinc in LB-grown heterologous cells. Similarly, MncA fromSynechocystisPCC 6803 is predicted to be correctly metalated with manganese inE. coligrown in M9 media, although mismetalated with iron in LB-grown heterologous cells. The M9 metalation calculator is available online and as a spreadsheet for use in optimising metalation in engineering biology. An MncA-refined metalation calculator for idealised cells, reflecting the midpoint of intracellular metal availabilities, is also included and available online.

Here we provide the background data supporting an additional web-based calculator to predict in-cell metalation of proteins within E. coli BW25113 grown in M9 media. Intracellular metal availabilities have been estimated from the calibrated responses of DNA-binding, metal-sensing, transcriptional regulators. We have previously tested and validated metalation calculator predictions. They confirm that when proteins are expressed heterologously in E. coli in LB medium, they can become mismatched to metal availability and be mismetalated. Mg2+GTP-CobW from Rhodobacter is predicted to be correctly metalated with cobalt in E. coli grown in M9 media, although mismetalated with zinc in LB-grown heterologous cells. Similarly, MncA from Synechocystis PCC 6803 is predicted to be correctly metalated with manganese in E. coli grown in M9 media, although mismetalated with iron in LB-grown heterologous cells. The M9 metalation calculator is available online and as a spreadsheet for use in optimising metalation in engineering biology. An MncA-refined metalation calculator for idealised cells, reflecting the midpoint of intracellular metal availabilities, is also included and available online. ### Competing Interest Statement The authors have declared no competing interest.

Bacillus subtilis MntR is a dual regulatory protein that responds to heightened Mn2+ availability in the cell by both repressing the expression of uptake transporters and activating the expression of efflux proteins. Recent work indicates that, in its role as an activator, MntR binds several sites upstream of the genes encoding Mn2+ exporters, leading to a cooperative response to manganese. Here, we use cryo-EM to explore the molecular basis of gene activation by MntR and report a structure of four MntR dimers bound to four 18-base pair sites across an 84-base pair regulatory region of the mneP promoter. Our structures, along with solution studies including mass photometry and in vivo transcription assays, reveal that MntR dimers employ polar and non-polar contacts to bind cooperatively to an array of low-affinity DNA-binding sites. These results reveal the molecular basis for cooperativity in the activation of manganese efflux.

P2X receptors are trimeric ATP-gated ion channels that activate diverse signaling cascades. Due to its role in apoptotic pathways, selective activation of P2X7 is a potential experimental tool and therapeutic approach in cancer biology. However, mechanisms of high-affinity P2X7 activation have not been defined. We report high-resolution cryo-EM structures of wild-type rat P2X7 bound to the high-affinity agonist BzATP as well as significantly improved apo receptor structures in the presence and absence of sodium. Apo structures define molecular details of pore architecture and reveal how a partially hydrated Na+ ion interacts with the conductance pathway in the closed state. Structural, electrophysiological, and direct binding data of BzATP reveal that three residues just outside the orthosteric ATP-binding site are responsible for its high-affinity agonism. This work provides insights into high-affinity agonism for any P2X receptor and lays the groundwork for development of subtype-specific agonists applicable to cancer therapeutics.

Protein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.

The association of proteins with metals, metalation, is challenging because the tightest binding metals are rarely the correct ones. Inside cells, correct metalation is enabled by controlled bioavailability plus extra mechanisms for tricky combinations such as iron and manganese.

AbstractStreptococcus mutans, one of ∼600 bacterial species in the human oral cavity, is among the most acidogenic constituents of the plaque biofilm. Considered to be the primary causative agent of dental caries,S. mutansharbors a 25kDa SloR metalloregulatory protein which controls metal ion transport across the bacterial cell membrane to maintain essential metal ion homeostasis. The expression of SloR derives, in part, from transcriptional readthrough of thesloABCoperon which encodes a Mn2+/Fe2+ABC transport system. Herein, we describe the details of thesloABCpromoter that drives this transcription, as well as a novel independent promoter in an intergenic region (IGR) that contributes to downstreamsloRexpression. RT-PCR studies supportsloRtranscription that is independent ofsloABCexpression, and the results of 5′ RACE revealed asloRtranscription start site in the IGR from which the −10 and −35 promoter regions were predicted. The results of gel mobility shift assays support direct SloR binding to the IGR, albeit with lower affinity than SloR binding to thesloABCRpromoter. Function of thesloRpromoter was validated in qRT-PCR experiments. Interestingly,sloRexpression was not significantly impacted when grown in the presence of high manganese, whereas expression of thesloABCoperon was repressed under these conditions. The results ofin vitrotranscription studies support SloR-mediated transcriptional-activation ofsloRand -repression ofsloABC.Taken together, these findings implicate SloR as a bifunctional regulator that repressessloABCpromoter activity and encouragessloRtranscription from an independent promoter.ImportanceTooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide.S. mutans-induced carious lesions cause functional, physical, and/or aesthetic impairment in the vast majority of adults, and in 60-90% of schoolchildren in industrialized countries. Billions of dollars are spent annually on caries treatment, and productivity losses due to absenteeism from the workplace are significant. Research aimed at alleviatingS. mutans-induced tooth decay is important because it can address the socioeconomic disparity that is associated with dental cavities and improve overall general health which is inextricably linked to oral health. Research focused on theS. mutansSloR metalloregulatory protein can guide the development of novel therapeutics and so alleviate the burden of dental cavities.

ABSTRACT Streptococcus mutans, one of ∼600 bacterial species in the human oral cavity, is among the most acidogenic constituents of the plaque biofilm. Considered to be the primary causative agent of dental caries, S. mutans harbors a 25-kDa SloR metalloregulatory protein which controls metal ion transport across the bacterial cell membrane to maintain essential metal ion homeostasis. The expression of SloR derives in part from transcriptional readthrough of the sloABC operon, which encodes a Mn2+/Fe2+ ABC transport system. Here we describe the details of the sloABC promoter that drives this transcription as well as those for a novel independent promoter in an intergenic region (IGR) that contributes to downstream sloR expression. Reverse transcriptase PCR (RT-PCR) studies support the occurrence of sloR transcription that is independent of sloABC expression, and the results of 5′ rapid amplification of cDNA ends (5′ RACE) revealed a sloR transcription start site in the IGR, from which the −10 and −35 promoter regions were predicted. The results of gel mobility shift assays support direct SloR binding to the IGR, albeit with a lower affinity than that for SloR binding to the sloABCR promoter. The function of the sloR promoter was validated by semiquantitative real-time PCR (qRT-PCR) experiments. Interestingly, sloR expression was not significantly affected when bacteria were grown in the presence of a high manganese concentration, whereas expression of the sloABC operon was repressed under these conditions. The results of in vitro transcription studies support the occurrence of SloR-mediated transcriptional activation of sloR and repression of sloABC. Taken together, these findings implicate SloR as a bifunctional regulator that represses sloABC promoter activity and encourages sloR transcription from an independent promoter. IMPORTANCE Tooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide. S. mutans-induced carious lesions cause functional, physical, and/or esthetic impairment in the vast majority of adults and in 60 to 90% of schoolchildren in industrialized countries. Billions of dollars are spent annually on caries treatment, and productivity losses due to absenteeism from the workplace are significant. Research aimed at alleviating S. mutans-induced tooth decay is important because it can address the socioeconomic disparity that is associated with dental cavities and improve overall general health, which is inextricably linked to oral health. Research focused on the S. mutans SloR metalloregulatory protein can guide the development of novel therapeutics and thus alleviate the burden of dental cavities.

ABSTRACT Streptococcus mutans is the causative agent of dental caries, a significant concern for human health, and therefore an attractive target for therapeutics development. Previous work in our laboratory has identified a homodimeric, manganese-dependent repressor protein, SloR, as an important regulator of cariogenesis and has used site-directed mutagenesis to map functions to specific regions of the protein. Here we extend those studies to better understand the structural interaction between SloR and its operator and its effector metal ions. The results of DNase I assays indicate that SloR protects a 42-bp region of DNA that overlaps the sloABC promoter on the S. mutans UA159 chromosome, while electrophoretic mobility shift and solution binding assays indicate that each of two SloR dimers binds to this region. Real-time semiquantitative reverse transcriptase PCR (real-time semi-qRT-PCR) experiments were used to determine the individual base pairs that contribute to SloR-DNA binding specificity. Solution studies indicate that Mn 2+ is better than Zn 2+ at specifically activating SloR to bind DNA, and yet the 2.8-Å resolved crystal structure of SloR bound to Zn 2+ provides insight into the means by which selective activation by Mn 2+ may be achieved and into how SloR may form specific interactions with its operator. Taken together, these experimental observations are significant because they can inform rational drug design aimed at alleviating and/or preventing S. mutans -induced caries formation. IMPORTANCE This report focuses on investigating the SloR protein as a regulator of essential metal ion transport and virulence gene expression in the oral pathogen Streptococcus mutans and on revealing the details of SloR binding to its metal ion effectors and binding to DNA that together facilitate this expression. We used molecular and biochemical approaches to characterize the interaction of SloR with Mn 2+ and with its SloR recognition element to gain a clearer picture of the regulatory networks that optimize SloR-mediated metal ion homeostasis and virulence gene expression in S. mutans . These experiments can have a significant impact on caries treatment and/or prevention by revealing the S. mutans SloR-DNA binding interface as an appropriate target for the development of novel therapeutic interventions.