Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes.

RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival1-3. RAF activity is tightly regulated and inappropriate activation is a frequent cause of cancer4-6; however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.

De novo mapping of α-helix recognition sites on protein surfaces using unbiased libraries.

The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered "undruggable", such as protein-protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein-protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.

Structure and RAF family kinase isoform selectivity of type II RAF inhibitors tovorafenib and naporafenib.

Upon activation by RAS, RAF family kinases initiate signaling through the MAP kinase cascade to control cell growth, proliferation, and differentiation. Among RAF isoforms (ARAF, BRAF, and CRAF), oncogenic mutations are by far most frequent in BRAF. The BRAFV600E mutation drives more than half of all malignant melanoma and is also found in many other cancers. Selective inhibitors of BRAFV600E (vemurafenib, dabrafenib, encorafenib) are used clinically for these indications, but they are not effective inhibitors in the context of oncogenic RAS, which drives dimerization and activation of RAF, nor for malignancies driven by aberrantly dimerized truncation/fusion variants of BRAF. By contrast, a number of "type II" RAF inhibitors have been developed as potent inhibitors of RAF dimers. Here, we compare potency of type II inhibitors tovorafenib (TAK-580) and naporafenib (LHX254) in biochemical assays against the three RAF isoforms and describe crystal structures of both compounds in complex with BRAF. We find that tovorafenib and naporafenib are most potent against CRAF but markedly less potent against ARAF. Crystal structures of both compounds with BRAFV600E or WT BRAF reveal the details of their molecular interactions, including the expected type II-binding mode, with full occupancy of both subunits of the BRAF dimer. Our findings have important clinical ramifications. Type II RAF inhibitors are generally regarded as pan-RAF inhibitors, but our studies of these two agents, together with recent work with type II inhibitors belvarafenib and naporafenib, indicate that relative sparing of ARAF may be a property of multiple drugs of this class.

A Comparative Study of AI-Based Automated and Manual Postprocessing of Head and Neck CT Angiography: An Independent External Validation with Multi-Vendor and Multi-Center Data.

PURPOSE: To externally validate the performance of automated postprocessing (AP) on head and neck CT Angiography (CTA) and compare it with manual postprocessing (MP). METHODS: This retrospective study included head and neck CTA-exams of patients from three tertiary hospitals acquired on CT scanners from five manufacturers. AP was performed by CerebralDoc. The image quality was assessed using Likert scales, and the qualitative and quantitative diagnostic performance of arterial stenosis and aneurysm, postprocessing time, and scanning radiation dose were also evaluated. RESULTS: A total of 250 patients were included. Among these, 55 patients exhibited significant stenosis (≥ 50%), and 33 patients had aneurysms, diagnosed using original CTA datasets and corresponding multiplanar reconstructions as the reference. While the scores of the V4 segment and the edge of the M1 segment on volume rendering (VR), as well as the C4 segment on maximum intensity projection (MIP), were significantly lower with AP compared to MP across vendors (all P < 0.05), most scores in AP demonstrated image quality that was either superior to or comparable with that of MP. Furthermore, the diagnostic performance of AP was either superior to or comparable with that of MP. Moreover, AP also exhibited advantages in terms of postprocessing time and radiation dose when compared to MP (P < 0.001). CONCLUSION: The AP of CerebralDoc presents clear advantages over MP and holds significant clinical value. However, further optimization is required in the image quality of the V4 and M1 segments on VR as well as the C4 segment on MIP.

Allosteric MEK inhibitors act on BRAF/MEK complexes to block MEK activation.

The RAF/MEK/ERK pathway is central to the control of cell physiology, and its dysregulation is associated with many cancers. Accordingly, the proteins constituting this pathway, including MEK1/2 (MEK), have been subject to intense drug discovery and development efforts. Allosteric MEK inhibitors (MEKi) exert complex effects on RAF/MEK/ERK pathway signaling and are employed clinically in combination with BRAF inhibitors in malignant melanoma. Although mechanisms and structures of MEKi bound to MEK have been described for many of these compounds, recent studies suggest that RAF/MEK complexes, rather than free MEK, should be evaluated as the target of MEKi. Here, we describe structural and biochemical studies of eight structurally diverse, clinical-stage MEKi to better understand their mechanism of action on BRAF/MEK complexes. We find that all of these agents bind in the MEK allosteric site in BRAF/MEK complexes, in which they stabilize the MEK activation loop in a conformation that is resistant to BRAF-mediated dual phosphorylation required for full activation of MEK. We also show that allosteric MEK inhibitors act most potently on BRAF/MEK complexes rather than on free active MEK, further supporting the notion that a BRAF/MEK complex is the physiologically relevant pharmacologic target for this class of compounds. Our findings provide a conceptual and structural framework for rational development of RAF-selective MEK inhibitors as an avenue to more effective and better-tolerated agents targeting this pathway.

Performance of deep learning-based autodetection of arterial stenosis on head and neck CT angiography: an independent external validation study.

PURPOSE: To externally validate the performance of automated stenosis detection on head and neck CT angiography (CTA) and investigate the impact factors using an independent bi-center dataset with digital subtraction angiography (DSA) as the ground truth. MATERIAL AND METHODS: Patients who underwent head and neck CTA and DSA between January 2019 and December 2021 were retrospectively included. The degree of stenosis was automatically evaluated using CerebralDoc based on CTA. The performance of CerebralDoc across levels (per-patient, per-region, per-vessel, and per-segment) and thresholds (≥ 50%, ≥ 70%, and = 100%) was evaluated. Logistic regression was performed to identify independent factors associated with false negative results. RESULTS: 296 patients were analyzed. Specificity across levels and thresholds was high, exceeding 92%. The area under the curve ranged from poor (0.615, 95% CI: 0.544, 0.686; at the region-based analysis for stenosis ≥ 70%) to excellent (0.945, 95% CI: 0.905, 0.985; at the patient-based analysis for stenosis ≥ 50%). Sensitivity ranged from 0.714 (95% CI: 0.675, 0.750) at the segment-based analysis for stenosis ≥ 70% to 0.895 (95% CI: 0.849, 0.919) at the patient-based analysis for stenosis ≥ 50%. The multiple logistic regression analysis revealed that false negative results were primarily more likely to specific stenosis locations (particularly the M2 segment and skull base segment of the internal carotid artery) and occlusion. CONCLUSIONS: CerebralDoc has the potential to automated stenosis detection on head and neck CTA, but further efforts are needed to optimize its performance.

A deep-learning model for identifying fresh vertebral compression fractures on digital radiography.

OBJECTIVES: To develop a deep-learning (DL) model for identifying fresh VCFs from digital radiography (DR), with magnetic resonance imaging (MRI) as the reference standard. METHODS: Patients with lumbar VCFs were retrospectively enrolled from January 2011 to May 2020. All patients underwent DR and MRI scanning. VCFs were categorized as fresh or old according to MRI results, and the VCF grade and type were assessed. The raw DR data were sent to InferScholar Center for annotation. A DL-based prediction model was built, and its diagnostic performance was evaluated. The DeLong test was applied to assess differences in ROC curves between different models. RESULTS: A total of 1877 VCFs in 1099 patients were included in our study and randomly divided into development (n = 824 patients) and test (n = 275 patients) datasets. The ensemble model identified fresh and old VCFs, reaching an AUC of 0.80 (95% confidence interval [CI], 0.77-0.83), an accuracy of 74% (95% CI, 72-77%), a sensitivity of 80% (95% CI, 77-83%), and a specificity of 68% (95% CI, 63-72%). Lateral (AUC, 0.83) views exhibited better performance than anteroposterior views (AUC, 0.77), and the best performance among respective subgroupings was obtained for grade 3 (AUC, 0.89) and crush-type (AUC, 0.87) subgroups. CONCLUSION: The proposed DL model achieved adequate performance in identifying fresh VCFs from DR. KEY POINTS: • The ensemble deep-learning model identified fresh VCFs from DR, reaching an AUC of 0.80, an accuracy of 74%, a sensitivity of 80%, and a specificity of 68% with the reference standard of MRI. • The lateral views (AUC, 0.83) exhibited better performance than anteroposterior views (AUC, 0.77). • The grade 3 (AUC, 0.89) and crush-type (AUC, 0.87) subgroups showed the best performance among their respective subgroupings.

The role of chest CT in management of asymptomatic SARS-CoV-2 infections: A longitudinal multi-center study in Chongqing, China.

Background: Multiple societies including the Fleischner Society do not recommend that CT is routinely used in asymptomatic SARS-CoV-2 infections; however, this advice is based on the limited evidence. In this study, we aim to confirm whether it is necessary to do CT scans in SARS-CoV-2 asymptomatic infections by summarizing the longitudinal chest CT and clinical features of asymptomatic SARS-CoV-2 infections. Methods: A total of 33 individuals (14 men and 19 women) with asymptomatic SARS-CoV-2 infections were retrospectively enrolled. Clinical data of CT positive and negative groups were compared. Longitudinal chest CT scans were reviewed for CT features and analyzed for temporal change. Results: Thirty-two (97%) individuals had positive results for first RT-PCR testing. For clinical data, only monocyte count showed a significant difference between CT positive and negative groups. For first chest CT, only eighteen (54.5%) individuals had abnormal manifestations, common CT features were GGO (88.9%) and consolidation (33.3%), the median number of segments involved was 3.0 (1.0-7.5). No case in CT negative group was abnormal on the follow-up CT. Three patterns of evolution throughout series of CT were observed in CT positive group, including gradual improvement (12, 66.7%), mismatch to improvement (3, 16.7%) and mild progression to improvement (3, 16.7%). On last CT scans, most cases had radiographic improvement but residual abnormalities. Significant differences were exhibited in density, long diameter, number of lung segments involved, and percentage of consolidation between the first and last CT scans. All cases had stable conditions and finally confirmed negative for SARS-CoV-2 RT-PCR tests without developing into severe pneumonia. Conclusion: Considering poor performance of CT in screening, stable conditions during followup, and good outcomes in asymptomatic SARS-CoV-2 infections, we confirm that it is unnecessary to do CT scans in asymptomatic SARS-CoV-2 infections.

Cytotoxic protein from the mushroom Coprinus comatus possesses a unique mode for glycan binding and specificity.

Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan-GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Coprinus comatus Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcß1-4(Fucα1-3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αßα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel ß-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment.

Invasive Pulmonary Adenocarcinomas Versus Preinvasive Lesions Appearing as Pure Ground-Glass Nodules: Differentiation Using Enhanced Dual-Source Dual-Energy CT.

OBJECTIVE. The objective of our study was to investigate the potentials of enhanced dual-source dual-energy CT (DECT) and three-planar measurements for differentiating invasive pulmonary adenocarcinomas (IPAs) from preinvasive lesions appearing as pure ground-glass nodules (pGGNs). MATERIALS AND METHODS. Thirty-nine patients with 53 pGGNs who underwent enhanced dual-source DECT were included in this retrospective study. All pGGNs were pathologically confirmed and categorized into two groups: preinvasive lesions or IPAs. The traditional CT features of the pGGNs were evaluated on unenhanced images. Quantitative parameters were measured on iodine-enhanced images of dual-source DECT in three planes, and both intra- and interobserver reproducibility analyses were performed to assess the measurement reproducibility of quantitative parameters. To identify significant factors for differentiating IPAs from preinvasive lesions, we performed logistic regression analysis and ROC curve analysis. RESULTS. For traditional CT features, only lesion size and unenhanced CT attenuation value showed significant differences between preinvasive lesions and IPAs (p < 0.05). Preinvasive lesions and IPAs exhibited significant differences in attenuation on virtual images, so-called "virtual HU" or "VHU," and the modified normalized iodine concentration (NIC) (p < 0.05), and both intra- and interobserver agreement for the quantitative measurements were excellent. Multivariate logistic regression analysis revealed that larger lesion size (adjusted odds ratio [OR], 3.65) and higher modified NIC (adjusted OR, 19.01) were significant differentiators of IPAs from preinvasive lesions (p < 0.05). ROC curve analysis revealed that modified NIC showed excellent performance (AUC, 0.924) and significantly higher performance than lesion size (AUC, 0.711) for differentiating IPAs from preinvasive lesions. CONCLUSION. In pGGNs, a lesion with a modified NIC value of more than 0.29 can be a very specific discriminator of IPAs from preinvasive lesions, and IPAs can be accurately and reliably differentiated from preinvasive lesions using enhanced dual-source DECT and three-planar measurements.

Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans.

Caenorhabditis elegans secretes ascarosides as pheromones to communicate with other worms and to coordinate the development and behavior of the population. Peroxisomal ß-oxidation cycles shorten the side chains of ascaroside precursors to produce the short-chain ascaroside pheromones. Acyl-CoA oxidases, which catalyze the first step in these ß-oxidation cycles, have different side chain-length specificities and enable C. elegans to regulate the production of specific ascaroside pheromones. Here, we determine the crystal structure of the acyl-CoA oxidase 1 (ACOX-1) homodimer and the ACOX-2 homodimer bound to its substrate. Our results provide a molecular basis for the substrate specificities of the acyl-CoA oxidases and reveal why some of these enzymes have a very broad substrate range, whereas others are quite specific. Our results also enable predictions to be made for the roles of uncharacterized acyl-CoA oxidases in C. elegans and in other nematode species. Remarkably, we show that most of the C. elegans acyl-CoA oxidases that participate in ascaroside biosynthesis contain a conserved ATP-binding pocket that lies at the dimer interface, and we identify key residues in this binding pocket. ATP binding induces a structural change that is associated with tighter binding of the FAD cofactor. Mutations that disrupt ATP binding reduce FAD binding and reduce enzyme activity. Thus, ATP may serve as a regulator of acyl-CoA oxidase activity, thereby directly linking ascaroside biosynthesis to ATP concentration and metabolic state.

Virulence and DNA fingerprinting analysis of Xanthomonas oryzae pv. oryzae identify a new pathotype in Guangxi, South China.

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases affecting rice worldwide. However, little is known about the population structure of this organism in Guangxi Zhuang Autonomous Region, South China. Here, pathotypic and DNA fingerprint analyses were conducted to characterize the isolates of Xoo collected from rice leaves in five districts of the region from 2013 to 2016. Their pathogenicity was tested by leaf clipping, and the DNA fingerprints were analyzed by repetitive sequence-based polymerase chain reaction and endogenous insertion sequence element-based polymerase chain reaction assays using the repetitive extragenic palindromic sequence and enterobacterial repetitive intergenic consensus primers, respectively. Pathogenicity assays of 70 representative isolates were conducted using a series of near-isogenic lines and two new pathotypes were identified. All the pathotypes were found to be incompatible with xa5 and Xa7. One pathotype was virulent to Xa14, Xa21, and Xa23, whereas another virulent to Xa21 and Xa23, but incompatible with Xa14. A dendrogram generated for the data sets obtained from DNA fingerprinting suggested the prevalence of high genetic diversity of Xoo throughout Guangxi, and no association between the molecular haplotypes and pathotypes was identified.

Structural basis for precursor protein-directed ribosomal peptide macrocyclization.

Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight into the unique protein-protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.

Association between media coverage and prevalence of idiopathic environmental intolerance attributed to electromagnetic field in Taiwan.

Idiopathic environmental intolerance attributed to electromagnetic fields (IEI-EMF) describes symptoms sufferers attribute to exposure to electromagnetic fields (EMF). In Taiwan, the prevalence rate of IEI-EMF was 13.3% in 2007, but a survey using the same method found the rate declined to 4.6% in 2012. Because media reports may encourage readers to attribute their symptoms to EMF, the change might be related to media coverage. We searched articles indexed in the largest newspaper database in Taiwan to evaluate the association between media coverage and the prevalence of IEI-EMF. We also assessed the effects of other potential affecting factors. The number of newspaper articles related to EMF and IEI-EMF increased from 2005 to 2007 and then has been decreasing until 2012, which is compatible with the change in the prevalence of IEI-EMF. However, from 2007 to 2012, the other potential affecting factors such as density of mobile phone base stations, number of mobile phone users, total mobile phone calling time, and number of text messages sent through mobile phones all increased in Taiwan. This finding indicated a positive association between media coverage and the prevalence of IEI-EMF in Taiwan, which might also be true in other countries.

Effects of Hinge-region Natural Polymorphisms on Human Immunodeficiency Virus-Type 1 Protease Structure, Dynamics, and Drug Pressure Evolution.

Multidrug resistance to current Food and Drug Administration-approved HIV-1 protease (PR) inhibitors drives the need to understand the fundamental mechanisms of how drug pressure-selected mutations, which are oftentimes natural polymorphisms, elicit their effect on enzyme function and resistance. Here, the impacts of the hinge-region natural polymorphism at residue 35, glutamate to aspartate (E35D), alone and in conjunction with residue 57, arginine to lysine (R57K), are characterized with the goal of understanding how altered salt bridge interactions between the hinge and flap regions are associated with changes in structure, motional dynamics, conformational sampling, kinetic parameters, and inhibitor affinity. The combined results reveal that the single E35D substitution leads to diminished salt bridge interactions between residues 35 and 57 and gives rise to the stabilization of open-like conformational states with overall increased backbone dynamics. In HIV-1 PR constructs where sites 35 and 57 are both mutated (e.g. E35D and R57K), x-ray structures reveal an altered network of interactions that replace the salt bridge thus stabilizing the structural integrity between the flap and hinge regions. Despite the altered conformational sampling and dynamics when the salt bridge is disrupted, enzyme kinetic parameters and inhibition constants are similar to those obtained for subtype B PR. Results demonstrate that these hinge-region natural polymorphisms, which may arise as drug pressure secondary mutations, alter protein dynamics and the conformational landscape, which are important thermodynamic parameters to consider for development of inhibitors that target for non-subtype B PR.

Crystal structure of the homocysteine methyltransferase MmuM from Escherichia coli.

Homocysteine S-methyltransferases (HMTs, EC 2.1.1.0) catalyse the conversion of homocysteine to methionine using S-methylmethionine or S-adenosylmethionine as the methyl donor. HMTs play an important role in methionine biosynthesis and are widely distributed among micro-organisms, plants and animals. Additionally, HMTs play a role in metabolite repair of S-adenosylmethionine by removing an inactive diastereomer from the pool. The mmuM gene product from Escherichia coli is an archetypal HMT family protein and contains a predicted zinc-binding motif in the enzyme active site. In the present study, we demonstrate X-ray structures for MmuM in oxidized, apo and metallated forms, representing the first such structures for any member of the HMT family. The structures reveal a metal/substrate-binding pocket distinct from those in related enzymes. The presented structure analysis and modelling of co-substrate interactions provide valuable insight into the function of MmuM in both methionine biosynthesis and cofactor repair.

Structure and functional analysis of the siderophore periplasmic binding protein from the fuscachelin gene cluster of Thermobifida fusca.

Iron acquisition is a complex, multicomponent process critical for most organisms' survival and virulence. Small iron chelating molecules, siderophores, mediate transport as key components of common pathways for iron assimilation in many microorganisms. The chemistry and biology of the extraordinary tight and specific metal binding siderophores is of general interest in terms of host/guest chemistry and is a potential target toward the development of therapeutic treatments for microbial virulence. The siderophore pathway of the moderate thermophile, Thermobifida fusca, is an excellent model system to study the process in Gram-positive bacteria. Here we describe the structure and characterization of the siderophore periplasmic binding protein, FscJ from the fuscachelin gene cluster of T. fusca. The structure shows a di-domain arrangement connected with a long α-helix hinge. Several X-ray structures detail ligand-free conformational changes at different pH values, illustrating complex interdomain flexibility of the siderophore receptors. We demonstrated that FscJ has a unique recognition mechanism and details the binding interaction with ferric-fuscachelin A through ITC and docking analysis. The presented work provides a structural basis for the complex molecular mechanisms of siderophore recognition and transportation.

Microbial siderophore-based iron assimilation and therapeutic applications.

Siderophores are structurally diverse, complex natural products that bind metals with extraordinary specificity and affinity. The acquisition of iron is critical for the survival and virulence of many pathogenic microbes and diverse strategies have evolved to synthesize, import and utilize iron. There has been a substantial increase of known siderophore scaffolds isolated and characterized in the past decade and the corresponding biosynthetic gene clusters have provided insight into the varied pathways involved in siderophore biosynthesis, delivery and utilization. Additionally, therapeutic applications of siderophores and related compounds are actively being developed. The study of biosynthetic pathways to natural siderophores augments the understanding of the complex mechanisms of bacterial iron acquisition, and enables a complimentary approach to address virulence through the interruption of siderophore biosynthesis or utilization by targeting the key enzymes to the siderophore pathways.

Structure and Mechanism of the Siderophore-Interacting Protein from the Fuscachelin Gene Cluster of Thermobifida fusca.

Microbial iron acquisition is a complex process and frequently a key and necessary step for survival. Among the several paths for iron assimilation, small molecule siderophore-mediated transport is a commonly employed strategy of many microorganisms. The chemistry and biology of the extraordinary tight and specific binding of siderophores to metal is also exploited in therapeutic treatments for microbial virulence and metal toxicity. The intracellular fate of iron acquired via the siderophore pathway is one of the least understood steps in the complex process at the molecular level. A common route to cellular incorporation is the single-electron reduction of ferric to ferrous iron catalyzed by specific and/or nonspecific reducing agents. The biosynthetic gene clusters for siderophores often contain representatives of one or two families of redox-active enzymes: the flavin-containing "siderophore-interacting protein" and iron-sulfur ferric siderophore reductases. Here we present the structure and characterization of the siderophore-interacting protein, FscN, from the fuscachelin siderophore gene cluster of Thermobifida fusca. The structure shows a flavoreductase fold with a noncovalently bound FAD cofactor along with an unexpected metal bound adjacent to the flavin site. We demonstrated that FscN is redox-active and measured the binding and reduction of ferric fuscachelin. This work provides a structural basis for the activity of a siderophore-interacting protein and further insight into the complex and important process of iron acquisition and utilization.