Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanism by which this class of enzymes facilitates Ub transfer remains enigmatic. Here, we ...present a structural model for a RING/U-box E3:E2∼Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2∼Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 side chain and an E2 backbone carbonyl, observed in all structures of active RING/U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2∼Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/U-box E3:E2∼Ub ligase complex and identify the long sought-after source of allostery for RING/U-Box activation of E2∼Ub conjugates.
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► Binding to RING/U-box E3s shifts UbcH5c∼Ub toward closed conformations ► Closed conformations are linked to E3 enhancement of E2∼Ub reactivity ► Induction of closed E2∼Ub conformations is a general mechanism of RING/U-box E3s ► Conserved E3:E2 hydrogen bond is required for allosteric activation of Ub transfer
The S100 family of EF-hand calcium (Ca2+)-binding proteins is essential for a wide range of cellular functions. During infection, certain S100 proteins act as damage-associated molecular patterns ...(DAMPs) and interact with pattern recognition receptors to modulate inflammatory responses. In addition, these inflammatory S100 proteins have potent antimicrobial properties and are essential components of the immune response to invading pathogens. In this review, we focus on S100 proteins that exhibit antimicrobial properties through the process of metal limitation, termed nutritional immunity, and discuss several recent advances in our understanding of S100 protein-mediated metal sequestration at the site of infection.
The EF hand, a helix−loop−helix structure, is one of the most common motifs found in animal genomes, and EF-hand Ca2+-binding proteins (EFCaBPs) are widely distributed throughout the cell. However, ...researchers remain confounded by a lack of understanding of how peptide sequences code for specific functions and by uncertainty about the molecular mechanisms that enable EFCaBPs to distinguish among many diverse cellular targets. Such knowledge could define the roles of EFCaBPs in health and disease and ultimately enable control or even design of Ca2+-dependent functions in medicine and biotechnology. In this Account, we describe our structural and biochemical research designed to understand the sequence-to-function relationship in EFCaBPs. The first structural goal was to define conformational changes induced by binding Ca2+, and our group and others established that solution NMR spectroscopy is well suited for this task. We pinpointed residues critical to the differences in Ca2+ response of calbindin D9k and calmodulin (CaM), homologous EFCaBPs from different functional classes, by using direct structure determination with site-directed mutagenesis and protein engineering. Structure combined with biochemistry provided the foundation for identifying the fundamental mechanism of cooperativity in the binding of Ca2+ ions: this cooperativity provides EFCaBPs with the ability to detect the relatively small changes in concentration that constitute Ca2+ signals. Using calbindin D9k as a model system, studies of the structure and fast time scale dynamics of each of the four ion binding states in a typical EF-hand domain provided direct evidence that site−site communication lowers the free energy cost of reorganization for binding the second ion. Our work has also extended models of how EFCaBPs interact with their cellular targets. We determined the unique dimeric architecture of S100 proteins, a specialized subfamily of EFCaBPs found exclusively in vertebrates. We described the implications for how these proteins transduce signals and went on to characterize interactions with peptide fragments of important cellular targets. Studies of the CaM homolog centrin revealed novel characteristics of its binding of Ca2+ and its interaction with its cellular target Kar1. These results provided clear examples of how subtle differences in sequence fine-tune EFCaBPs to interact with their specific targets. The structural approach stands at a critical crossroad, shifting in emphasis from descriptive structural biochemistry to integrated biology and medicine. We present our dual-molecular-switch model for Ca2+ regulation of gating functions of voltage-gated sodium channels in which both CaM and an intrinsic EF-hand domain serve as coupled Ca2+ sensors. A second example involves novel EFCaBP extracellular function, that is, the role of S100A8/S100A9 heterodimer in the innate immune response to bacterial pathogens. A mechanism for the antimicrobial activity of S100A8/S100A9 was discovered. We describe interactions of S100A8/S100A9 and S100B with the cell surface receptor for advanced glycation end products. Biochemical and structural studies are now uncovering the mechanisms by which EFCaBPs work and are helping to define their biological activities, while simultaneously expanding knowledge of the roles of these proteins in normal cellular physiology and the pathology of disease.
RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis but also generate ...hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks, where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.
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•RADX (CXorf57) is a single-strand DNA binding protein related to RPA•RADX prevents MUS81-dependent replication fork collapse•RADX antagonizes RAD51 to prevent excessive replication fork remodeling•RADX deletion restores fork protection and chemoresistance to BRCA2-deficient cells
Dungrawala et al. identify RADX as an RPA-like single-strand DNA binding protein enriched at replication forks. RADX antagonizes RAD51 accumulation at forks, thereby preventing aberrant fork remodeling. RADX deletion confers fork protection and chemoresistance to BRCA2 mutant cells without affecting homologous recombination repair of double-strand breaks.
By sequestering manganese and zinc, the neutrophil protein calprotectin plays a crucial role in host defense against bacterial and fungal pathogens. However, the essential processes disrupted by ...calprotectin remain unknown. We report that calprotectin enhances the sensitivity of Staphylococcus aureus to superoxide through inhibition of manganese-dependent bacterial superoxide defenses, thereby increasing superoxide levels within the bacterial cell. Superoxide dismutase activity is required for full virulence in a systemic model of S. aureus infection, and disruption of staphylococcal superoxide defenses by calprotectin augments the antimicrobial activity of neutrophils promoting in vivo clearance. Calprotectin mutated in two transition metal binding sites and therefore defective in binding manganese and zinc does not inhibit microbial growth, unequivocally linking the antimicrobial properties of calprotectin to metal chelation. These results suggest that calprotectin contributes to host defense by rendering bacterial pathogens more sensitive to host immune effectors and reducing bacterial growth.
The ATR checkpoint kinase coordinates cellular responses to DNA replication stress. Budding yeast contain three activators of Mec1 (the ATR orthologue); however, only TOPBP1 is known to activate ATR ...in vertebrates. We identified ETAA1 as a replication stress response protein in two proteomic screens. ETAA1-deficient cells accumulate double-strand breaks, sister chromatid exchanges, and other hallmarks of genome instability. They are also hypersensitive to replication stress and have increased frequencies of replication fork collapse. ETAA1 contains two RPA-interaction motifs that localize ETAA1 to stalled replication forks. It also interacts with several DNA damage response proteins including the BLM/TOP3α/RMI1/RMI2 and ATR/ATRIP complexes. It binds ATR/ATRIP directly using a motif with sequence similarity to the TOPBP1 ATR-activation domain; and like TOPBP1, ETAA1 acts as a direct ATR activator. ETAA1 functions in parallel to the TOPBP1/RAD9/HUS1/RAD1 pathway to regulate ATR and maintain genome stability. Thus, vertebrate cells contain at least two ATR-activating proteins.
The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous ...recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes.
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•DSS1 works in conjunction with BRCA2 to facilitate RPA-RAD51 exchange on ssDNA•DSS1 interacts with RPA via its solvent-exposed acidic loop•DSS1 binds the RPA70 subunit of RPA and attenuates ssDNA binding by it•DSS1 could also function as a nucleic acid mimic in other biological processes
How RPA is displaced from DNA to enable RAD51 recombinase loading in homologous recombination (HR) is not known. Zhao et al. show that DSS1 targets RPA and mimics DNA to facilitate RPA displacement. This identifies DSS1 as an essential component of a HR mediator complex with the tumor suppressor BRCA2.
Calprotectin, the most abundant cytoplasmic protein in neutrophils, suppresses the growth of Staphylococcus aureus by sequestering the nutrient metal ions Zn and Mn. Here we show that calprotectin ...can also enhance the activity of the SaeRS two component system (TCS), a signaling system essential for production of over 20 virulence factors in S. aureus. The activity of the SaeRS TCS is repressed by certain divalent ions found in blood or neutrophil granules; however, the Zn bound-form of calprotectin relieves this repression. During staphylococcal encounter with murine neutrophils or staphylococcal infection of the murine peritoneal cavity, calprotectin increases the activity of the SaeRS TCS as well as the production of proinflammatory cytokines such as IL-1β and TNF-α, resulting in higher murine mortality. These results suggest that, under certain conditions, calprotectin can be exploited by S. aureus to increase bacterial virulence and host mortality.
The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosis-inducing activity against various cells, especially tumor cells. Here, we present evidence ...that S100A8/A9 also has cell growth-promoting activity at low concentrations. Receptor of advanced glycation end product (RAGE) gene silencing and cotreatment with a RAGE-specific blocking antibody revealed that this activity was mediated via RAGE ligation. To investigate the signaling pathways, MAPK phosphorylation and NF-kappaB activation were characterized in S100A8/A9-treated cells. S100A8/A9 caused a significant increase in p38 MAPK and p44/42 kinase phosphorylation, and the status of stress-activated protein kinase/JNK phosphorylation remained unchanged. Treatment of cells with S100A8/A9 also enhanced NF-kappaB activation. RAGE small interfering RNA pretreatment abrogated the S100A8/A9-induced NF-kappaB activation. Our data indicate that S100A8/A9-promoted cell growth occurs through RAGE signaling and activation of NF-kappaB.
Neutrophils are innate immune cells that counter pathogens by many mechanisms, including release of antimicrobial proteins such as calprotectin to inhibit bacterial growth. Calprotectin sequesters ...essential micronutrient metals such as zinc, thereby limiting their availability to microbes, a process termed nutritional immunity. We find that while calprotectin is induced by neutrophils during infection with the gut pathogen Salmonella Typhimurium, calprotectin-mediated metal sequestration does not inhibit S. Typhimurium proliferation. Remarkably, S. Typhimurium overcomes calprotectin-mediated zinc chelation by expressing a high affinity zinc transporter (ZnuABC). A S. Typhimurium znuA mutant impaired for growth in the inflamed gut was rescued in the absence of calprotectin. ZnuABC was also required to promote the growth of S. Typhimurium over that of competing commensal bacteria. Thus, our findings indicate that Salmonella thrives in the inflamed gut by overcoming the zinc sequestration of calprotectin and highlight the importance of zinc acquisition in bacterial intestinal colonization.
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► The ZnuABC transporter mediates resistance to calprotectin ► The ZnuABC transporter enhances zinc acquisition by Salmonella in the inflamed gut ► Zinc promotes the growth of Salmonella over competing microbes ► Salmonella overcomes zinc sequestration by calprotectin to colonize the gut
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