Bacterial Polysaccharide Specificity of the Pattern Recognition Receptor Langerin Is Highly Species-dependent.

The recognition of pathogen surface polysaccharides by glycan-binding proteins is a cornerstone of innate host defense. Many members of the C-type lectin receptor family serve as pattern recognition receptors facilitating pathogen uptake, antigen processing, and immunomodulation. Despite the high evolutionary pressure in host-pathogen interactions, it is still widely assumed that genetic homology conveys similar specificities. Here, we investigate the ligand specificities of the human and murine forms of the myeloid C-type lectin receptor langerin for simple and complex ligands augmented by structural insight into murine langerin. Although the two homologs share the same three-dimensional structure and recognize simple ligands identically, a screening of more than 300 bacterial polysaccharides revealed highly diverging avidity and selectivity for larger and more complex glycans. Structural and evolutionary conservation analysis identified a highly variable surface adjacent to the canonic binding site, potentially forming a secondary site of interaction for large glycans.

Calcium-Independent Activation of an Allosteric Network in Langerin by Heparin Oligosaccharides.

The C-type lectin receptor Langerin is a glycan-binding protein that serves as an uptake receptor on Langerhans cells and is essential for the formation of Birbeck granules. Whereas most Langerin ligands are recognized by a canonical Ca2+ -dependent binding site, heparins have been proposed to make additional contacts to a secondary, Ca2+ -independent site. Glycan array screening and biomolecular NMR spectroscopy were employed to investigate the molecular mechanism of these interactions. We observed that binding of heparin hexasaccharides to a secondary site did not require the presence of Ca2+ and activated a previously identified intradomain allosteric network of Langerin (thus far only associated with Ca2+ affinity and release). We propose a communication hub between these two binding sites, which sheds new light on modulatory functions of Langerin-heparin interactions.

Identification of Multiple Druggable Secondary Sites by Fragment Screening against DC-SIGN.

DC-SIGN is a cell-surface receptor for several pathogenic threats, such as HIV, Ebola virus, or Mycobacterium tuberculosis. Multiple attempts to develop inhibitors of the underlying carbohydrate-protein interactions have been undertaken in the past fifteen years. Still, drug-like DC-SIGN ligands are sparse, which is most likely due to its hydrophilic, solvent-exposed carbohydrate-binding site. Herein, we report on a parallel fragment screening against DC-SIGN applying SPR and a reporter displacement assay, which complements previous screenings using 19 F NMR spectroscopy and chemical fragment microarrays. Hit validation by SPR and 1 H-15 N HSQC NMR spectroscopy revealed that although no fragment bound in the primary carbohydrate site, five secondary sites are available to harbor drug-like molecules. Building on key interactions of the reported fragment hits, these pockets will be targeted in future approaches to accelerate the development of DC-SIGN inhibitors.

Intradomain Allosteric Network Modulates Calcium Affinity of the C-Type Lectin Receptor Langerin.

Antigen uptake and processing by innate immune cells is crucial to initiate the immune response. Therein, the endocytic C-type lectin receptors serve as pattern recognition receptors, detecting pathogens by their glycan structures. Herein, we studied the carbohydrate recognition domain of Langerin, a C-type lectin receptor involved in the host defense against viruses such as HIV and influenza as well as bacteria and fungi. Using a combination of nuclear magnetic resonance and molecular dynamics simulations, we unraveled the molecular determinants underlying cargo capture and release encoded in the receptor architecture. Our findings revealed receptor dynamics over several time scales associated with binding and release of the essential cofactor Ca(2+) controlled by the coupled motions of two loops. Applying mutual information theory and site-directed mutagenesis, we identified an allosteric intradomain network that modulates the Ca(2+) affinity depending on the pH, thereby promoting fast ligand release.

Conformational properties of cardiolipin-bound cytochrome c.

Interactions of cytochrome c (cyt c) with cardiolipin (CL) are important for both electron transfer and apoptotic functions of this protein. A sluggish peroxidase in its native state, when bound to CL, cyt c catalyzes CL peroxidation, which contributes to the protein apoptotic release. The heterogeneous CL-bound cyt c ensemble is difficult to characterize with traditional structural methods and ensemble-averaged probes. We have employed time-resolved FRET measurements to evaluate structural properties of the CL-bound protein in four dansyl (Dns)-labeled variants of horse heart cyt c. The Dns decay curves and extracted Dns-to-heme distance distributions P(r) reveal a conformational diversity of the CL-bound cyt c ensemble with distinct populations of the polypeptide structures that vary in their degree of protein unfolding. A fraction of the ensemble is substantially unfolded, with Dns-to-heme distances resembling those in the guanidine hydrochloride-denatured state. These largely open cyt c structures likely dominate the peroxidase activity of the CL-bound cyt c ensemble. Site variations in P(r) distributions uncover structural features of the CL-bound cyt c, rationalize previous findings, and implicate the prime role of electrostatic interactions, particularly with the protein C terminus, in the CL-induced unfolding.

Redox Modulation of PTEN Phosphatase Activity by Hydrogen Peroxide and Bisperoxidovanadium Complexes.

PTEN is a dual-specificity protein tyrosine phosphatase. As one of the central tumor suppressors, a thorough regulation of its activity is essential for proper cellular homeostasis. The precise implications of PTEN inhibition by reactive oxygen species (e.g. H2 O2 ) and the subsequent structural consequences remain elusive. To study the effects of PTEN inhibition, bisperoxidovanadium (bpV) complexes serve as important tools with the potential for the treatment of nerve injury or cardiac ischemia. However, their mode of action is unknown, hampering further optimization and preventing therapeutic applications. Based on protein crystallography, mass spectrometry, and NMR spectroscopy, we elucidate the molecular basis of PTEN inhibition by H2O2 and bpV complexes. We show that both molecules inhibit PTEN via oxidative mechanisms resulting in the formation of the same intramolecular disulfide, therefore enabling the reactivation of PTEN under reductive conditions.

Superior fluorescent probe for detection of cardiolipin.

Cardiolipin (CL) is a unique phospholipid found in mitochondrial inner membrane. It is a key component for mitochondrial function in both respiration and apoptosis. The level of CL is an important parameter for investigating these intracellular events and is a critical indicator of a number of diseases associated with mitochondrial respiratory functions. 10-Nonyl acridine orange (NAO) is the only fluorescent dye currently available for CL detection. However, the performance of NAO is far from satisfactory in terms of selectivity and sensitivity. In this work, we report an aggregation-induced emission-active fluorogen, TTAPE-Me, for CL detection and quantification. With improved sensitivity and excellent selectivity to CL over other major mitochondrial membrane lipids, TTAPE-Me could serve as a valuable fluorescent sensor for CL quantification. The use of TTAPE-Me for the quantification of isolated mitochondria is also demonstrated.

Recombinant expression, biophysical characterization, and cardiolipin-induced changes of two Caenorhabditis elegans cytochrome c proteins.

Cytochrome c (cyt c) is one of the most widely studied biomolecules, but not much is known about this protein from nematodes. Recombinant expression of Caenorhabditis elegans CYC-2.1 and CYC-2.2 allowed for detailed characterization of their structural features, redox properties, stabilities, and interactions with cardiolipin (CL)-containing liposomes. Using a variety of spectroscopic tools, we show that CYC-2.1 and CYC-2.2 adopt a globular α-helical fold with His/Met heme ligation. The longer CYC-2.2 has a lower thermodynamic stability than CYC-2.1 and lacks His residues to misligate to the heme in the protein's denatured state. Both C. elegans proteins bind to CL-containing liposomes, and these interactions promote the proteins' peroxidase activity but to a much greater degree for CYC-2.2. Dye-to-heme distance distributions from time-resolved fluorescence resonance energy transfer in bimane-labeled CYC-2.1 and CYC-2.2 revealed similar populations of extended and compact conformers for CL-bound proteins, suggesting that their distinct peroxidase activities in the presence of CL arise from differences in the local heme environments for the two polypeptide ensembles. Without inhibition from His misligation, a less stable and more prone to unfolding CYC-2.2 allows for better access of substrates to the heme and thus exhibits higher peroxidase activity. Similar features of the conformational ensembles of CYC-2.1 and CYC-2.2 to those of mammalian cyt c suggest that C. elegans proteins, particularly the former, could serve as useful models for examining the mechanism of cyt c-CL interactions in live organisms.

The cryo-EM structure of a 12-subunit variant of RNA polymerase I reveals dissociation of the A49-A34.5 heterodimer and rearrangement of subunit A12.2.

RNA polymerase (Pol) I is a 14-subunit enzyme that solely transcribes pre-ribosomal RNA. Cryo-electron microscopy (EM) structures of Pol I initiation and elongation complexes have given first insights into the molecular mechanisms of Pol I transcription. Here, we present cryo-EM structures of yeast Pol I elongation complexes (ECs) bound to the nucleotide analog GMPCPP at 3.2 to 3.4 Å resolution that provide additional insight into the functional interplay between the Pol I-specific transcription-like factors A49-A34.5 and A12.2. Strikingly, most of the nucleotide-bound ECs lack the A49-A34.5 heterodimer and adopt a Pol II-like conformation, in which the A12.2 C-terminal domain is bound in a previously unobserved position at the A135 surface. Our structural and biochemical data suggest a mechanism where reversible binding of the A49-A34.5 heterodimer could contribute to the regulation of Pol I transcription initiation and elongation.

Langerhans Cells Sense Staphylococcus aureus Wall Teichoic Acid through Langerin To Induce Inflammatory Responses.

Staphylococcus aureus is a major cause of skin and soft tissue infections and aggravator of the inflammatory skin disease atopic dermatitis (AD [eczema]). Epicutaneous exposure to S. aureus induces Th17 responses through skin Langerhans cells (LCs), which paradoxically contribute to host defense but also to AD pathogenesis. The molecular mechanisms underlying the interaction between S. aureus and LCs are poorly understood. Here we demonstrate that human LCs directly interact with S. aureus through the pattern recognition receptor langerin (CD207). Human, but not mouse, langerin interacts with S. aureus through the conserved ß-N-acetylglucosamine (GlcNAc) modifications on wall teichoic acid (WTA), thereby discriminating S. aureus from other staphylococcal species. Importantly, the specific S. aureus WTA glycoprofile strongly influences the level of proinflammatory cytokines that are produced by in vitro-generated LCs. Finally, in a murine epicutaneous infection model, S. aureus strongly upregulated transcripts of Cxcl1, Il6, and Il17, which required the presence of both human langerin and WTA ß-GlcNAc. Our findings provide molecular insight into the unique proinflammatory capacities of S. aureus in relation to skin inflammation.IMPORTANCE The bacterium Staphylococcus aureus is an important cause of skin infections and is also associated with the occurrence and severity of eczema. Langerhans cells (LCs), a specific subset of skin immune cells, participate in the immune response to S. aureus, but it is yet unclear how LCs recognize S. aureus Therefore, we investigated the molecular mechanism underlying the interaction between LCs and S. aureus We identified that wall teichoic acid, an abundant polymer on the S. aureus surface, is recognized by langerin, a receptor unique to LCs. This interaction allows LCs to discriminate S. aureus from other related staphylococcal species and initiates a proinflammatory response similar to that observed in patients with eczema. Our data therefore provide important new insights into the relationship between S. aureus, LCs, and eczema.

The cryo-EM resolution revolution and transcription complexes.

Direct electron detector technology combined with improved imaging processing procedures has dramatically increased the resolution that can be obtained by single-particle cryo-electron microscopy and cryo-electron tomography. These developments-often referred to as the `resolution revolution' in cryo-EM-have had a profound impact on the structural biology of transcription as they allow the determination of atomic or near-atomic resolution structures of very large, flexible and often transient transcription complexes that in many cases had resisted crystal structure determination for decades. In this review, we will discuss recent advances and breakthroughs in the structural biology of transcription complexes enabled by the revolution in cryo-electron microscopy with particular focus on eukaryotic RNA polymerases and their pre-initiation complexes, but also chromatin remodelers and epigenetic regulators.

(19)F NMR-Guided Design of Glycomimetic Langerin Ligands.

C-type lectin receptors (CLRs) play a pivotal role in pathogen defense and immune homeostasis. Langerin, a CLR predominantly expressed on Langerhans cells, represents a potential target receptor for the development of anti-infectives or immunomodulatory therapies. As mammalian carbohydrate binding sites typically display high solvent exposure and hydrophilicity, the recognition of natural monosaccharide ligands is characterized by low affinities. Consequently, glycomimetic ligand design poses challenges that extend to the development of suitable assays. Here, we report the first application of (19)F R2-filtered NMR to address these challenges for a CLR, i.e., Langerin. The homogeneous, monovalent assay was essential to evaluating the in silico design of 2-deoxy-2-carboxamido-α-mannoside analogs and enabled the implementation of a fragment screening against the carbohydrate binding site. With the identification of both potent monosaccharide analogs and fragment hits, this study represents an important advancement toward the design of glycomimetic Langerin ligands and highlights the importance of assay development for other CLRs.

Multivalent display of minimal Clostridium difficile glycan epitopes mimics antigenic properties of larger glycans.

Synthetic cell-surface glycans are promising vaccine candidates against Clostridium difficile. The complexity of large, highly antigenic and immunogenic glycans is a synthetic challenge. Less complex antigens providing similar immune responses are desirable for vaccine development. Based on molecular-level glycan-antibody interaction analyses, we here demonstrate that the C. difficile surface polysaccharide-I (PS-I) can be resembled by multivalent display of minimal disaccharide epitopes on a synthetic scaffold that does not participate in binding. We show that antibody avidity as a measure of antigenicity increases by about five orders of magnitude when disaccharides are compared with constructs containing five disaccharides. The synthetic, pentavalent vaccine candidate containing a peptide T-cell epitope elicits weak but highly specific antibody responses to larger PS-I glycans in mice. This study highlights the potential of multivalently displaying small oligosaccharides to achieve antigenicity characteristic of larger glycans. The approach may result in more cost-efficient carbohydrate vaccines with reduced synthetic effort.

Computational and experimental prediction of human C-type lectin receptor druggability.

Mammalian C-type lectin receptors (CTLRS) are involved in many aspects of immune cell regulation such as pathogen recognition, clearance of apoptotic bodies, and lymphocyte homing. Despite a great interest in modulating CTLR recognition of carbohydrates, the number of specific molecular probes is limited. To this end, we predicted the druggability of a panel of 22 CTLRs using DoGSiteScorer. The computed druggability scores of most structures were low, characterizing this family as either challenging or even undruggable. To further explore these findings, we employed a fluorine-based nuclear magnetic resonance screening of fragment mixtures against DC-SIGN, a receptor of pharmacological interest. To our surprise, we found many fragment hits associated with the carbohydrate recognition site (hit rate = 13.5%). A surface plasmon resonance-based follow-up assay confirmed 18 of these fragments (47%) and equilibrium dissociation constants were determined. Encouraged by these findings we expanded our experimental druggability prediction to Langerin and MCL and found medium to high hit rates as well, being 15.7 and 10.0%, respectively. Our results highlight limitations of current in silico approaches to druggability assessment, in particular, with regard to carbohydrate-binding proteins. In sum, our data indicate that small molecule ligands for a larger panel of CTLRs can be developed.

Epitope recognition of antibodies against a Yersinia pestis lipopolysaccharide trisaccharide component.

Today, the process of selecting carbohydrate antigens as a basis for active vaccination and the generation of antibodies for therapeutic and diagnostic purposes is based on intuition combined with trial and error experiments. In efforts to establish a rational process for glycan epitope selection, we employed glycan array screening, surface plasmon resonance, and saturation transfer difference (STD)-NMR to elucidate the interactions between antibodies and glycans representing the Yersinia pestis lipopolysaccharide (LPS). A trisaccharide epitope of the LPS inner core glycan and different LPS-derived oligosaccharides from various Gram-negative bacteria were analyzed using this combination of techniques. The antibody-glycan interaction with a heptose substructure was determined at atomic-level detail. Antibodies specifically recognize the Y. pestis trisaccharide and some substructures with high affinity and specificity. No significant binding to LPS glycans from other bacteria was observed, which suggests that the epitopes for just one particular bacterial species can be identified. On the basis of these results we are beginning to understand the rules for structure-based design and selection of carbohydrate antigens.