The structure of the second CysD domain of MUC2 and role in mucin organization by transglutaminase-based cross-linking.

The MUC2 mucin protects the colonic epithelium by a two-layered mucus with an inner attached bacteria-free layer and an outer layer harboring commensal bacteria. CysD domains are 100 amino-acid-long sequences containing 10 cysteines that separate highly O-glycosylated proline, threonine, serine (PTS) regions in mucins. The structure of the second CysD, CysD2, of MUC2 is now solved by nuclear magnetic resonance. CysD2 shows a stable stalk region predicted to be partly covered by adjacent O-glycans attached to neighboring PTS sequences, whereas the CysD2 tip with three flexible loops is suggested to be well exposed. It shows transient dimer interactions at acidic pH, weakened at physiological pH. This transient interaction can be stabilized in vitro and in vivo by transglutaminase 3-catalyzed isopeptide bonds, preferring a specific glutamine residue on one flexible loop. This covalent dimer is modeled suggesting that CysD domains act as connecting hubs for covalent stabilization of mucins to form a protective mucus.

Global Lrp regulator protein from Haloferax mediterranei: Transcriptional analysis and structural characterization.

Haloferax mediterranei, an extreme halophilic archaeon thriving in hypersaline environments, has acquired significant attention in biotechnological and biochemical research due to its remarkable ability to flourish in extreme salinity conditions. Transcription factors, essential in regulating diverse cellular processes, have become focal points in understanding its adaptability. This study delves into the role of the Lrp transcription factor, exploring its modulation of glnA, nasABC, and lrp gene promoters in vivo through ß-galactosidase assays. Remarkably, our findings propose Lrp as the pioneering transcriptional regulator of nitrogen metabolism identified in a haloarchaeon. This study suggests its potential role in activating or repressing assimilatory pathway enzymes (GlnA and NasA). The interaction between Lrp and these promoters is analyzed using Electrophoretic Mobility Shift Assay and Differential Scanning Fluorimetry, highlighting l-glutamine's indispensable role in stabilizing the Lrp-DNA complex. Our research uncovers that halophilic Lrp forms octameric structures in the presence of l-glutamine. The study reveals the three-dimensional structure of the Lrp as a homodimer using X-ray crystallography, confirming this state in solution by Small-Angle X-ray Scattering. These findings illuminate the complex molecular mechanisms driving Hfx. mediterranei's nitrogen metabolism, offering valuable insights about its gene expression regulation and enriching our comprehension of extremophile biology.

The Underrated Gut Microbiota Helminths, Bacteriophages, Fungi, and Archaea.

The microbiota inhabits the gastrointestinal tract, providing essential capacities to the host. The microbiota is a crucial factor in intestinal health and regulates intestinal physiology. However, microbiota disturbances, named dysbiosis, can disrupt intestinal homeostasis, leading to the development of diseases. Classically, the microbiota has been referred to as bacteria, though other organisms form this complex group, including viruses, archaea, and eukaryotes such as fungi and protozoa. This review aims to clarify the role of helminths, bacteriophages, fungi, and archaea in intestinal homeostasis and diseases, their interaction with bacteria, and their use as therapeutic targets in intestinal maladies.

Survivin prevents the polycomb repressor complex 2 from methylating histone 3 lysine 27.

This study investigates the role of survivin in epigenetic control of gene transcription through interaction with the polycomb repressive complex 2 (PRC2). PRC2 is responsible for silencing gene expression by trimethylating lysine 27 on histone 3. We observed differential expression of PRC2 subunits in CD4+ T cells with varying levels of survivin expression, and ChIP-seq results indicated that survivin colocalizes with PRC2 along DNA. Inhibition of survivin resulted in a significant increase in H3K27 trimethylation, implying that survivin prevents PRC2 from functioning. Peptide microarray showed that survivin interacts with peptides from PRC2 subunits, and machine learning revealed that amino acid composition contains relevant information for predicting survivin interaction. NMR and BLI experiments supported the interaction of survivin with PRC2 subunit EZH2. Finally, protein-protein docking revealed that the survivin-EZH2 interaction interface overlaps with catalytic residues of EZH2, potentially inhibiting its H3K27 methylation activity. These findings suggest that survivin inhibits PRC2 function.

The intestinal MUC2 mucin C-terminus is stabilized by an extra disulfide bond in comparison to von Willebrand factor and other gel-forming mucins.

The MUC2 mucin polymer is the main building unit of the intestinal mucus layers separating intestinal microbiota from the host epithelium. The MUC2 mucin is a large glycoprotein with a C-terminal domain similar to the MUC5AC and MUC5B mucins and the von Willebrand factor (VWF). A structural model of the C-terminal part of MUC2, MUC2-C, was generated by combining Cryo-electron microscopy, AlphaFold prediction, information of its glycosylation, and small angle X-ray scattering information. The globular VWD4 assembly in the N-terminal of MUC2-C is followed by 3.5 linear VWC domains that form an extended flexible structure before the C-terminal cystine-knot. All gel-forming mucins and VWF form tail-tail disulfide-bonded dimers in their C-terminal cystine-knot domain, but interestingly the MUC2 mucin has an extra stabilizing disulfide bond on the N-terminal side of the VWD4 domain, likely essential for a stable intestinal mucus barrier.

Survivin promotes a glycolytic switch in CD4+ T cells by suppressing the transcription of PFKFB3 in rheumatoid arthritis.

In this study, we explore the role of nuclear survivin in maintaining the effector phenotype of IFNγ-producing T cells acting through the transcriptional control of glucose utilization. High expression of survivin in CD4+T cells was associated with IFNγ-dependent phenotype and anaerobic glycolysis. Transcriptome of CD4+ cells and sequencing of survivin-bound chromatin showed that nuclear survivin had a genome-wide and motif-specific binding to regulatory regions of the genes controlling cell metabolism. Survivin coprecipitates with transcription factors IRF1 and SMAD3, which repressed the transcription of the metabolic check-point enzyme phosphofructokinase 2 gene PFKFB3 and promoted anaerobic glycolysis. Combining transcriptome analyses of CD4+ cells and functional studies in glucose metabolism, we demonstrated that the inhibition of survivin reverted PFKFB3 production, inhibited glucose uptake, and reduces interferon effects in CD4+ cells. These results present a survivin-dependent mechanism in coordinating the metabolic adaptation of CD4+T cells and propose an attractive strategy to counteract IFNγ-dependent inflammation in autoimmunity.

Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre.

Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25 Šresolution when exposed to XFEL radiation, which is an improvement of 0.15 Šover previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile QB pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand.

Cohesin-Mediated Chromatin Interactions and Autoimmunity.

Proper physiological functioning of any cell type requires ordered chromatin organization. In this context, cohesin complex performs important functions preventing premature separation of sister chromatids after DNA replication. In partnership with CCCTC-binding factor, it ensures insulator activity to organize enhancers and promoters within regulatory chromatin. Homozygous mutations and dysfunction of individual cohesin proteins are embryonically lethal in humans and mice, which limits in vivo research work to embryonic stem cells and progenitors. Conditional alleles of cohesin complex proteins have been generated to investigate their functional roles in greater detail at later developmental stages. Thus, genome regulation enabled by action of cohesin proteins is potentially crucial in lineage cell development, including immune homeostasis. In this review, we provide current knowledge on the role of cohesin complex in leukocyte maturation and adaptive immunity. Conditional knockout and shRNA-mediated inhibition of individual cohesin proteins in mice demonstrated their importance in haematopoiesis, adipogenesis and inflammation. Notably, these effects occur rather through changes in transcriptional gene regulation than through expected cell cycle defects. This positions cohesin at the crossroad of immune pathways including NF-kB, IL-6, and IFNγ signaling. Cohesin proteins emerged as vital regulators at early developmental stages of thymocytes and B cells and after antigen challenge. Human genome-wide association studies are remarkably concordant with these findings and present associations between cohesin and rheumatoid arthritis, multiple sclerosis and HLA-B27 related chronic inflammatory conditions. Furthermore, bioinformatic prediction based on protein-protein interactions reveal a tight connection between the cohesin complex and immune relevant processes supporting the notion that cohesin will unearth new clues in regulation of autoimmunity.

The IgGFc-binding protein FCGBP is secreted with all GDPH sequences cleaved but maintained by interfragment disulfide bonds.

Mucus forms an important protective barrier that minimizes bacterial contact with the colonic epithelium. Intestinal mucus is organized in a complex network with several specific proteins, including the mucin-2 (MUC2) and the abundant IgGFc-binding protein, FCGBP. FCGBP is expressed in all intestinal goblet cells and is secreted into the mucus. It is comprised of repeated von Willebrand D (vWD) domain assemblies, most of which have a GDPH amino acid sequence that can be autocatalytically cleaved, as previously observed in the mucins MUC2 and mucin-5AC. However, the functions of FCGBP in the mucus are not understood. We show that all vWD domains of FCGBP with a GDPH sequence are cleaved and that these cleavages occur early during biosynthesis in the endoplasmic reticulum. All cleaved fragments, however, remain connected via a disulfide bond within each vWD domain. This cleavage generates a C-terminal-reactive Asp-anhydride that could react with other molecules, such as MUC2, but this was not observed. Quantitative analyses by MS showed that FCGBP was mainly soluble in chaotropic solutions, whereas MUC2 was insoluble, and most of the secreted FCGBP was not covalently bound to MUC2. Although FCGBP has been suggested to bind immunoglobulin G, we were unable to reproduce this binding in vitro using purified proteins. In conclusion, while the function of FCGBP is still unknown, our results suggest that it does not contribute to covalent crosslinking in the mucus, nor incorporate immunoglobulin G into mucus, instead the single disulfide bond linking each fragment could mediate controlled dissociation.

Analysis of Haloferax mediterranei Lrp Transcriptional Regulator.

Haloferax mediterranei is an extremely halophilic archaeon, able to live in hypersaline environments with versatile nutritional requirements, whose study represents an excellent basis in the field of biotechnology. The transcriptional machinery in Archaea combines the eukaryotic basal apparatus and the bacterial regulation mechanisms. However, little is known about molecular mechanisms of gene expression regulation compared with Bacteria, particularly in Haloarchaea. The genome of Hfx. mediterranei contains a gene, lrp (HFX_RS01210), which encodes a transcriptional factor belonging to Lrp/AsnC family. It is located downstream of the glutamine synthetase gene (HFX_RS01205), an enzyme involved in ammonium assimilation and amino acid metabolism. To study this transcriptional factor more deeply, the lrp gene has been homologously overexpressed and purified under native conditions by two chromatographic steps, namely nickel affinity and gel filtration chromatography, showing that Lrp behaves asa tetrameric protein of approximately 67 kDa. Its promoter region has been characterized under different growth conditions using bgaH as a reporter gene. The amount of Lrp protein was also analyzed by Western blotting in different nitrogen sources and under various stress conditions. To sum up, regarding its involvement in the nitrogen cycle, it has been shown that its expression profile does not change in response to the nitrogen sources tested. Differences in its expression pattern have been observed under different stress conditions, such as in the presence of hydrogen peroxide or heavy metals. According to these results, the Lrp seems to be involved in a general response against stress factors, acting as a first-line transcriptional regulator.

High-resolution macromolecular crystallography at the FemtoMAX beamline with time-over-threshold photon detection.

Protein dynamics contribute to protein function on different time scales. Ultrafast X-ray diffraction snapshots can visualize the location and amplitude of atom displacements after perturbation. Since amplitudes of ultrafast motions are small, high-quality X-ray diffraction data is necessary for detection. Diffraction from bovine trypsin crystals using single femtosecond X-ray pulses was recorded at FemtoMAX, which is a versatile beamline of the MAX IV synchrotron. The time-over-threshold detection made it possible that single photons are distinguishable even under short-pulse low-repetition-rate conditions. The diffraction data quality from FemtoMAX beamline enables atomic resolution investigation of protein structures. This evaluation is based on the shape of the Wilson plot, cumulative intensity distribution compared with theoretical distribution, I/σ, Rmerge/Rmeas and CC1/2 statistics versus resolution. The FemtoMAX beamline provides an interesting alternative to X-ray free-electron lasers when studying reversible processes in protein crystals.

Clustering of atomic displacement parameters in bovine trypsin reveals a distributed lattice of atoms with shared chemical properties.

Low-frequency vibrations are crucial for protein structure and function, but only a few experimental techniques can shine light on them. The main challenge when addressing protein dynamics in the terahertz domain is the ubiquitous water that exhibit strong absorption. In this paper, we observe the protein atoms directly using X-ray crystallography in bovine trypsin at 100 K while irradiating the crystals with 0.5 THz radiation alternating on and off states. We observed that the anisotropy of atomic displacements increased upon terahertz irradiation. Atomic displacement similarities developed between chemically related atoms and between atoms of the catalytic machinery. This pattern likely arises from delocalized polar vibrational modes rather than delocalized elastic deformations or rigid-body displacements. The displacement correlation between these atoms were detected by a hierarchical clustering method, which can assist the analysis of other ultra-high resolution crystal structures. These experimental and analytical tools provide a detailed description of protein dynamics to complement the structural information from static diffraction experiments.

Bayesian machine learning improves single-wavelength anomalous diffraction phasing.

Single-wavelength X-ray anomalous diffraction (SAD) is a frequently employed technique to solve the phase problem in X-ray crystallography. The precision and accuracy of recovered anomalous differences are crucial for determining the correct phases. Continuous rotation (CR) and inverse-beam geometry (IBG) anomalous data collection methods have been performed on tetragonal lysozyme and monoclinic survivin crystals and analysis carried out of how correlated the pairs of Friedel's reflections are after scaling. A multivariate Bayesian model for estimating anomalous differences was tested, which takes into account the correlation between pairs of intensity observations and incorporates the a priori knowledge about the positivity of intensity. The CR and IBG data collection methods resulted in positive correlation between I(+) and I(-) observations, indicating that the anomalous difference dominates between these observations, rather than different levels of radiation damage. An alternative pairing method based on near simultaneously observed Bijvoet's pairs displayed lower correlation and it was unsuccessful for recovering useful anomalous differences when using the multivariate Bayesian model. In contrast, multivariate Bayesian treatment of Friedel's pairs improved the initial phasing of the two tested crystal systems and the two data collection methods.

A practical guide to developing virtual and augmented reality exercises for teaching structural biology.

Although virtual and augmented reality (VR and AR) techniques have been used extensively in specialized laboratories, only recently did they become affordable, reaching wider consumer markets. With increased availability, it is timely to examine the roles that VR and AR may play in teaching structural biology and in experiencing complex data sets such as macromolecular structures. This guide is suitable for those teachers of structural biology who do not have a deep knowledge of information technologies. This study focuses on three questions: 1) How can teachers of structural biology produce and disseminate VR/AR-ready educational material with established and user-friendly software tools?; 2) What are the positive and negative experiences reported by test participants when performing identical learning tasks in the VR and AR environments?; and 3) How do the test participants perceive prerecorded narration during VR/AR exploration? © 2018 International Union of Biochemistry and Molecular Biology, 47(1):16-24, 2018.

Bayesian Analysis of MicroScale Thermophoresis Data to Quantify Affinity of Protein:Protein Interactions with Human Survivin.

A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners. By applying Bayesian machine learning principles, binding curves can be autonomously analyzed without manual intervention and without introducing subjective bias by outlier rejection. We demonstrate the Bayesian inference protocol on the known survivin:borealin interaction and on the putative protein-protein interactions between human survivin and two members of the human Shugoshin-like family (hSgol1 and hSgol2). These interactions were identified in a protein microarray binding assay against survivin and confirmed by MicroScale Thermophoresis.

Estimating the difference between structure-factor amplitudes using multivariate Bayesian inference.

In experimental research referencing two or more measurements to one another is a powerful tool to reduce the effect of systematic errors between different sets of measurements. The interesting quantity is usually derived from two measurements on the same sample under different conditions. While an elaborate experimental design is essential for improving the estimate, the data analysis should also maximally exploit the covariance between the measurements. In X-ray crystallography the difference between structure-factor amplitudes carries important information to solve experimental phasing problems or to determine time-dependent structural changes in pump-probe experiments. Here a multivariate Bayesian method was used to analyse intensity measurement pairs to determine their underlying structure-factor amplitudes and their differences. The posterior distribution of the model parameter was approximated with a Markov chain Monte Carlo algorithm. The described merging method is shown to be especially advantageous when systematic and random errors result in recording negative intensity measurements.