60 research outputs found
Structural glycobiology by NMR and molecular modelling: ligand recognition by the carbohydrate active proteins LYVE-1, SseK1/2 and PsLBP
The term carbohydrate-active protein (CAP) encompasses the group of proteins that either act as carbohydrate receptors (e.g. lectins) or enzymatically catalyse reactions involving carbohydrates as at least one of their substrates (e.g. glycosyltransferases). The fundamental importance of CAPs has only been realised over the past few decades, with carbohydrates playing a profound role in cellular signalling, adhesion and migration. Furthermore, carbohydrates are routinely used by pathogens in immune evasion and to modify host function, and changes in glycosylation patterns are associated with a range of disease states, including cancer and inflammation.
However, the study of CAP-carbohydrate interactions has been challenging, in part due to the inherently low affinity of many CAP-carbohydrate interactions that precluded detection by many techniques and make it difficult to obtained experimentally derived structures of their complexes. Fortunately, STD NMR spectroscopy is ideally suited to detecting weak interactions of this nature and provides structural information about the interaction through ligand epitope mapping. Furthermore, quantitative analysis of STD intensities allows three-dimensional models of the validated in the solution state, whether these models be derived from experiment or molecular modelling.
Within this thesis, a combination of STD NMR spectroscopy and molecular modelling has been used to unravel structural and dynamic detail of CAP-ligand interactions in three biologically or industrially relevant systems - (1) CD44/LYVE-1 which may play a role in cell trafficking across the lymphatics in cancer; (2) PsLBP which may lead to new developments in the field of enzymatic carbohydrate synthesis; (3) SseK1/2 which exhibit a novel enzymatic mechanism involving glycosylation of arginine residues and may be involved in Salmonella virulence
Deriving ligand orientation on weak protein-ligand complexes by DEEP-STD NMR in the absence of protein chemical shift assignment
Differential epitope mapping saturation transfer difference (DEEP-STD) NMR spectroscopy is a recently developed powerful approach for elucidating the structure and pharmacophore of weak proteinâligand interactions, as it reports key information on the orientation of the ligand and the architecture of the binding pocket. The method relies on selective saturation of protein residues in the binding site and the generation of a differential epitope map by observing the ligand, which depicts the nature of the protein residues making contact with the ligand in the bound state. Selective saturation requires knowledge of the chemical-shift assignment of the protein residues, which can be obtained either experimentally by NMR spectroscopy or predicted from 3D structures. Herein, we propose a simple experimental procedure to expand the DEEP-STD NMR methodology to proteinâligand cases in which the spectral assignment of the protein is not available. This is achieved by experimentally identifying the chemical shifts of the residues present in binding hot-spots on the surface of the receptor protein by using 2D NMR experiments combined with a paramagnetic probe
Bug Off Pain: An Educational Virtual Reality Game on Spider Venoms and Chronic Pain for Public Engagement
Virtual reality (VR) technology has been capturing the public imagination for decades. VR software applications that allow for interactive immersion are emerging as a renowned medium in many areas, including educating the public in biochemistry-related subjects via public engagement events. This report provides information about an immersive, interactive and educational virtual reality (VR) game named Bug Off Pain that increases scientific literacy about chronic pain and spider venoms among the public and high school students. Here, VR was shown to be an innovative and fun approach to learning and public engagement in biochemistry. Bug Off Pain places the viewer inside the brain and shows the molecular system that allows people to sense pain. After securing three (learning) points via the multimedia-based clips, this experience translates to the interactive game. Here, a player has to choose a venom that shuts down the pain until that results in âpain overâ. Bug Off Pain can be played (free of charge) on two different VR platforms: Oculus Rift and Android devices
Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7
P2X7 receptors are important in the regulation of inflammatory responses and immune responses to intracellular pathogens such as Mycobacterium tuberculosis and Toxoplasma gondii. Enhancement of P2X7 receptor responses may be useful in pathogen clearance particularly in individuals with defective microbial killing mechanisms. Ginsenosides from Panax ginseng have been discovered to act as positive allosteric modulators of P2X7. Here we describe a novel modulator binding site identified by computational docking located in the central vestibule of P2X7 involving S60, D318, and L320 in the lower body ÎČ-sheets lining the lateral portals. Potentiation of ATP-mediated responses by ginsenosides CK and Rd caused enhanced ionic currents, Ca2+ influx and YOPRO-1 uptake in stably transfected HEK-293 cells (HEK-hP2X7) plus enhanced cell death responses. Potentiation of ATP responses by CK and Rd was markedly reduced by mutations S59A, S60A, D318L and L320A supporting the proposed allosteric modulator binding site. Furthermore, mutation of the conserved residues S60 and D318 led to alterations in P2X7 response and a higher sensitivity to ATP in the absence of modulators suggesting residues in the connecting rods play an important role in regulating P2X7 gating. Identification of this novel binding site location in the central vestibule may also be relevant for structurally similar channels
Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YMâ1 towards Donor Substrates Glucose/Mannose 1âPhosphate by Using Xâray Crystallography and Saturation Transfer Difference NMR Spectroscopy
Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1âphosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YMâ1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 (ÎČâdâmannopyranosylâ(1â3)âdâglucopyranose) by using a natural acceptor glucose and noncognate donor substrate αâmannose 1âphosphate (Man1P). To investigate how the enzyme recognises different sugar 1âphosphates, the Xâray crystal structures of PsLBP in complex with Glc1P and Man1P have been solved, providing the first molecular detail of the recognition of a noncognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxy groups at C2, C4, and C6 of sugar 1âphosphates. Furthermore, we used saturation transfer difference NMR spectroscopy to support crystallographic studies on the sugar 1âphosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and disaccharide products
The intrinsically disordered Tarp protein from chlamydia binds actin with a partially preformed helix
Tarp (translocated actin recruiting phosphoprotein) is an effector protein common to all chlamydial species that functions to remodel the host-actin cytoskeleton during the initial stage of infection. In C. trachomatis, direct binding to actin monomers has been broadly mapped to a 100-residue region (726-825) which is predicted to be predominantly disordered, with the exception of a ~10-residue α helical patch homologous to other WH2 actin-binding motifs. Biophysical investigations demonstrate that a Tarp726-825 construct behaves as a typical intrinsically disordered protein; within it, NMR relaxation measurements and chemical shift analysis identify the ten residue WH2-homologous region to exhibit partial α-helix formation. Isothermal titration calorimetry experiments on the same construct in the presence of monomeric G-actin show a well defined binding event with a 1:1 stoichiometry and Kd of 102 nM, whilst synchrotron radiation circular dichroism spectroscopy suggests the binding is concomitant with an increase in helical secondary structure. Furthermore, NMR experiments in the presence of G-actin indicate this interaction affects the proposed WH2-like α-helical region, supporting results from in silico docking calculations which suggest that, when folded, this α helix binds within the actin hydrophobic cleft as seen for other actin-associated proteins
The human gut symbiont Ruminococcus gnavus shows specificity to blood group A antigen during mucin glycan foraging: Implication for niche colonisation in the gastrointestinal tract
AU The:human Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly gut symbiont Ruminococcus gnavus displays strain-specific : repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-ÎČ-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.Innovate UK Biocatalyst BB/M029042Institute Strategic Programme Gut Microbes and Health BB/R012490/1Biotechnology and Biological Sciences Research Council BB/ P010660/1Ministerio de Ciencia, InnovaciĂłn y Universidades PID2019-109395GB-I0European Union 814102Protein-Glycan Interaction Resource of the Consortium for Functional Glycomics R24 GM098791Harvard Medical School P41 GM1036
Ginsenosides act as positive modulators of P2X4 receptors
We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by âŒtwofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/ Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP 2 or ATP 1 ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain
Structural basis for the role of Serine-Rich Repeat Proteins from Lactobacillus reuteri in gut microbe-host interactions
Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens but no structural information is available in commensal bacteria. Here we report the 2.00 Ă
and 1.92 Ă
crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique âÎČ-solenoidâ fold in this important adhesin family. BRSRRP53608 boundto host epithelial cells and DNA at neutral pH and recognised polygalacturonic acid (PGA), rhamnogalacturonan I or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of BRSRRP53608 with PGA. Long molecular dynamics simulations showed that SRRP53608 undergoes a pH-dependent conformational change. Together, these findings shed new mechanistic insights into the role of SRRPs in host-microbe interactions and open new avenues of research into the use of biofilm-forming probiotics against clinically important pathogens
Fucosidases from the human gut symbiont Ruminococcus gnavus
The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3'-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers
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