Fast Quantitative Validation of 3D Models of Low-Affinity Protein-Ligand Complexes by STD NMR Spectroscopy.

Low-affinity protein-ligand interactions are important for many biological processes, including cell communication, signal transduction, and immune responses. Structural characterization of these complexes is also critical for the development of new drugs through fragment-based drug discovery (FBDD), but it is challenging due to the low affinity of fragments for the binding site. Saturation transfer difference (STD) NMR spectroscopy has revolutionized the study of low-affinity receptor-ligand interactions enabling binding detection and structural characterization. Comparison of relaxation and exchange matrix calculations with 1H STD NMR experimental data is essential for the validation of 3D structures of protein-ligand complexes. In this work, we present a new approach based on the calculation of a reduced relaxation matrix, in combination with funnel metadynamics MD simulations, that allows a very fast generation of experimentally STD-NMR-validated 3D structures of low-affinity protein-ligand complexes.

Speeding-up the Determination of Protein-Ligand Affinities by STD NMR: The Reduced Data Set STD NMR Approach (rd-STD NMR).

STD NMR spectroscopy is a powerful ligand-observed NMR tool for screening and characterizing the interactions of small molecules and low molecular weight fragments with a given macromolecule, identifying the main intermolecular contacts in the bound state. It is also a powerful analytical technique for the accurate determination of protein-ligand dissociation constants (KD) of medium-to-weak affinity, of interest in the pharmaceutical industry. However, accurate KD determination and epitope mapping requires a long series of experiments at increasing saturation times to carry out a full analysis using the so-called STD NMR build-up curve approach and apply the "initial slopes approximation". Here, we have developed a new protocol to bypass this important limitation, which allows us to obtain initial slopes by using just two saturation times and, hence, to very quickly determine precise protein-ligand dissociation constants by STD NMR.

Cross-reactivity of glycan-reactive HIV-1 broadly neutralizing antibodies with parasite glycans.

The HIV-1 Envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs). Env is heavily glycosylated with host-derived N-glycans, and many bnAbs bind to, or are dependent upon, Env glycans for neutralization. Although glycan-binding bnAbs are frequently detected in HIV-infected individuals, attempts to elicit them have been unsuccessful because of the poor immunogenicity of Env N-glycans. Here, we report cross-reactivity of glycan-binding bnAbs with self- and non-self N-glycans and glycoprotein antigens from different life-stages of Schistosoma mansoni. Using the IAVI Protocol C HIV infection cohort, we examine the relationship between S. mansoni seropositivity and development of bnAbs targeting glycan-dependent epitopes. We show that the unmutated common ancestor of the N332/V3-specific bnAb lineage PCDN76, isolated from an HIV-infected donor with S. mansoni seropositivity, binds to S. mansoni cercariae while lacking reactivity to gp120. Overall, these results present a strategy for elicitation of glycan-reactive bnAbs which could be exploited in HIV-1 vaccine development.

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.

The human 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-ß-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.

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.

Exploring Multi-Subsite Binding Pockets in Proteins: DEEP-STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin†B.

Ligand-based NMR techniques to study protein-ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi-frequency STD NMR approach, differential epitope mapping (DEEP)-STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so-called DEEP-STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP-STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi-subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.

Identification of selective protein-protein interaction inhibitors using efficient in silico peptide-directed ligand design.

The development of protein-protein interaction (PPI) inhibitors with therapeutic value is of increasing importance as the first clinical agent has now been approved, but PPIs remain difficult targets for the development of small molecule ligands. This article describes a highly efficient approach to the development of inhibitors of the p53/hDMX or hDM2 interaction that involves the design of small molecules in silico based upon a peptide/protein structure. The process for molecule design, starting from a virtual library of just over 1200 fragments, led to the eventual synthesis of twenty compounds, of which ten bound to either hDM2, hDMX or both in in vitro binding assays. This 50% success rate is extremely efficient compared to traditional high throughput screening. The identification of two selective hDMX inhibitors from twenty compounds highlights this efficiency as, to date, only two other hDMX-selective agents exist in the literature. Preliminary biological studies show that 20% of the compounds identified have cellular activity and activate downstream pathways associated with p53 activation.

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 ß-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.

STD NMR as a Technique for Ligand Screening and Structural Studies.

STD NMR is a powerful ligand-based tool for screening small molecules and low molecular weight fragments for their interaction with a given macromolecule. Such information is invaluable both in the drug discovery sector and in understanding fundamental biological interactions. Recently, powerful methods have been developed to extract a greater wealth of information from the STD NMR experiment, including ligand binding epitopes, dissociation constant determination, and mapping of binding site properties. Herein we describe these STD NMR experiments, giving practical examples for each approach, and highlight the important parameters and common pitfalls that must be considered for a successful experiment.

Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YM-1 towards Donor Substrates Glucose/Mannose 1-Phosphate by Using X-ray Crystallography and Saturation Transfer Difference NMR Spectroscopy.

Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1-phosphates. 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. YM-1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 (ß-d-mannopyranosyl-(1→3)-d-glucopyranose) by using a natural acceptor glucose and noncognate donor substrate α-mannose 1-phosphate (Man1P). To investigate how the enzyme recognises different sugar 1-phosphates, the X-ray 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 1-phosphates. Furthermore, we used saturation transfer difference NMR spectroscopy to support crystallographic studies on the sugar 1-phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and disaccharide products.

Deriving Ligand Orientation in Weak Protein-Ligand Complexes by DEEP-STD NMR Spectroscopy 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 protein-ligand 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 protein-ligand 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.

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 ∼twofold, 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- or ATP+ 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 arginine glycosylation of host substrates by bacterial effector proteins.

The bacterial effector proteins SseK and NleB glycosylate host proteins on arginine residues, leading to reduced NF-κB-dependent responses to infection. Salmonella SseK1 and SseK2 are E. coli NleB1 orthologs that behave as NleB1-like GTs, although they differ in protein substrate specificity. Here we report that these enzymes are retaining glycosyltransferases composed of a helix-loop-helix (HLH) domain, a lid domain, and a catalytic domain. A conserved HEN motif (His-Glu-Asn) in the active site is important for enzyme catalysis and bacterial virulence. We observe differences between SseK1 and SseK2 in interactions with substrates and identify substrate residues that are critical for enzyme recognition. Long Molecular Dynamics simulations suggest that the HLH domain determines substrate specificity and the lid-domain regulates the opening of the active site. Overall, our data suggest a front-face SNi mechanism, explain differences in activities among these effectors, and have implications for future drug development against enteric pathogens.

Discovery of Small Molecule WWP2 Ubiquitin Ligase Inhibitors.

We have screened small molecule libraries specifically for inhibitors that target WWP2, an E3 ubiquitin ligase associated with tumour outgrowth and spread. Selected hits demonstrated dose-dependent WWP2 inhibition, low micromolar IC50 values, and inhibition of PTEN substrate-specific ubiquitination. Binding to WWP2 was confirmed by ligand-based NMR spectroscopy. Furthermore, we used a combination of STD NMR, the recently developed DEEP-STD NMR approach, and docking calculations, to propose for the first time an NMR-validated 3D molecular model of a WWP2-inhibitor complex. These first generation WWP2 inhibitors provide a molecular framework for informing organic synthetic approaches to improve activity and selectivity.

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-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique ß-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized 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 SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host-microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.

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.