GH18 endo-ß-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans.

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-ß-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.

Exploring the excited-state charge transfer fluorescence profile of 7-hydroxycoumarin and 2-methylimidazole - a combined X-ray diffraction and theoretical approach.

This study investigated the effect of 2-methylimidazole (2-MIM) addition on the fluorescence of ethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate using low-cost density functional theory (DFT) and Time-Dependent DFT calculations on single crystal X-ray geometries of ethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate hydrate (1), 2-MIM (2), and the 1 : 1 co-crystal of (1) and (2), (3). At low concentrations (1 : 1-1 : 10) of 2-MIM, the fluorophore shows a decrease in the fluorescence intensity, but at higher concentrations (above 1 : 10) the fluorescence excitation maximum shifted from 354 nm to 405 nm, with a significant emission intensity increase. The changed excitation and emission profile at high concentrations is due to the deprotonation of the coumarin's phenolic group, which was confirmed by the increased shielding of the aromatic protons in the titration 1H NMR spectra. The experimental fluorescence data between the 1 : 1 and 1 : 10 ratios agreed with the theoretical fluorescence data, with a redshift and decreased intensity when comparing (1) and (3). The data indicated that combining the fluorophore with 2-MIM increased levels of vibronic coupling between 2-MIM and the fluorophore decreasing de-excitation efficiency. These increased vibronic changes were due to charge transfer between the fluorophore and 2-MIM in (3). The subtle movement of the proton, H(5) toward N(2') (0.07 Å) caused a significant decrease in fluorescence due to electron density distribution (EDD) changes. This was identified by comparison of the EDD in the excited (S1) and ground (S0) states plotted as an isosurface of EDD difference. For the higher concentrations, an alternative excitation pathway was explored by modifying the crystal geometry of (3) based on 1H NMR spectroscopy data to resemble excitoplexes. Theses excitoplex geometries reflected the fluorescence profile of the fluorophore with high concentrations of 2-MIM; there were dramatic changes in the theoretical fluorescence pathway, which was 100% vibronic coupling compared to 15.31% in the free fluorophore. At this concentration, the de-excitation pathway causes remodelling of the lactone ring via stretching/breaking the CO bond in the S1 causing increased fluorescence by movement of the transition dipole moment. These results reflect previous studies, but the methods used are less experimentally and computationally expensive. This study is among the first to explain charge transfer fluorescence using crystalline geometries. This study will be of interest to the fields of crystal engineering and fluorescence spectroscopy.

Understanding Hygroscopicity of Theophylline via a Novel Cocrystal Polymorph: A Charge Density Study.

The charge density distribution in a novel cocrystal (1) complex of 1,3-dimethylxanthine (theophylline) and propanedioic acid (malonic acid) has been determined. The molecules crystallize in the triclinic, centrosymmetric space group P1̅, with four independent molecules (Z = 4) in the asymmetric unit (two molecules each of theophylline and malonic acid). Theophylline has a notably high hygroscopic nature, and numerous cocrystals have shown a significant improvement in stability to humidity. A charge density study of the novel polymorph has identified interesting theoretical results correlating the stability enhancement of theophylline via cocrystallization. Topological analysis of the electron density highlighted key differences (up to 17.8) in Laplacian (∇2ρ) between the experimental (EXP) and single-point (SP) models, mainly around intermolecular-bonded carbonyls. Further investigation via molecular electrostatic potential maps reaffirmed that the charge redistribution enhanced intramolecular hydrogen bonding, predominantly for N(2') and N(2) (61.2 and 61.8 kJ mol-1, respectively). An overall weaker lattice energy of the triclinic form (-126.1 kJ mol-1) compared to that of the monoclinic form (-133.8 kJ mol-1) suggests a lower energy threshold to overcome to initiate dissociation. Future work via physical testing of the novel cocrystal in both dissolution and solubility will further solidify the correlation between theoretical and experimental results.

Structural insights into the mechanisms and specificities of IgG-active endoglycosidases.

The conserved N-glycan on Asn297 of immunoglobulin G (IgG) has significant impacts on antibody effector functions, and is a frequent target for antibody engineering. Chemoenzymatic synthesis has emerged as a strategy for producing antibodies with homogenous glycosylation and improved effector functions. Central to this strategy is the use of enzymes with activity on the Asn297 glycan. EndoS and EndoS2, produced by Streptococcus pyogenes, are endoglycosidases with remarkable specificity for Asn297 glycosylation, making them ideal tools for chemoenzymatic synthesis. Although both enzymes are specific for IgG, EndoS2 recognizes a wider range of glycans than EndoS. Recent progress has been made in understanding the structural basis for their activities on antibodies. In this review, we examine the molecular mechanism of glycosidic bond cleavage by these enzymes and how specific point mutations convert them into glycosynthases. We also discuss the structural basis for differences in the glycan repertoire that IgG-active endoglycosidases recognize, which focuses on the structure of the loops within the glycoside hydrolase (GH) domain. Finally, we discuss the important contributions of carbohydrate binding modules (CBMs) to endoglycosidase activity, and how CBMs work in concert with GH domains to produce optimal activity on IgG.

Exploring the Binding of Barbital to a Synthetic Macrocyclic Receptor. A Charge Density Study.

Experimental charge density distribution studies, complemented by quantum mechanical theoretical calculations, of a host-guest system composed of a macrocycle (1) and barbital (2) in a 1:1 ratio (3) have been carried out via high-resolution single-crystal X-ray diffraction. The data were modeled using the conventional multipole model of electron density according to the Hansen-Coppens formalism. The asymmetric unit of macrocycle 1 contained an intraannular ethanol molecule and an extraannular acetonitrile molecule, and the asymmetric unit of 3 also contained an intraannular ethanol molecule. Visual comparison of the conformations of the macrocyclic ring shows the rotation by 180° of an amide bond attributed to competitive hydrogen bonding. It was found that the intraannular and extraannular molecules inside were orientated to maximize the number of hydrogen bonds present, with the presence of barbital in 3 resulting in the greatest stabilization. Hydrogen bonds ranging in strength from 4 to 70 kJ mol-1 were the main stabilizing force. Further analysis of the electrostatic potential among 1, 2, and 3 showed significant charge redistribution when cocrystallization occurred, which was further confirmed by a comparison of atomic charges. The findings presented herein introduce the possibility of high-resolution X-ray crystallography playing a more prominent role in the drug design process.

A comparison of the experimental and theoretical charge density distributions in two polymorphic modifications of piroxicam.

Experimental charge density distribution studies of two polymorphic forms of piroxicam, ß-piroxicam (1) and piroxicam monohydrate (2), were carried out via high-resolution single crystal X-ray diffraction experiments and multipole refinement. The asymmetric unit of (2) consists of two discrete piroxicam molecules, (2a) and (2b), and two water molecules. Geometry differs between (1) and (2) due to the zwitterionic nature of (2) which results in the rotation of the pyridine ring around the C(10)-N(2) bond by approximately 180°. Consequently, the pyridine and amide are no longer co-planar and (2) forms two exclusive, strong hydrogen bonds, H(3)O(4) and H(2)O(3), with bond energies of 66.14 kJ mol-1 and 112.82 kJ mol-1 for (2a), and 58.35 kJ mol-1 and 159.51 kJ mol-1 for (2b), respectively. Proton transfer between O(3) and N(3) in (2) results in significant differences in surface electrostatic potentials. This is clarified by the calculation of atomic charges in the zwitterion that shows the formally positive charge of the pyridyl nitrogen which is redistributed over the whole of the pyridine ring instead of concentrating at N-H. Similarly, the negative charge of the oxygen is distributed across the benzothiazine carboxamide moiety. The multipole derived lattice energy for (1) is -304 kJ mol-1 and that for (2) is -571 kJ mol-1, which is in agreement with the experimentally determined observations of higher solubility and dissolution rates of (1) compared to (2).

Experimental and theoretical charge density distribution in Pigment Yellow 101.

The charge density distribution in 2,2'-dihydroxy-1,1'-naphthalazine (Pigment Yellow 101; P.Y.101) has been determined using high-resolution X-ray diffraction and multipole refinement, along with density functional theory calculations. Topological analysis of the resulting densities highlights the localisation of single/double bonds in the central C=N-N=C moiety of the molecule in its ground state. The density in the N-N is examined in detail, where we show that very small differences between experiment and theory are amplified by use of the Laplacian of the density. Quantification of hydrogen bonds highlights the importance of the intramolecular N-H···O interaction, known to be vital for retention of fluorescence in the solid state, relative to the many but weak intermolecular contacts located. However, a popular method for deriving H-bond strengths from density data appears to struggle with the intramolecular N-H···O interaction. We also show that theoretical estimation of anisotropic displacements for hydrogen atoms brings little benefit overall, and degrades agreement with experiment for one intra-molecular contact.

Correction: Experimental and theoretical charge density distribution in Pigment Yellow 101.

Correction for 'Experimental and theoretical charge density distribution in Pigment Yellow 101' by Jonathan J. Du et al., Phys. Chem. Chem. Phys., 2015, DOI: 10.1039/c4cp04302b.

Combinatorially restricted computational design of protein-protein interfaces to produce IgG heterodimers.

Redesigning protein-protein interfaces is an important tool for developing therapeutic strategies. Interfaces can be redesigned by in silico screening, which allows for efficient sampling of a large protein space before experimental validation. However, computational costs limit the number of combinations that can be reasonably sampled. Here, we present combinatorial tyrosine (Y)/serine (S) selection (combYSelect), a computational approach combining in silico determination of the change in binding free energy (ΔΔG) of an interface with a highly restricted library composed of just two amino acids, tyrosine and serine. We used combYSelect to design two immunoglobulin G (IgG) heterodimers-combYSelect1 (L368S/D399Y-K409S/T411Y) and combYSelect2 (D399Y/K447S-K409S/T411Y)-that exhibit near-optimal heterodimerization, without affecting IgG stability or function. We solved the crystal structures of these heterodimers and found that dynamic π-stacking interactions and polar contacts drive preferential heterodimeric interactions. Finally, we demonstrated the utility of our combYSelect heterodimers by engineering both a bispecific antibody and a cytokine trap for two unique therapeutic applications.

Modulating antibody effector functions by Fc glycoengineering.

Antibody based drugs, including IgG monoclonal antibodies, are an expanding class of therapeutics widely employed to treat cancer, autoimmune and infectious diseases. IgG antibodies have a conserved N-glycosylation site at Asn297 that bears complex type N-glycans which, along with other less conserved N- and O-glycosylation sites, fine-tune effector functions, complement activation, and half-life of antibodies. Fucosylation, galactosylation, sialylation, bisection and mannosylation all generate glycoforms that interact in a specific manner with different cellular antibody receptors and are linked to a distinct functional profile. Antibodies, including those employed in clinical settings, are generated with a mixture of glycoforms attached to them, which has an impact on their efficacy, stability and effector functions. It is therefore of great interest to produce antibodies containing only tailored glycoforms with specific effects associated with them. To this end, several antibody engineering strategies have been developed, including the usage of engineered mammalian cell lines, in vitro and in vivo glycoengineering.

Mass Spectrometry-Based Methods to Determine the Substrate Specificities and Kinetics of N-Linked Glycan Hydrolysis by Endo-ß-N-Acetylglucosaminidases.

Glycosylation is a common posttranslational modification of proteins and refers to the covalent addition of glycans, chains of polysaccharides, onto proteins producing glycoproteins. The glycans influence the structure, function, and stability of proteins. They also play an integral role in the immune system, and aberrantly glycosylated proteins have wide ranging effects, including leading to diseases such as autoimmune conditions and cancer. Carbohydrate-active enzymes (CAZymes) are produced in bacteria, fungi, and humans and are enzymes which modify glycans via the addition or subtraction of individual or multiple saccharides from glycans. One of the hurdles in studying these enzymes is determining the types of substrates each enzyme is specific for and the kinetics of enzymatic activity. In this chapter, we discuss methods which are currently used to study the substrate specificity and kinetics of CAZymes and introduce a novel mass spectrometry-based technique which enables the specificity and kinetics of CAZymes to be determined accurately and efficiently.

Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases.

Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-ß-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications.

A Guide to In Silico Drug Design.

The drug discovery process is a rocky path that is full of challenges, with the result that very few candidates progress from hit compound to a commercially available product, often due to factors, such as poor binding affinity, off-target effects, or physicochemical properties, such as solubility or stability. This process is further complicated by high research and development costs and time requirements. It is thus important to optimise every step of the process in order to maximise the chances of success. As a result of the recent advancements in computer power and technology, computer-aided drug design (CADD) has become an integral part of modern drug discovery to guide and accelerate the process. In this review, we present an overview of the important CADD methods and applications, such as in silico structure prediction, refinement, modelling and target validation, that are commonly used in this area.

Sculpting therapeutic monoclonal antibody N-glycans using endoglycosidases.

Immunoglobulin G (IgG) monoclonal antibodies are a prominent and expanding class of therapeutics used for the treatment of diverse human disorders. The chemical composition of the N-glycan on the fragment crystallizable (Fc) region determines the effector functions through interaction with the Fc gamma receptors and complement proteins. The chemoenzymatic synthesis using endo-ß-N-acetylglucosaminidases (ENGases) emerged as a strategy to obtain antibodies with customized glycoforms that modulate their therapeutic activity. We discuss the molecular mechanism by which ENGases recognize different N-glycans and protein substrates, especially those that are specific for IgG antibodies, in order to rationalize the glycoengineering of immunotherapeutic antibodies, which increase the impact on the treatment of myriad diseases.

An experimental and theoretical charge density study of theophylline and malonic acid cocrystallization.

The pharmaceutical agent theophylline (THEO) is primarily used as a bronchodilator and is commercially available in both tablet and liquid dosage forms. THEO is highly hygroscopic, reducing its stability, overall shelf-life, and therefore usage as a drug. THEO and dicarboxylic acid cocrystals were designed by Trask et al. in an attempt to decrease the hygroscopic behaviour of THEO; cocrystallisation of THEO with malonic acid (MA) did not improve the hygroscopic stability of THEO in simulated atmospheric humidity testing. The current study employed high-resolution X-ray crystallography, and Density Functional Theory (DFT) calculations to examine the electron density distribution (EDD) changes between the cocrystal and its individual components. The EED changes identified the reasons why the THEO:MA cocrystal did not alter the hygroscopic profile of THEO. The cocrystal was equally porous, with atomic packing factors (APF) similar to those of THEO 0.73 vs. 0.71, respectively. The THEO:MA (1) cocrystal structure is held together by an array of interactions; a heterogeneous synthon between the imidazole and a carboxylic fragment stabilising the asymmetric unit, a pyrimidine-imidazole homosynthon, and an aromatic cycle stack between two THEO moieties have been identified, providing 9.7-12.9 kJ mol-1 of stability. These factors did not change the overall relative stability of the cocrystal relative to its individual THEO and MA components, as shown by cocrystal (1) and THEO being equally stable, with calculated lattice energies within 2.5 kJ mol-1 of one other. The hydrogen bond analysis and fragmented atomic charge analysis highlighted that the formation of (1) combined both the EDD of THEO and MA with no net chemical change, suggesting that the reverse reaction - (1) back to THEO and MA - is of equal potential, ultimately producing THEO hydrate formation, in agreement with the work of Trask et al. These results highlight that a review of the EDD change associated with a chemical reaction can aid in understanding cocrystal design. In addition, they indicate that cocrystal design requires further investigation before becoming a reliable process, with particular emphasis on identifying the appropriate balance of synthon engineering, weak interactions, and packing dynamics.

Mechanism of cooperative N-glycan processing by the multi-modular endoglycosidase EndoE.

Bacteria produce a remarkably diverse range of glycoside hydrolases to metabolize glycans from the environment as a primary source of nutrients, and to promote the colonization and infection of a host. Here we focus on EndoE, a multi-modular glycoside hydrolase secreted by Enterococcus faecalis, one of the leading causes of healthcare-associated infections. We provide X-ray crystal structures of EndoE, which show an architecture composed of four domains, including GH18 and GH20 glycoside hydrolases connected by two consecutive three α-helical bundles. We determine that the GH20 domain is an exo-ß-1,2-N-acetylglucosaminidase, whereas the GH18 domain is an endo-ß-1,4-N-acetylglucosaminidase that exclusively processes the central core of complex-type or high-mannose-type N-glycans. Both glycoside hydrolase domains act in a concerted manner to process diverse N-glycans on glycoproteins, including therapeutic IgG antibodies. EndoE combines two enzyme domains with distinct functions and glycan specificities to play a dual role in glycan metabolism and immune evasion.

The role of adjuvants in overcoming antibacterial resistance due to enzymatic drug modification.

Antibacterial resistance is a prominent issue with monotherapy often leading to treatment failure in serious infections. Many mechanisms can lead to antibacterial resistance including deactivation of antibacterial agents by bacterial enzymes. Enzymatic drug modification confers resistance to ß-lactams, aminoglycosides, chloramphenicol, macrolides, isoniazid, rifamycins, fosfomycin and lincosamides. Novel enzyme inhibitor adjuvants have been developed in an attempt to overcome resistance to these agents, only a few of which have so far reached the market. This review discusses the different enzymatic processes that lead to deactivation of antibacterial agents and provides an update on the current and potential enzyme inhibitors that may restore bacterial susceptibility.

Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides.

The human gut microbiota plays a central role not only in regulating the metabolism of nutrients but also promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. EndoBT-3987 is a key endo-ß-N-acetylglucosaminidase (ENGase) that initiates the degradation/processing of mammalian high-mannose-type (HM-type) N-glycans in the intestine. Here, we provide structural snapshots of EndoBT-3987, including the unliganded form, the EndoBT-3987-Man9GlcNAc2Asn substrate complex, and two EndoBT-3987-Man9GlcNAc and EndoBT-3987-Man5GlcNAc product complexes. In combination with alanine scanning mutagenesis and activity measurements we unveil the molecular mechanism of HM-type recognition and specificity for EndoBT-3987 and an important group of the GH18 ENGases, including EndoH, an enzyme extensively used in biotechnology, and for which the mechanism of substrate recognition was largely unknown.

Tricyclic heterocycles display diverse sensitivity to the A147T TSPO polymorphism.

The 18 kDa translocator protein (TSPO) is a target for the development of imaging agents to detect neuroinflammation. The clinical utility of second-generation TSPO ligands has been hindered by the presence of a polymorphism, rs6971, which causes a non-conservative substitution of alanine for threonine at amino acid residue 147 (TSPO A147T). Given the complex nature of TSPO binding, and the lack of non-discriminating high-affinity ligands at both wild type and A147T forms of TSPO, a series of novel TSPO ligands containing various heterocyclic scaffolds was developed to explore the pharmacophoric drivers of affinity loss at TSPO A147T. In general, N-benzyl-N-methyl-substituted amide ligands showed increased affinity at TSPO A147T, and a pyrazolopyrimidine acetamide containing this motif displayed low nanomolar binding affinities to both TSPO forms.