Protein NMR Studies of Substrate Binding to Human Blood Group†A and B Glycosyltransferases.

Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly 2 H,15 N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1 H,15 N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[13 C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic.

Thermodynamic signature of substrates and substrate analogs binding to human blood group B galactosyltransferase from isothermal titration calorimetry experiments.

It has been observed earlier that human blood group B galactosyltransferase (GTB) hydrolyzes its donor substrate UDP-Galactose (UDP-Gal) in the absence of acceptor substrate, and that this reaction is promoted by the presence of an acceptor substrate analog, α-L-Fuc-(1,2)-ß-D-3-deoxy-Gal-O-octyl (3DD). This acceleration of enzymatic hydrolysis of UDP-Gal was traced back to an increased affinity of GTB toward the donor substrate in the presence of 3DD. Herein, we present new thermodynamic data from isothermal titration calorimetry (ITC) on the binding of donor and acceptor substrates and analogs to GTB. ITC data are supplemented by surface plasmon resonance and STD-NMR titration experiments. These new data validate mutual allosteric control of binding of donor and acceptor substrates to GTB. It is of note that ITC experiments reveal significant differences in enthalpic and entropic contributions to binding of the natural donor substrate UDP-Gal, when compared with its analog UDP-Glucose (UDP-Glc). This may reflect different degrees of ordering of an internal loop (amino acids 176-194) and the C-terminus (amino acids 346-354), which close the binding pocket on binding of UDP-Gal or UDP-Glc. As both ligands have rather similar dissociation constants KD and almost identical modes of binding this finding is unexpected. Another surprising finding is that an acceptor analog, α-L-Fuc-(1,2)-ß-D-3-amino-3-deoxy-Gal-O-octyl (3AD) as well as the constituent monosaccharide ß-D-3-amino-3-deoxy-Gal-O-octyl (3AM) effectively inhibit enzymatic hydrolysis of UDP-Gal. This is unexpected, too, because in analogy to the effects of 3DD one would have predicted acceleration of enzymatic hydrolysis of UDP-Gal. It is difficult to explain these observations based on structural data alone. Therefore, our results highlight that there is an urgent need of experimental studies into the dynamic properties of GTB.

A post-translational modification of human Norovirus capsid protein attenuates glycan binding.

Attachment of human noroviruses to histo blood group antigens (HBGAs) is essential for infection, but how this binding event promotes the infection of host cells is unknown. Here, we employ protein NMR experiments supported by mass spectrometry and crystallography to study HBGA binding to the P-domain of a prevalent virus strain (GII.4). We report a highly selective transformation of asparagine 373, located in an antigenic loop adjoining the HBGA binding site, into an iso-aspartate residue. This spontaneous post-translational modification (PTM) proceeds with an estimated half-life of a few days at physiological temperatures, independent of the presence of HBGAs but dramatically affecting HBGA recognition. Sequence conservation and the surface-exposed position of this PTM suggest an important role in infection and immune recognition for many norovirus strains.

STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease.

West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3pro, we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3pro. Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC50 ∼ 500 µM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3pro. To identify the matching protein binding site, chemical shift perturbation studies employing 1H,15N-TROSY-HSQC experiments with uniformly 2H,15N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3pro inhibitors.

Galectin-3 and aldosterone as potential tandem biomarkers in pulmonary arterial hypertension.

BACKGROUND: Several studies have identified circulating biomarkers to be associated with the presence and severity of pulmonary arterial hypertension (PAH). Recent evidence supports a role for galectin-3 (Gal-3) and the mineralcorticoid aldosterone in left ventricular failure. However, studies on aldosterone together with Gal-3 in PAH are lacking. OBJECTIVE: We investigated a novel Aldosterone-galectin-3 (Gal-3) tandem and several other potential PAH biomarkers and their association with the disease severity. METHODS: A total of 57 patients, 41 with idiopathic PAH. (IPAH) and 16 with PAH associated with connective tissue disease (CTD), and 8 age-matched, non-relative controls were studied. Gal-3, aldosterone and other potential protein plasma concentrations were measured by single ELISA and multi-array MSD (Meso Scale Discovery) technology. RESULTS: Gal-3 values were increased in both patients with IPAH (12.2±0.6 ng/mL; p<0.05) and with PAH-CTD (14.1±1.6 ng/mL; p<0.05) versus control (8.5±0.9 ng/mL), while aldosterone was significantly elevated in IPAH only (248.5±38.8 pg/mL vs control 71.9±18.2 pg/mL; p<0.05). In addition, aldosterone, Gal-3, and N-terminal pro-brain natriuretic peptide (NT-proBNP) values were all higher in patients in WHO functional class II-III versus PAH functional class I or controls. The vascular injury marker intercellular adhesion molecule 1 (ICAM-1) was increased in IPAH and PAH-CTD versus controls (559.5±18.2 pg/mL and 734.1±59.4 pg/mL vs controls 394.8±39.3 pg/mL, p<0.05, p<0.0001, respectively), whereas vascular cell adhesion molecule 1 (VCAM-1) and proinflammatory, anti-angiogenic interleukin-12 (IL-12) were elevated in PAH-CTD only (879.5±110.0 pg/mL and 391.2±70.3 pg/mL vs controls 489.8±44.6 pg/mL, p<0.01, and 102.1±15.2 pg/mL, p<0.01, respectively). CONCLUSIONS: Heightened Gal-3 and aldosterone plasma concentrations in PAH patients indicate a role for Gal-3 signalling in the pathobiology of IPAH and PAH-CTD, and may serve as biomarkers for functional status and progression of disease.

Weaponization of a Hormone: Convergent Recruitment of Hyperglycemic Hormone into the Venom of Arthropod Predators.

Arthropod venoms consist primarily of peptide toxins that are injected into their prey with devastating consequences. Venom proteins are thought to be recruited from endogenous body proteins and mutated to yield neofunctionalized toxins with remarkable affinity for specific subtypes of ion channels and receptors. However, the evolutionary history of venom peptides remains poorly understood. Here we show that a neuropeptide hormone has been convergently recruited into the venom of spiders and centipedes and evolved into a highly stable toxin through divergent modification of the ancestral gene. High-resolution structures of representative hormone-derived toxins revealed they possess a unique structure and disulfide framework and that the key structural adaptation in weaponization of the ancestral hormone was loss of a C-terminal α helix, an adaptation that occurred independently in spiders and centipedes. Our results raise a new paradigm for toxin evolution and highlight the value of structural information in providing insight into protein evolution.

Basic fibroblast and epidermal growth factors stimulate survival in adult porcine photoreceptor cell cultures.

PURPOSE: To investigate the effects of basic fibroblast and epidermal growth factor (FGF2 and EGF, respectively) on the survival and phenotypic expression of photoreceptors isolated from adult mammalian retinas. METHODS: Primary cultures highly enriched in photoreceptors were prepared from adult domestic pig retinas and maintained in chemically defined medium. Cell culture composition was characterized through the use of specific antibody markers of retinal neurons, and neuronal survival was quantified through viability assays as a function of time in the presence or absence of different doses of FGF2 and EGF. Western blot analysis of phosphotyrosine residues was used to monitor activation of FGF2 and EGF signaling pathways. RESULTS: Reproducible survival of adult pig rod and cone photoreceptors was obtained for approximately 2 weeks in vitro, with the continued expression of rod opsin, recoverin, S-antigen, cone arrestin, and neuron-specific enolase. Purity of cultures was routinely more than 95% photoreceptors, with a rod-cone ratio of 2:3.1. Photoreceptor survival was stable for the initial week, decreasing slowly during the second, with rapid cell loss occurring thereafter. In the presence of FGF2 (20 ng/mL), the percentage of photoreceptor survival during the second week in culture was statistically significantly different, at least two times higher than in control experiments. Photoreceptor survival correlated directly with increasing concentrations of FGF2, and also of EGF. Combined treatment with FGF2 and EGF did not induce higher survival than either factor alone. There was no detectable selective loss of rods or cones in the experimental model. Phosphotyrosine immunoblots after stimulation of cultures with FGF2 and EGF revealed time-dependent appearance of multiple immunoreactive bands. CONCLUSIONS: The adult pig photoreceptor culture in the current study exhibits reproducible neuronal survival in vitro and represents a useful novel experimental system for the study of potential neuroprotective effects and signaling pathways of neurotrophic factors such as FGF2 and EGF in fully adult higher mammalian retina.