Saturation Transfer Difference Research Articles

Studying the interaction of glycans with intact virions and virus-like particles by ligand-observed NMR spectroscopy.

Virus-glycan interactions play a crucial role in the infection process of many viruses. NMR spectroscopy has emerged as a powerful tool for studying these interactions at the molecular level. In this article, we review several published papers and reports that have highlighted the application of NMR spectroscopy in understanding the complex questions of how viruses engage with and bind to receptor glycans. The use of saturation transfer difference (STD) NMR spectroscopy has demonstrated itself as highly advantageous in investigating the interaction between glycans and intact virions or virus-like particles (VLPs). The broad NMR signal linewidth of virions and VLPs allows efficient saturation without affecting the glycan signals. The advantage of this approach is that the viral capsid environment in protein organization and function is not ignored and therefore provides a more biologically relevant model for exploring the interactions between the virus and the host cell glycans. We will review some examples of using NMR spectroscopy to study influenza cell tropism, rotaviruses, and noroviruses.

Design, synthesis and biological evaluation of novel 2,4-thiazolidinedione derivatives able to target the human BAG3 protein

The Bcl-2-associated athanogene 3 (BAG3) protein plays multiple roles in controlling cellular homeostasis, and it has been reported to be deregulated in many cancers, leading tumor cell apoptosis escape. BAG3 protein is then an emerging target for its oncogenic activities in both leukemia and solid cancers, such as medulloblastoma. In this work a series of forty-four compounds were designed and successfully synthesized by the modification and optimization of a previously reported 2,4-thiazolidinedione derivative 28. Using an efficient cloning and transfection in human embryonic kidney HEK-293T cells, BAG3 was collected and purified by chromatographic techniques such as IMAC and SEC, respectively. Subsequently, through Surface Plasmon Resonance (SPR) all the compounds were evaluated for their binding ability to BAG3, highlighting the compound FB49 as the one having the greatest affinity for the protein (Kd = 45 ± 6 μM) also against the reference compound 28. Further analysis carried out by Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) spectroscopy further confirmed the highest affinity of FB49 for the protein. In vitro biological investigation showed that compound FB49 is endowed with an antiproliferative activity in the micromolar range in three human tumoral cell lines and more importantly is devoid of toxicity in human peripheral mononuclear cell deriving from healthy donors. Moreover, FB49 was able to block cell cycle in G1 phase and to induce apoptosis as well as autophagy in medulloblastoma HD-MB03 treated cells. In addition, FB49 demonstrated a synergistic effect when combined with a chemotherapy cocktail of Vincristine, Etoposide, Cisplatin, Cyclophosphamide (VECC). In conclusion we have demonstrated that FB49 is a new derivative able to bind human BAG3 with high affinity and could be used as BAG3 modulator in cancers correlated with overexpression of this protein.

Tumor Carbohydrate Associated Antigen Analogs as Potential Binders for Siglec‐7

AbstractWe investigated two recently synthesized and characterized sialyl derivatives, bearing the Neu5Ac‐α‐(2‐6)‐Gal epitope, as promising binders for Siglec‐7, an inhibitory Siglec mainly found on natural killer cells. A variety of sialoglycan structures can be recognized by Siglec‐7 with implications in the modulation of immune responses. Notably, overexpression of sialylated glycans recognized by Siglec‐7 can be associated with the progression of several tumors, including melanoma and renal cell carcinoma. NOE‐based NMR techniques, including Saturation Transfer Difference and transferred‐NOESY NMR, together with molecular docking and dynamic simulations were combined to shed light on the molecular basis of Siglec‐7 recognition of two conformationally constrained Sialyl‐Tn antigen analogs. We, therefore, identify the ligands epitope mapping and their conformational features and propose 3D models accurately describing the protein‐ligand complexes. We found that the binding site of Siglec‐7 can accommodate both synthetic analogs, with the sialic acid mainly involved in the interaction. Moreover, the flexibility of Siglec‐7 loops allows a preferred accommodation of the more rigid compound bearing a biphenyl moiety at position 9 of the sialic acid that contributed to the interaction to a large extent. Our findings provided insights for developing potential novel high affinity ligands for Siglec‐7 to hinder tumor evasion.

Binding Affinity Studies of Nicotinamide N-methyltransferase and Ligands by Saturation Transfer Difference NMR.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide with S-adenosine-L-methionine (SAM) as the methyl donor. Abnormal expression of NNMT is associated with many diseases (such as multiple cancers and metabolic and liver diseases), making NNMT a potential therapeutic target. Limited studies concerning the enzymesubstrate/ inhibitor interactions could be found to fully understand the detailed reaction mechanism. The binding affinity and ligand binding epitopes of nicotinamide or SAH for binding NNMT and its mutants were determined using saturated transfer difference (STD) nuclear magnetic resonance (NMR) techniques combined with site-directed mutagenesis. The average dissociation constant of WT NNMT with nicotinamide and S-adenosine homocysteine (SAH) was 5.5 ± 0.9 mM and 1.2 ± 0.3 mM, respectively, while the mutants Y20F and Y20G with nicotinamide were up to nearly 4 times and 20 times that of WT and with SAH nearly 2 times and 5 times that of WT. The data suggested that WT had the highest binding affinity for nicotinamide or SAH, followed by Y20F and Y20G, which was consistent with its catalytic activity. The binding affinity of nicotinamide and SAH to NNMT and its mutants were obtained by STD NMR in this study. It was found that nicotinamide and SAH bind to WT in a particular orientation, and Y20 is critical for their binding orientation and affinity to NNMT.

Molecular insights on the binding of chlortetracycline to bovine casein and its effect on the thermostability of chlortetracycline

Bovine casein was selected as a model protein to evaluate the impact of food matrix on the thermal degradation of antibiotics. Fluorescence quenching and isothermal titration calorimetry experiments revealed that chlortetracycline (CTC) could spontaneously bind to casein via hydrogen bonding and hydrophobic interactions. The amino acid residues forming the binding pocket were further identified using molecular docking, while saturation transfer difference NMR deciphered that the binding of CTC engages its -N(CH3)2 group. Moreover, the degradation behavior of free CTC versus that bound in casein-CTC complex was compared during thermal treatment. Compared with free CTC, a lower first-order rate constant was observed in the presence of casein. Removal of casein shortened the half-life of CTC by at least 48.1% at low concentrations. Elucidating that the formation of protein-antibiotic complexes alters the amenability of antibiotics to degradative reactions, which could help eliminate residual antibiotics and guarantee the safety of dairy products.

Molecular Mechanisms Underlying Detection Sensitivity in Nanoparticle-Assisted NMR Chemosensing.

Nanoparticle-assisted nuclear magnetic resonance (NMR) chemosensing exploits monolayer-protected nanoparticles as supramolecular hosts to detect small molecules in complex mixtures via nuclear Overhauser effect experiments with detection limits down to the micromolar range. Still, the structure-sensitivity relationships at the basis of such detection limits are little understood. In this work, we integrate NMR spectroscopy and atomistic molecular dynamics simulations to examine the covariates that affect the sensitivity of different NMR chemosensing experiments [saturation transfer difference (STD), water STD, and high-power water-mediated STD]. Our results show that the intensity of the observed signals correlates with the number and duration of the spin-spin interactions between the analytes and the nanoparticles and/or between the analytes and the nanoparticles' solvation molecules. In turn, these parameters depend on the location and dynamics of each analyte inside the monolayer. This insight will eventually facilitate the tailoring of experimental and computational setups to the analyte's chemistry, making NMR chemosensing an even more effective technique in practical use.

Imaging Saturation Transfer Difference (STD) NMR: Affinity and Specificity of Protein-Ligand Interactions from a Single NMR Sample.

We have combined saturation transfer difference NMR (STD NMR) with chemical shift imaging (CSI) and controlled concentration gradients of small molecule ligands to develop imaging STD NMR, a new tool for the assessment of protein-ligand interactions. Our methodology allows the determination of protein-ligand dissociation constants (KD) and assessment of the binding specificity in a single NMR tube, avoiding time-consuming titrations. We demonstrate the formation of suitable and reproducible concentration gradients of ligand along the vertical axis of the tube, against homogeneous protein concentration, and present a CSI pulse sequence for the acquisition of STD NMR experiments at different positions along the sample tube. Compared to the conventional methodology in which the [ligand]/[protein] ratio is increased manually, we can perform STD NMR experiments at a greater number of ratios and construct binding epitopes in a fraction (∼20%) of the experimental time. Second, imaging STD NMR also allows us to screen for non-specific binders, by monitoring any variation of the binding epitope map at increasing [ligand]/[protein] ratios. Hence, the proposed method does carry the potential to speed up and smooth out the drug discovery process.

N-Substituted 3-Aminooxindoles and N-Propargyl Derivatives: Potential Biological Activities against Alzheimer’s Disease

The oxindole core is an important structural motif in many natural and synthetic substances with various biological activities including anticancer, antineurodegenerative, and antimicrobial properties. This report focuses on the synthesis and biological activity of a series of novel N-substituted 3-aminooxindoles and their assessment in cholinesterase (ChE) and monoamine oxidase (MAO) inhibition. With regard to MAO inhibition, a series of N-propargyl containing derivatives was synthesized and screened. Despite being weak inhibitors of MAO-A and MAO-B, the compounds were selective for butyrylcholinesterase (BuChE) over acetylcholinesterase (AChE). Most of them were strong inhibitors of BuChE with IC50's of less than 1 µM, and one compound showed an IC50 = 27 nM. The mechanism of action of the inhibition was pin-pointed through molecular modeling, and was validated using saturation-transfer-difference (STD) NMR. Some of the compounds were screened for anti-oxidant properties, but showed no activity. The same compounds were screened in the neurodegenerative disease model cell-line SH-SY5Y and although some were found to be non-cytotoxic, others were moderately cytotoxic. Continuous live cell imaging experiments showed that the compounds do not induce relevant cell damage and thus, the compounds might be interesting drug candidates for Alzheimer’s disease. Furthermore, the most active compounds showed excellent drug-likeness and pharmacological properties predicted using Swiss-ADME, and the pharmacokinetic simulations indicated that all these compounds cross the blood-brain-barrier.

Structural and Functional Analyses of the Acidic and Basic Amino Acid Repeat Sequence (DDRK) in Pif 80 from Pinctada fucata on the Aragonite Crystal Surface Using NMR

Shellfish and crustaceans use calcium carbonate (CaCO3) as a protective shell and exoskeleton. CaCO3 is a major biomineral component in invertebrates. The Pinctada fucata shell contains two crystal polymorphs, with outer prismatic and inner nacreous layers. Diverse shell organic materials play significant roles in microstructure formation with organic–inorganic interactions. Pif 80 has a repeated acidic and basic amino acid low-complexity region that specifically binds to aragonite crystals. In this study, we aimed to elucidate the microstructure formation mechanism by analyzing the interaction between the low-complexity region and CaCO3. We used solution NMR to evaluate the structure and activity of the Pif 80 acidic region. We designed synthesis methods for aragonite nanoparticles that can be dispersed and rotated in solution. The results revealed that the “GDDRKDDRKG” peptide has the appropriate structure for aragonite surface binding, wherein the four carboxy groups of the Asp side chain are in the same plane, supported by Arg and Lys residues. The saturation transfer difference also supported this structure. These results suggest that the Pif 80 acidic region binds the aragonite crystal surface via Asp side chains with basic residues, providing new insights into the function of acidic and basic amino acids in biomineral formation.

Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b

Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.

NMR proven albumin interaction with metabolites in complex mixtures

One critical step in new drugs development is the investigation of the interactions between drug candidate and target protein. Nuclear Magnetic Resonance Spectroscopy (NMR) is a well-established technique for studding these interactions. Due to its availability and structural similarities to human albumin, bovine serum albumin (BSA) is widely accepted as a model for investigating the binding of small molecules to serum albumin. We report here on the evaluation of binding interactions between BSA and 18 metabolites using saturation transfer difference (STD) NMR experiments. Positive STD signals that indicate metabolite-protein interactions were obtained for leucine, pyruvic acid, valine, threonine, alanine, 4-aminohippuric acid and tryptophan.

Molecular Basis for the Selectivity of DHA and EPA in Sudlow's Drug Binding Sites in Human Serum Albumin with the Combined Use of NMR and Docking Calculations.

Medium- and long-chain saturated and unsaturated free fatty acids (FFAs) are known to bind to human serum albumin (HSA), the main plasma carrier protein. Atomic-level structural data regarding the binding mode in Sudlow's sites I (FA7) and II (FA4, FA3) of the polyunsaturated ω-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), however, are largely unknown. Herein, we report the combined use of saturation transfer difference (STD) and Interligand NOEs for Pharmacophore Mapping (INPHARMA) NMR techniques and molecular docking calculations to investigate the binding mode of DHA and EPA in Sudlow's sites Ι and ΙΙ of HSA. The docking calculations and the significant number of interligand NOEs between DHA and EPA and the drugs warfarin and ibuprofen, which are stereotypical ligands for Sudlow's sites I and II, respectively, were interpreted in terms of competitive binding modes and the presence of two orientations of DHA and EPA at the binding sites FA7 and FA4. The exceptional flexibility of the long-chain DHA and EPA and the formation of strongly folded structural motives are the key properties of HSA-PUFA complexes.

Enhanced and suppressed phosphorus mineralization by Ca complexation: NMR and CD spectroscopy investigation

With the increasing demand for P fertilizer for world food production, the use of soil organic P fraction via mineralization could become an important P resource in agricultural soils. However, the predominant organic P species, phytic acid, has been considered rather recalcitrant to mineralization due to its active interaction with dissolved metals like Ca2+ in soil pore water. Calcium ions can be an inhibitor to many phytases, yet the mechanism was not clear. The objective of this study was to understand the effects of Ca2+(aq) on the phytase activity and inhibitory mechanisms using batch degradation kinetic experiments, Nuclear Magnetic Resonance (NMR) spectroscopy, Saturation Transfer Difference (STD) NMR, and Circular dichroism (CD) spectroscopy. The phytase activity followed Michaelis-Menten kinetics and increased Michaelis constant Km and decreased Vmax with Ca2+ addition were observed at pH 6. Therefore, mixed inhibition was the inhibition mechanism which was likely a result of the allosteric effect of Ca2+. The near-UV CD spectra supported phytase secondary conformational change upon the interaction between Ca2+ and the enzyme. It was found that phytase initially reacted with the D/L-3 phosphate of phytic acid at pH 6. At pH 8, the overall phytase activity decreased, yet the effect of Ca2+ on phytase activity was the opposite of that of pH 6. Enhanced phytase activity with Ca2+ addition was attributed to the structural change of phytic acid upon the Ca2+ complexation, which was confirmed by NOE spectra. The Ca2+-phytic acid complex might be a more favorable substrate than the free phytic acid. Unlike the findings from pH 6, Ca2+ didn't induce significant changes in either the near- or far-UV region of the CD spectra at pH 8. Furthermore, P5 was found to be the target of phytase at pH 8. The study revealed the pH-specific effects of Ca2+ on the mineralization of phytic acid

Elucidation of the binding behavior between tetracycline and bovine casein by multi-spectroscopic and molecular simulation methods

Bovine casein was used as a model protein to explore the binding behavior of food matrix with residual antibiotics. The interaction mechanism between casein and tetracycline (TC) was characterized by fluorescence quenching and isothermal titration calorimetry. Through forming the non-fluorescent ground-state complex, TC effectively quenched the endogenous fluorescence of casein. The formed casein-TC complex was maintained by hydrogen bonding, electrostatic and hydrophobic interactions (ΔH < 0, ΔS > 0). Circular dichroism and infrared spectroscopic studies indicated that the complexation of TC did not affect the secondary structure of casein. Molecular docking further theoretically validated the main interaction forces and deciphered the amino acid residues surrounding the antibiotic in the casein binding pocket. Interestingly, 1H NMR results demonstrated the π-π stacking interaction formed between PHE-189 and the benzene ring of TC, which was consistent with the docking findings. Besides, saturation transfer difference (STD) NMR revealed that the binding of TC engages its –N(CH3)2 group. This study sheds new light on the transition of residual antibiotics from the free to the bound state in foodstuffs.

Abstract 6359: Structural insights into Siglec-15 reveal glycosylation dependency for interaction with T cells through integrin CD11b

Abstract Sialic acid-binding Ig-like lectin 15 (Siglec-15) is emerging as an immune modulator and target for cancer immunotherapy. However, limited knowledge regarding the mechanism of action, structure and binding partners on T cells restrains the development of drug candidates that unleash its full therapeutic potential. Here, we solve the crystal structure of Siglec-15 and delineate its binding epitope by co-crystallization with an anti-Siglec-15 blocking antibody. Saturation transfer-difference (STD) nuclear magnetic resonance (NMR) spectroscopy assisted with molecular dynamic simulations revealed the binding mode of Siglec-15 to the cancer-associated sialyl-Tn (sTn) glycoform. Binding of Siglec-15 to T cells, which lack sTn expression, depends on the presence of sialic acid. We identified the leukocyte integrin CD11b as a binding partner of Siglec-15 on human T cells. Collectively, these data provide an integrative understanding of the structural features of Siglec-15 and underscore glycosylation as an additional layer of control of T cell responses. Citation Format: Maria Pia Lenza, Leire Egia-Mendikute, Asier Antoñana-Vildosola, Cátia O. Soares, Francisco Corzana, Iker Oyenarte, Filipa Marcelo, Jesús Jiménez-Barbero, June Ereño-Orbea, Asis Palazon. Structural insights into Siglec-15 reveal glycosylation dependency for interaction with T cells through integrin CD11b [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6359.

PH-Induced Biophysical Perspectives of Binding of Surface-Active Ionic Liquid [BMIM][OSU] with HSA and Dynamics of the Formed Complex.

The influence of pH on the human serum albumin (HSA) interaction with ionic liquid (IL)1-butyl 3-methylimidazolium octyl sulfate ([BMIM][OSU]) at its sub-micellar concentration of 5 mM (well below CMC ∼31 mM at 25 °C) in aqueous solution has been monitored employing different methods, viz., circular dichroism (CD), fluorescence, electrokinetic determination of the zeta potential (ZP), nuclear magnetic resonance (NMR), small-angle neutron scattering (SANS), and molecular docking (MD). CD analysis indicated a noticeable reduction of the α-helical content of HSA by IL at pH 3. A significant interaction of the anionic part of IL with HSA was evident from the 1H chemical shifts and saturation transfer difference (STD) NMR. A strong binding between IL and HSA was observed at pH 3 relative to pH 5, revealing the importance of electrostatic and hydrophobic interactions assessed from global binding affinities and molecular correlation times derived from STD NMR and a combined selective/nonselective spin-relaxation analysis, respectively. ZP data supported the electrostatic interaction between HSA and the anionic part of IL. The nature of IL self-diffusion with HSA was assessed from the translational self-diffusion coefficients by pulse field gradient NMR. SANS results revealed the formation of prolate ellipsoidal geometry of the IL-HSA complex. MD identified the preferential binding sites of IL to the tryptophan centers on HSA. The association of IL with HSA was supported by fluorescence measurements, in addition to the structural changes that occurred in the protein by the interaction with IL. The anionic part of IL contributed a major interaction with HSA at the pH levels of study (3, 5, 8, and 11.4); at pH > 8 (effectively 11.4), the protein also interacted weakly with the cationic component of IL.

Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) quantification of transient ischemia using a combination method of 5-pool Lorentzian fitting and inverse Z-spectrum analysis.

Chemical exchange saturation transfer (CEST) is a promising method for the detection of biochemical alterations in cancers and neurological diseases. However, the sensitivity of the currently existing quantitative method for detecting ischemia needs further improvement. To further improve the quantification of the CEST signal and enhance the CEST detection for ischemia, we used a quantitative analysis method that combines an inverse Z-spectrum analysis and a 5-pool Lorentzian fitting. Specifically, a 5-pool Lorentzian simulation was conducted with the following brain tissue parameters: water, amide (3.5 ppm), amine (2.2 ppm), magnetization transfer (MT), and nuclear Overhauser enhancement (NOE; -3.5 ppm). The parameters were first calculated offline and stored as the initial value of the Z-spectrum fitting. Then, the measured Z-spectrum with the peak value set to 0 was fitted via the stored initial value, which yielded the reference Z-spectrum. Finally, the difference between the inverse of the Z-spectrum and the inverse of the reference Z-spectrum was used as the CEST definite spectrum. The simulation results demonstrated that the Z-spectra of the rat brain were well simulated by a 5-pool Lorentzian fitting. Further, the proposed method detected a larger difference than did either the saturation transfer difference or the 5-pool Lorentzian fitting, as demonstrated by simulations. According to the results of the cerebral ischemia rat model, the proposed method provided the highest contrast-to-noise ratio (CNR) between the contralateral and the ipsilateral striatum under various acquisition conditions. The results indicated that the difference of fitted amplitudes generated with a 5-pool Lorentzian fitting in amide at 3.5 ppm (6.04%±0.39%; 6.86%±0.39%) was decreased in a stroke lesion compared to the contralateral normal tissue. Moreover, the difference of the residual of inversed Z-spectra in which 5-pool Lorentzian fitting was used to calculate the reference Z-spectra ( ) amplitudes in amide at 3.5 ppm (13.83%±2.20%, 15.69%±1.99%) was reduced in a stroke lesion compared to the contralateral normal tissue. is predominantly pH-sensitive and is suitable for detecting tissue acidosis following an acute stroke.

Phosphorylated Peptide Derived from the Myosin Phosphatase Target Subunit Is a Novel Inhibitor of Protein Phosphatase-1

Identification of specific protein phosphatase-1 (PP1) inhibitors is of special importance regarding the study of its cellular functions and may have therapeutic values in diseases coupled to signaling processes. In this study, we prove that a phosphorylated peptide of the inhibitory region of myosin phosphatase (MP) target subunit (MYPT1), R690QSRRS(pT696)QGVTL701 (P-Thr696-MYPT1690-701), interacts with and inhibits the PP1 catalytic subunit (PP1c, IC50 = 3.84 µM) and the MP holoenzyme (Flag-MYPT1-PP1c, IC50 = 3.84 µM). Saturation transfer difference NMR measurements established binding of hydrophobic and basic regions of P-Thr696-MYPT1690-701 to PP1c, suggesting interactions with the hydrophobic and acidic substrate binding grooves. P-Thr696-MYPT1690-701 was dephosphorylated by PP1c slowly (t1/2 = 81.6-87.9 min), which was further impeded (t1/2 = 103 min) in the presence of the phosphorylated 20 kDa myosin light chain (P-MLC20). In contrast, P-Thr696-MYPT1690-701 (10-500 µM) slowed down the dephosphorylation of P-MLC20 (t1/2 = 1.69 min) significantly (t1/2 = 2.49-10.06 min). These data are compatible with an unfair competition mechanism between the inhibitory phosphopeptide and the phosphosubstrate. Docking simulations of the PP1c-P-MYPT1690-701 complexes with phosphothreonine (PP1c-P-Thr696-MYPT1690-701) or phosphoserine (PP1c-P-Ser696-MYPT1690-701) suggested their distinct poses on the surface of PP1c. In addition, the arrangements and distances of the surrounding coordinating residues of PP1c around the phosphothreonine or phosphoserine at the active site were distinct, which may account for their different hydrolysis rate. It is presumed that P-Thr696-MYPT1690-701 binds tightly at the active center but the phosphoester hydrolysis is less preferable compared to P-Ser696-MYPT1690-701 or phosphoserine substrates. Moreover, the inhibitory phosphopeptide may serve as a template to synthesize cell permeable PP1-specific peptide inhibitors.

Identification of New L-Fucosyl and L-Galactosyl Amides as Glycomimetic Ligands of TNF Lectin Domain of BC2L-C from Burkholderia cenocepacia.

The inhibition of carbohydrate-lectin interactions is being explored as an efficient approach to anti adhesion therapy and biofilm destabilization, two alternative antimicrobial strategies that are being explored against resistant pathogens. BC2L-C is a new type of lectin from Burkholderia cenocepacia that binds (mammalian) fucosides at the N-terminal domain and (bacterial) mannosides at the C-terminal domain. This double carbohydrate specificity allows the lectin to crosslink host cells and bacterial cells. We have recently reported the design and generation of the first glycomimetic antagonists of BC2L-C, β-C- or β-N-fucosides that target the fucose-specific N-terminal domain (BC2L-C-Nt). The low water solubility of the designed N-fucosides prevented a full examination of this promising series of ligands. In this work, we describe the synthesis and biophysical evaluation of new L-fucosyl and L-galactosyl amides, designed to be water soluble and to interact with BC2L-C-Nt. The protein-ligand interaction was investigated by Saturation Transfer Difference NMR, Isothermal Titration Calorimetry and crystallographic studies. STD-NMR experiments showed that both fucosyl and galactosyl amides compete with α-methyl fucoside for lectin binding. A new hit compound was identified with good water solubility and an affinity for BC2L-C-Nt of 159 μM (ITC), which represents a one order of magnitude gain over α-methyl fucoside. The x-ray structure of its complex with BC2L-C-Nt was solved at 1.55 Å resolution.

Molecular Recognition Patterns between Vitamin B12 and Proteins Explored through STD-NMR and In Silico Studies.

Ligand-receptor molecular recognition is the basis of biological processes. The Saturation Transfer Difference-NMR (STD-NMR) technique has been recently used to gain qualitative and quantitative information about physiological interactions at an atomic resolution. The molecular recognition patterns between the cyanocobalamin (CNBL)/aqua cobalamin (OHBL) and different plant and animal proteins were investigated via STD-NMR supplemented by molecular docking. This study demonstrates that myoglobin has the highest binding affinity and that gluten has the lowest affinity. Casein also shows a higher binding affinity for cyanocobalamin when compared with that of plant-based proteins. STD-NMR results showed the moderate binding capability of casein with both CNBL and OHBL. Computer simulation confirmed the recognition mode in theory and was compared with the experiments. This work is beneficial for understanding the binding affinity and biological action of cyanocobalamin and will attract researchers to use NMR technology to link the chemical and physiological properties of nutrients.