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 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

ANTIOXIDANTS IN FOOD AND PHARMACEUTICAL RESEARCH

In this work we provide a general overview about the natural and synthetic antioxidants and studies carried out to improve their potential. These compounds are able to scavenge free radicals that cause deterioration of food and pharmaceutical products during processing and storage. Today there are several known natural compounds with antioxidant properties that are extracted from plants, which are mainly phenols and polyphenols. The limit for the use of these compounds in the food and pharmaceutical industry as antioxidants concerns their poor solubility in hydrophobic environment. This limitation was overcame in the past years by the introduction of synthetic antioxidants, such as BHA (butylated hidroxy anisole), BHT (butylated hydroxyl toluene), TBHQ (tert butyl hydroquinone) and PG (propyl gallate). Unfortunately several authors showed that BHA, BHT, TBHQ and PG may present adverse effects on the health of living organisms. In recent years novel antioxidants have been synthesized from natural polyphenols in order to modify the hydrophilicity/lipophilicity balance and increase their biological functionalities arising from insertion of new functional groups or molecule moieties in pre-existing natural polyphenols. The aim of these modifications was to avoid the major adverse effects associated with the use of BHT, BHA, TBHQ and PG

Spatially Resolved STD-NMR Applied to the Study of Solute Transport in Biphasic Systems. Application to Protein-Ligand Interactions

Fluid biphasic systems are one of the most interesting dynamic systems in chemistry and biochemistry. In NMR spectroscopy, the study of the solute dynamics across fluid biphasic systems requires the introduction of dedicated NMR methods, due to their intrinsic heterogeneity. Diffusion and spatially resolved NMR techniques represent a useful approach for dealing with the study of solutes in biphasic systems and have been applied lately with success. Nevertheless, other potential applications of NMR spectroscopy for biphasic systems remain to be explored. In this proof of concept communication, we specifically aimed to investigate whether solute exchange between two immiscible phases can be followed by NMR experiments involving transfer of magnetization. To that aim, we have used Spatially Resolved Saturation Transfer Difference NMR (SR-STD NMR) experiments to analyze solute exchange by transfer of saturation from one phase to the other in a biphasic system and have explored which are the underlying mechanisms leading to the transfer of magnetization between phases and the limits of the approach. We hereby demonstrate that SR-STD NMR is feasible and that it might be implemented in pharmacological screening for binders of biological receptors or in the study of chemical and biochemical reactions occurring at interfaces

CUBAN, a Case Study of Selective Binding:Structural Details of the Discrimination between Ubiquitin and NEDD8

The newly identified CUBAN (Cullin binding domain associating with NEDD8) domain recognizes both ubiquitin and the ubiquitin-like NEDD8. Despite the high similarity between the two molecules, CUBAN shows a clear preference for NEDD8, free and conjugated to cullins. We previously characterized the domain structure, both alone and in complex with NEDD8. The results here reported are addressed to investigate the determinants that drive the selective binding of CUBAN towards NEDD8 and ubiquitin. The 15N HSQC NMR perturbation pattern of the labeled CUBAN domain, when combined with either NEDD8 or ubiquitin, shows a clear involvement of hydrophobic residues that characterize the early stages of these interactions. After a slow conformational selection step, hydrophobic and then neutral and polar interactions take place, which drive the correct orientation of the CUBAN domain, leading to differences in the recognition scheme of NEDD8 and ubiquitin. As a result, a cascade of induced fit steps seems to determine the structural preference shown for NEDD8 and therefore the basis of the selectivity of the CUBAN domain. Finally, molecular dynamics analysis was performed to determine by fluctuations the internal flexibility of the CUBAN/NEDD8 complex. We consider that our results, based on a structural investigation mainly focused on the early stages of the recognition, provide a fruitful opportunity to report the different behavior of the same protein with two highly similar binding partners

Thymosin α1 Interacts with Hyaluronic Acid Electrostatically by Its Terminal Sequence LKEKK

Thymosin α1 (Tα1), is a peptidic hormone, whose immune regulatory properties have been demonstrated both in vitro and in vivo and approved in different countries for treatment of several viral infections and cancers. Tα1 assumes a conformation in negative membranes upon insertion into the phosphatidylserine exposure as found in several pathologies and in apoptosis. These findings are in agreement with the pleiotropy of Tα1, which targets both normal and tumor cells, interacting with multiple cellular components, and have generated renewed interest in the topic. Hyaluronan (HA) occurs ubiquitously in the extracellular matrix and on cell surfaces and has been related to a variety of diseases, and developmental and physiological processes. Proteins binding HA, among them CD44 and the Receptor for HA-mediated motility (RHAMM) receptors, mediate its biological effects. NMR spectroscopy indicated preliminarily that an interaction of Tα1 with HA occurs specifically around lysine residues of the sequence LKEKK of Tα1 and is suggestive of a possible interference of Tα1 in the binding of HA with CD44 and RHAMM. Further studies are needed to deepen these observations because Tα1 is known to potentiate the T-cell immunity and anti-tumor effect. The binding inhibitory activity of Tα1 on HA-CD44 or HA-RHAMM interactions can suppress both T-cell reactivity and tumor progression

Multifrequency STD NMR unveils the interactions of antibiotics with Burkholderia multivorans biofilm exopolysaccharide

Biofilms confine bacterial cells within self-produced matrices, offering advantages such as protection from antibiotics and entrapment of nutrients. Polysaccharides are major components in these macromolecular assemblies, and their interactions with other chemicals are of high relevance for the benefits provided by the biofilm 3D molecular matrix. NMR is a powerful technique for the study and characterization of the interactions between molecules of biological relevance. In this study, we have applied multifrequency saturation transfer difference (STD) NMR and DOSY NMR approaches to elucidate the interactions between the exopolysaccharide produced by Burkholderia multivorans C1576 (EpolC1576) and the antibiotics kanamycin and ceftadizime. The NMR strategies presented here allowed for an extensive characterization at an atomic level of the mechanisms behind the implication of the EpolC1576 in the recalcitrance phenomena, which is the ability of bacteria in biofilms to survive in the presence of antibiotics. Our results suggest an active role for EpolC1576 in the recalcitrance mechanisms toward kanamycin and ceftadizime, though through two different mechanisms

CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44, including residues Leu8, Ile44, His68, and Val70. A variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD specifically contacts ubiquitin. Here, we describe the structural model of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis, and nuclear magnetic resonance (NMR) spectroscopy of the 15N isotopically labeled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the coupling of ubiquitin conjugation to endoplasmic-reticulum (ER) degradation (CUE) domain family, where an invariant proline, usually following a phenylalanine, is required for ubiquitin binding. Interestingly, this novel UBD, which is not evolutionary related to CUE domains, shares a 40% identity and 47% similarity with cullin binding domain associating with NEDD8 (CUBAN), a protein module that also recognizes the ubiquitin-like NEDD8. Based on these features, we dubbed the region spanning the C-terminal 50 residues of N4BP1 the CoCUN domain, for Cousin of CUBAN. By performing circular dichroism and 15N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the CoCUN domain lacks the NEDD8 binding properties observed in CUBAN. We also show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both CUBAN and CoCUN are poly-ubiquitinated in cells. The structural and the functional characterization of this novel UBD can contribute to a deeper understanding of the molecular mechanisms governing N4BP1 function, providing at the same time a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved

CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44, including residues Leu8, Ile44, His68, and Val70. A variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD specifically contacts ubiquitin. Here, we describe the structural model of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis, and nuclear magnetic resonance (NMR) spectroscopy of the 15N isotopically labeled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the coupling of ubiquitin conjugation to endoplasmic-reticulum (ER) degradation (CUE) domain family, where an invariant proline, usually following a phenylalanine, is required for ubiquitin binding. Interestingly, this novel UBD, which is not evolutionary related to CUE domains, shares a 40% identity and 47% similarity with cullin binding domain associating with NEDD8 (CUBAN), a protein module that also recognizes the ubiquitin-like NEDD8. Based on these features, we dubbed the region spanning the C-terminal 50 residues of N4BP1 the CoCUN domain, for Cousin of CUBAN. By performing circular dichroism and 15N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the CoCUN domain lacks the NEDD8 binding properties observed in CUBAN. We also show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both CUBAN and CoCUN are poly-ubiquitinated in cells. The structural and the functional characterization of this novel UBD can contribute to a deeper understanding of the molecular mechanisms governing N4BP1 function, providing at the same time a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved

Structural basis of glycerophosphodiester recognition by the Mycobacterium tuberculosis substrate-binding protein UgpB

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and has evolved an incredible ability to survive latently within the human host for decades. The Mtb pathogen encodes for a low number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates that may reflect the nutrient poor environment within the host macrophages. Mtb UgpB (Rv2833) is the substrate binding domain of the UgpABCE transporter that recognises glycerophosphocholine (GPC), indicating that this transporter has a role in recycling glycerophospholipid metabolites. By using a combination of saturation transfer difference (STD) NMR and X-ray crystallography we report the structural analysis of Mtb UgpB complexed with GPC and have identified that Mtb UgpB does not only recognise GPC but that it is promiscuous for a broad range of glycerophosphodiesters. Complementary biochemical analyses and site-directed mutagenesis precisely define the molecular basis and specificity of glycerophosphodiester recognition. Our results provide critical insights into the structural and functional role of the Mtb UgpB transporter and reveal that the specificity of this ABC-transporter is not limited to GPC therefore optimising the ability of Mtb to scavenge scarce nutrients and essential glycerophospholipid metabolites via a single transporter during intracellular infection

Selectivity of the CUBAN domain in the recognition of ubiquitin and NEDD8

Among the members of the Ubiquitin‐like (Ubl) protein family, Neural precursor cell expressed developmentally down‐regulated protein 8 (NEDD8) is the closest in sequence to ubiquitin (57% identity). The two modification mechanisms and their functions, however, are highly distinct and the two Ubls are not interchangeable. A complex network of interactions between modifying enzymes and adaptors, most of which are specific while others are promiscuous, ensures selectivity. Many domains that bind the ubiquitin hydrophobic patch also bind NEDD8 while no domain that specifically binds NEDD8 has yet been described. Here we report an unbiased selection of domains that bind ubiquitin and/or NEDD8 and we characterize their specificity/promiscuity. Many ubiquitin binding domains bind ubiquitin preferentially and, to a lesser extent, NEDD8. In a few cases, the affinity of these domains for NEDD8 can be increased by substituting the alanine at position 72 with arginine, as in ubiquitin. We have also identified a unique domain, mapping to the carboxyl‐end of the protein KHNYN, which has a starkly preference for NEDD8. Given its ability to bind neddylated cullins we have named this domain CUBAN (Cullin Binding domain Associating with NEDD8). We present here the solution structure of the CUBAN domain both in the isolated form and in complex with NEDD8. The results contribute to the understanding of the discrimination mechanism between ubiquitin and the Ubl. They also provide new insights on the biological role of a ill‐defined protein, whose function is hitherto only predicted