The Nature and Sequence of the Amino Acid Aglycone Strongly Modulates the Conformation and Dynamics Effects of Tn Antigen's Clusters

All in a cluster : The selected sequence has a crucial influence on both the orientation and flexibility of the carbohydrate moiety in mucin‐type glycopeptides (see picture). This feature can be explained through the varied conformational behavior of the glycosidic linkage of the Thr residue when compared with the Ser residue (see figure). On this basis, and taking into account that these carbohydrates presumably interact with components of the immune system, these findings could be helpful in the design of new cancer vaccines.

     

Serine versus Threonine Glycosylation with α‐O‐GalNAc: Unexpected Selectivity in Their Molecular Recognition with Lectins

The molecular recognition of several glycopeptides bearing Tn antigen (α‐O‐GalNAc‐Ser or α‐O‐GalNAc‐Thr) in their structure by three lectins with affinity for this determinant has been analysed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the molecular recognition. In fact, while Soybean agglutinin and Vicia villosa agglutinin lectins prefer Tn‐threonine, Helix pomatia agglutinin shows a higher affinity for the glycopeptides carrying Tn‐serine. The different conformational behaviour of the two Tn biological entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topology of the binding site of the lectins are at the origin of these differences.     

Conformational Dynamics of the Single Lipopolysaccharide O‐Antigen in Solution

The O‐antigen is the most variable and highly immunogenic part of the lipopolysaccharide molecule that covers the surface of Gram‐negative bacteria and makes up the first line of cellular defense. To provide insight into the details of the O‐antigen arrangement on the membrane surface, we simulated its behavior in solution by molecular dynamics. We developed the energetically favorable O‐antigen conformation by analyzing free‐energy distributions for its disaccharide fragments. Starting from this conformation, we simulated the behavior of the O‐antigen chain on long timescales. Depending on the force field and temperature, the single molecule can undergo reversible or irreversible coil‐to‐globule transitions. The mechanism of these transitions is related either to the rotation of the carbohydrate residues around O‐glycosidic bonds or to flips of the pyranose rings. We found that the presence of rhamnose in the O‐antigen chain crucially increases its conformational mobility.     

Strong Positive Allosterism which Appears in Molecular Recognition with Cerium(IV) Double Decker Porphyrins: Correlation between the Number of Binding Sites and Hill Coefficients

Abstract

Cerium(IV) double decker porphyrins bearing one-to-four pairs of 4-pyridyl groups (3a, 3a′, 3bp, 3bd, 3c, and 3d) were synthesized from tetraarylporphyrins bearing mono-, bis-, tris-, and tetrakis(4-pyridyl) groups. In 3b bearing two pairs of 4-pyridyl groups, there exist two isomers in which the 4-pyridyl groups are either proximal or distal (3bp and 3bd, respectively). In a mixed solvent of dichloromethane: ethyl acetate (30:1 v/v), 3a′ bearing one pair of 4-pyridyl groups and three pairs of phenyl groups did not interact with any dicarboxylic acids whereas 3d bearing four pairs of 4-pyridyl groups interacted only with dicarboxylic acid guests with a dimethylene spacer [e.g., BOC-L-aspartic acid (L-4) and (1R,2R)-cyclohexane-1,2-dicarboxylic acid ((1R,2R)-5)]. Interestingly, the complexation process monitored by CD spectroscopy showed a positive homotropic allosterism which satisfied the Hill equation giving constants K = 2.63 × 1011M-4 and n = 3.9 for L-4 and K = 2.75 × 109 M-4 and n = 4.0 for (1R,2R)-5. The continuous variation plots (Job plots) also supported the formation of the 1:4 3d/dicarboxylic acid guest complexes. The results consistently indicate that four pairs of 4-pyridyl groups in 3d allosterically bind these guests. In 3d, the two porphyrin rings can still rotate, but once the rotation is suppressed by the first guest binding, the subsequent binding of the second, third and fourth guests can occur cooperatively. This is the origin of the present positive homotoropic allosterism. A similar positive homotropic allosterism was also observed for 3bp and 3bd with n = 1.5 and 1.7, respectively and 3c with n = 3.0. The X-ray crystallographic study of the 3d·[(1R,2R)-5]4 complex showed that the two porphyrin planes are warped outward to relax the electrostatic repulsion and chirally twisted. The two carboxylic acid groups form intermolecular hydrogen bonds (but not intramolecular bridge-type hydrogen bonds) with the pyridyl groups because of the close packing effect of rigid host 3d and rigid guest (1R,2R)-5. In conclusion, this is a rare example of positive homotropic allosterism in an artificial system which is frequently seen in nature where the biological events must be efficiently regulated in response to signals.     

Identification of Two Distinct Sites for Antagonist and Biased Agonist Binding to the Human Chemokine Receptor CXCR3

The chemokine receptor CXCR3 is a G protein‐coupled receptor that conveys extracellular signals into cells by changing its conformation upon ligand binding. We previously hypothesized that small‐molecule allosteric CXCR3‐agonists do not bind to the same allosteric binding pocket as 8‐azaquinazolinone‐based negative allosteric modulators. We have now performed molecular‐dynamics (MD) simulations with metadynamics enhanced sampling on the CXCR3 system to refine structures and binding modes and to predict the CXCR3‐binding affinities of the biased allosteric agonist FAUC1036 and the negative allosteric modulator RAMX3. We have identified two distinct binding sites; a “shallow” and a second “deeper” pocket to which the biased allosteric agonist FAUC1036 and negative allosteric modulator RAMX3 bind, respectively.     

Conformational selection of the AGA*IA(M) heparin pentasaccharide when bound to the fibroblast growth factor receptor

The interaction of the synthetic pentasaccharide AGA*IA(M) (GlcNS,6S-GlcA-GlcNS,3S,6S-IdoA2S-GlcNS,6S-Me) with the extracellular Ig2 domain of the fibroblast growth factor receptor (FGFR2) has been studied by NMR and computational methods. Analysis of the heparin pentasaccharide in the free state and in the complex indicates the existence of a conformational selection process. Although an equilibrium exists between the (1)C(4) and (2)S(0) conformers (ratio 60:40) of the 2-O-sulfo-α-L-iduronate ring (IdoA2S) in the free state, FGFR2 selects only the unique twisted-boat (2)S(0) conformation of this IdoA2S residue. In addition, the protein residues involved in the binding with AGA*IA(M) have also been characterized. The NMR results obtained, from both the ligand and protein perspective, were employed to model the bound conformation of the pentasaccharide by a combined docking and molecular dynamic simulation approach.     

Computational Insights into the Allosteric Effect and Dynamic Structural Features of the SARS-COV-2 Spike Protein

COVID-19 caused by SARS-COV-2 is continuing to surge globally. The spike (S) protein is the key protein of SARS-COV-2 that recognizes and binds to the host target ACE2. In this study, molecular dynamics simulation was used to elucidate the allosteric effect of the S protein. Binding of ACE2 caused a centripetal movement of the receptor-binding domain of the S protein. The dihedral changes in Phe329 and Phe515 played a key role in this process. Two potential cleavage sites S1/S2 and S2′ were exposed on the surface after the binding of ACE2. The binding affinity of SARS-COV-2 S protein and ACE2 was higher than that of SARS-COV. This was mainly due to the mutation of Asp480 in SARS-COV to Ser494 in SARS-COV-2, which greatly weakened the electrostatic repulsion. The result provides a theoretical basis for the SARS-COV-2 infection and aids the development of biosensors and detection reagents.     

The bound conformation of microtubule-stabilizing agents: NMR insights into the bioactive 3D structure of discodermolide and dictyostatin

A protocol based on a combination of NMR experimental data with molecular mechanics calculations and docking procedures has been employed to determine the microtubule-bound conformation of two microtubule-stabilizing agents, discodermolide (DDM) and dictyostatin (DCT). The data indicate that tubulin in assembled microtubules recognizes DDM through a conformational selection process, with minor changes in the molecular skeleton between the major conformer in water solution and that bound to assembled microtubules. For DCT, the deduced bound geometry presents some key conformation differences around certain torsion angles, with respect to the major conformer in solution, and still displays mobility even when bound. The bound conformer of DCT resembles that of DDM and provides very similar contacts with the receptor. Competition experiments indicate that both molecules compete with the taxane-binding site. A model of the binding mode of DDM and DCT to tubulin is proposed.     

Engineering O-glycosylation points in non-extended peptides: implications for the molecular recognition of short tumor-associated glycopeptides

The ties that bind: The incorporation of non-natural residues in the peptide backbone allows the design of O-glycosylation points in helical segments. This strategy could help to modulate the binding properties between glycopeptides and their protein receptors, such as lectins and antibodies.     

Conformational Selection in Glycomimetics: Human Galectin‐1 Only Recognizes syn‐Ψ‐Type Conformations of β‐1,3‐Linked Lactose and Its C‐Glycosyl Derivative

The human lectin galectin‐1 (hGal‐1) translates sugar signals, that is, β‐galactosides, into effects on the level of cells, for example, growth regulation, and has become a model for studying binding of biopharmaceutically relevant derivatives. Bound‐state conformations of Galβ‐C‐(1→3)‐Glcβ‐OMe ( 1 ) and its βGal‐(1→3)‐βGlc‐OMe disaccharide parent compound were studied by using NMR spectroscopy (transferred (TR)‐NOESY data), assisted by docking experiments and molecular dynamics (MD) simulations. The molecular recognition process involves a conformational selection event. Although free C‐glycoside access four distinct conformers in solution, hGal‐1 recognizes shape of a local minimum of compound 1 , the syn‐Φ/syn‐Ψ conformer, not the structure at global minimum. MD simulations were run to explain, in structural terms, the observed geometry of the complex.     

Mimicking Chitin: Chemical Synthesis,Conformational Analysis,and Molecular Recognition of the β(1→3) N‐Acetylchitopentaose Analogue

Mimicking Nature by using synthetic molecules that resemble natural products may open avenues to key knowledge that is difficult to access by using substances from natural sources. In this context, a novel N‐acetylchitooligosaccharide analogue, β‐1,3‐N‐acetamido‐gluco‐pentasaccharide, has been designed and synthesized by using aminoglucose as the starting material. A phthalic group has been employed as the protecting group of the amine moiety, whereas a thioalkyl was used as the leaving group on the reducing end. The conformational properties of this new molecule have been explored and compared to those of the its chito analogue, with the β‐1,3 linkages, by a combined NMR spectroscopic/molecular modeling approach. Furthermore, the study of its molecular recognition properties towards two proteins, a lectin (wheat germ agglutinin) and one enzyme (a chitinase) have also been performed by using NMR spectroscopy and docking protocols. There are subtle differences in the conformational behavior of the mimetic versus the natural chitooligosaccharide, whereas this mimetic is still recognized by these two proteins and can act as a moderate inhibitor of chitin hydrolysis.     

A Disulfide Bridge Allows for Site‐Selective Binding in Liver Bile Acid Binding Protein Thereby Stabilising the Orientation of Key Amino Acid Side Chains

The presence of a disulfide bridge in liver bile acid binding protein (L‐BABP/S‐S) allows for site‐selective binding of two bile acids, glycochenodeoxycholic (GCDA) and glycocholic acid (GCA), differing only in the presence of a hydroxyl group. The protein form devoid of the disulfide bridge (L‐BABP) binds both bile salts without discriminating ability. We investigate the determinants of the molecular recognition process in the formation of the heterotypic L‐BABP/S‐S complex with GCA and GCDA located in the superficial and inner protein sites, respectively. The comparison of the NMR spectroscopy structure of heterotypic holo L‐BABP/S‐S, the first reported for this protein family, with that of the homotypic L‐BABP complex demonstrates that the introduction of a S–S link between adjacent strands changes the conformation of three key residues, which function as hot‐spot mediators of molecular discrimination. The favoured χ₁ rotameric states (t, g⁺ and g^? for E99, Q100 and E109 residues, respectively) allow the onset of an extended intramolecular hydrogen‐bond network and the consequent stabilisation of the side‐chain orientation of a buried histidine, which is capable of anchoring a specific ligand.     

Effect of the Substituents of the Neighboring Ring in the Conformational Equilibrium of Iduronate in Heparin‐like Trisaccharides

Based on the structure of the regular heparin, we have prepared a smart library of heparin‐like trisaccharides by incorporating some sulfate groups in the sequence α‐D ‐GlcNS‐ (1‐4)‐α‐L ‐Ido2S‐(1‐4)‐α‐D ‐GlcN. According to the 3D structure of heparin, which features one helix turn every four residues, this fragment corresponds to the minimum binding motif. We have performed a complete NMR study and found that the trisaccharides have a similar 3D structure to regular heparin itself, but their spectral properties are such that allow to extract very detailed information about distances and coupling constants as they are isotropic molecules. The characteristic conformational equilibrium of the central iduronate ring has been analyzed combining NMR and molecular dynamics and the populations of the conformers of the central iduronate ring have been calculated. We have found that in those compounds lacking the sulfate group at position 6 of the reducing end glucosamine, the population of ²S₀ of the central iduronate residue is sensitive to the temperature decreasing to 19 % at 278 K. On the contrary, the trisaccharides with 6‐O‐sulfate in the reducing end glucosamine keep the level of population constant with temperature circa 40 % of ²S₀ similar to that observed at room temperature. Another structural feature that has been revealed through this analysis is the larger flexibility of the L ‐IdoAS‐ D ‐GlcN glycosidic linkage, compared with the D ‐GlcNS‐L ‐IdoA. We propose that this is the point where the heparin chain is bended to form structures far from the regular helix known as kink that have been proposed to play an important role in the specificity of the heparin–protein interaction.     

1H‐Azepine‐4‐amino‐4‐carboxylic Acid: A New α,α‐Disubstituted Ornithine Analogue Capable of Inducing Helix Conformations in Short Ala‐Aib Pentapeptides

A very efficient synthesis of orthogonally protected 1H‐azepine‐4‐amino‐4‐carboxylic acid, abbreviated as Azn, a conformationally restricted analogue of ornithine, was realized. It was obtained on a gram scale in good overall yield in five steps, three of which did not require isolation of the intermediates, starting from the readily available 1‐amino‐4‐oxo‐cyclohexane‐4‐carboxylic acid. Both enantiomers were used for the preparation of pentapeptide models containing Ala, Aib, and Azn. Conformational studies using both spectroscopic techniques (NMR, CD) and molecular dynamics on model 5‐mer peptides showed that the (R)‐Azn isomer possesses a marked helicogenic effect.     

Janus‐Like Squaramide‐Based Hosts: Dual Mode of Binding and Conformational Transitions Driven by Ion‐Pair Recognition

New tripodal squaramide‐based hosts have been synthesised and structurally characterised by spectroscopic methods. In 2.5 % (v/v) [D₆]DMSO in CDCl₃, compound 4 formed dimeric assemblies [log K_dim=3.68(8)] as demonstrated by ¹H NMR spectroscopy and UV dilution experiments. AFM and SEM analyses revealed the formation of a network of bundled fibres, which indicates a preferential mechanism for aggregation. These C₃‐symmetric tripodal hosts exhibited two different and mutually exclusive modes of binding, each one easily accessible by simultaneous reorientation of the squaramide groups. In the first, a convergent disposition of the NH squaramide protons allowed the formation of an array of N? H???X^? hydrogen bonds with anions. In the second mode, reorientation of carbonyl squaramide groups allowed multiple C?O???H interactions with ammonium cations. The titration of 4 with different tetraalkylammonium iodides persistently showed the formation of 1:1 complexes, as well as 1:2 and 1:3 complexes. The corresponding stoichiometries and binding affinities of the complexes were evaluated by multi‐regression analysis. The formation of high‐order complexes, supported by ROESY, NOESY and mass spectrometry experiments, has been attributed to the insertion of NR₄I ion pairs between the carbonyl and NH protons of the squaramide groups located in adjacent arms of 4 . The observed effects reflect the induction of significant conformational changes in the hosts, mainly in relation to the relative orientation of the squaramide groups adapting their geometries to incoming ion‐pair complementary substrates. The results presented herein identify and fully describe two different modes of ion‐pair recognition aimed at directing conformational transitions in the host, therefore establishing a base for controlling more elaborate movements of molecular devices through ion‐pair recognition.     

Shapes and Internal Dynamics of the 1:1 Adducts of Ammonia with trans and gauche Ethanol: A Rotational Study

Two 1:1 adducts of ammonia with ethanol have been characterized by using pulsed‐jet FT microwave spectroscopy. They are formed with two different (trans and gauche), stable conformers of ethanol. Several internal‐dynamics effects are reflected in the features of the rotational spectra. The trans complex shows the tunneling effects owing to internal rotation of both ammonia and the methyl group. The rotational transitions of the gauche species exhibit a small splitting that is related to tunneling through the potential‐energy barrier between the two equivalent minima.     

Effect of beta-O-glucosylation on L-Ser and L-Thr diamides: a bias toward alpha-helical conformations

Beta-D-O-glucosylation produces a remarkable effect on the peptide backbone of the model peptides derived from serine and threonine. Consequently, this type of glycosylation is responsible for the experimentally observed shift from extended conformations (model peptides) towards the folded conformations (model glycopeptides). The conclusion has been solidly assessed by a combined NMR/MD protocol. Interestingly, the MD (molecular dynamics) results for the glycopeptides point towards the existence of water-bridging molecules between the sugar and peptide moieties, which could explain the stabilization of the folded conformers in aqueous solution.