Prediction of protein structure and AI.

AlphaFold, an artificial intelligence (AI)-based tool for predicting the 3D structure of proteins, is now widely recognized for its high accuracy and versatility in the folding of human proteins. AlphaFold is useful for understanding structure-function relationships from protein 3D structure models and can serve as a template or a reference for experimental structural analysis including X-ray crystallography, NMR and cryo-EM analysis. Its use is expanding among researchers, not only in structural biology but also in other research fields. Researchers are currently exploring the full potential of AlphaFold-generated protein models. Predicting disease severity caused by missense mutations is one such application. This article provides an overview of the 3D structural modeling of AlphaFold based on deep learning techniques and highlights the challenges in predicting the pathogenicity of missense mutations.

NMR characterization of uniformly 13C- and/or 15N-labeled, unsulfated chondroitins with high molecular weights.

Solution nuclear magnetic resonance (NMR) analysis of polysaccharides can provide valuable information not only on their primary structures but also on their conformation, dynamics, and interactions under physiological conditions. One of the main problems is that non-anomeric 1H signals typically overlap, and this often hinders detailed NMR analysis. Isotope enrichment, such as with 13C and 15N, will add a new dimension to the NMR spectra of polysaccharides, and spectral analysis can be performed with enhanced sensitivity using isolated peaks. For this purpose, here we have prepared uniformly 13C- and/or 15N-labeled chondroitin polysaccharides -4)-ß-D-glucuronopyranosyl-(1-3)-2-acetamido-2-deoxy-ß-D-galactopyranosyl-(1- with molecular weights in the range from 310 to 460 k by bacterial fermentation. The enrichment ratios for 13C and 15N were 98.9 and 99.8%, respectively, based on the mass spectrometric analysis of the constituent chondroitin disaccharides. 1H and 13C NMR signals were assigned mainly based on HSQC and 13C-detection experiments including INADEQUATE, HETCOR, and HETCOR-TOCSY. The carbonyl carbon signal of the N-acetyl-ß-D-galactosamine residue was unambiguously distinguished from the C6 carbon of the ß-D-glucuronic acid residue by the observation of 13C peak splitting due to 1JCN coupling in 13C- and 15N-labeled chondroitin. The T2* and T1 were measured and indicate that both rigid and mobile sites are present in the long sequence of chondroitin. The conformation, dynamics, and interactions of chondroitin and its derivatives will be further analyzed based on the results obtained in this study.

Comparative Conformational Analysis of Acyclic Sugar Alcohols Ribitol, Xylitol and d-Arabitol by Solution NMR and Molecular Dynamics Simulations.

Ribitol (C5H12O5) is an acyclic sugar alcohol that was recently identified in O-mannose glycan on mammalian α-dystroglycan. The conformation and dynamics of acyclic sugar alcohols such as ribitol are dependent on the stereochemistry of the hydroxyl groups; however, the dynamics are not fully understood. To gain insights into the conformation and dynamics of sugar alcohols, we carried out comparative analyses of ribitol, d-arabitol and xylitol by a crystal structure database search, solution NMR analysis and molecular dynamics (MD) simulations. The crystal structures of the sugar alcohols showed a limited number of conformations, suggesting that only certain stable conformations are prevalent among all possible conformations. The three-bond scholar coupling constants and exchange rates of hydroxyl protons were measured to obtain information on the backbone torsion angle and possible hydrogen bonding of each hydroxyl group. The 100 ns MD simulations indicate that the ribitol backbone has frequent conformational transitions with torsion angles between 180∘ and ±60∘, while d-arabitol and xylitol showed fewer conformational transitions. Taking our experimental and computational data together, it can be concluded that ribitol is more flexible than d-arabitol or xylitol, and the flexibility is at least in part defined by the configuration of the OH groups, which may form intramolecular hydrogen bonds.

Functional validation of novel variants in B4GALNT1 associated with early-onset complex hereditary spastic paraplegia with impaired ganglioside synthesis.

Childhood-onset forms of hereditary spastic paraplegia are ultra-rare diseases and often present with complex features. Next-generation-sequencing allows for an accurate diagnosis in many cases but the interpretation of novel variants remains challenging, particularly for missense mutations. Where sufficient knowledge of the protein function and/or downstream pathways exists, functional studies in patient-derived cells can aid the interpretation of molecular findings. We here illustrate the case of a 13-year-old female who presented with global developmental delay and later mild intellectual disability, progressive spastic diplegia, spastic-ataxic gait, dysarthria, urinary urgency, and loss of deep tendon reflexes of the lower extremities. Exome sequencing showed a novel splice-site variant in trans with a novel missense variant in B4GALNT1 [NM_001478.5: c.532-1G>C/c.1556G>C (p.Arg519Pro)]. Functional studies in patient-derived fibroblasts and cell models of GM2 synthase deficiency confirmed a loss of B4GALNT1 function with no synthesis of GM2 and other downstream gangliosides. Collectively these results established the diagnosis of B4GALNT1-associated HSP (SPG26). Our approach illustrates the importance of careful phenotyping and functional characterization of novel gene variants, particularly in the setting of ultra-rare diseases, and expands the clinical and molecular spectrum of SPG26, a disorder of complex ganglioside biosynthesis.

Synthesis of the matriglycan hexasaccharide, -3Xylα1-3GlcAß1-trimer and its interaction with laminin.

Matriglycan, a polysaccharide that is a pivotal part of the core M3 O-mannosyl glycan composed of the repeating disaccharide -3Xylα1-3GlcAß1-, interacts with laminin to stabilize muscle tissue. We herein report the synthesis of matriglycan-repeating hexasaccharides equipped with an alkyne linker to form glycoconjugates. The key step in the formation of an α-linked xylosyl glycoside was resolved by solvent-specific separation from an anomeric mixture. Successful glycan elongation was regio- and stereoselectively performed to obtain (-3Xylα1-3GlcAß1)3-O(C2H4O)3CH2CCH and the biotin conjugate. We also investigated interactions between matriglycan hexasaccharides and laminin-G-like domains 4 and 5 of laminin-α2 using saturation transfer difference-NMR. The dissociation constant obtained from bio-layer interferometry was estimated to be 7.5 × 10-8 M. These results indicate that a chemical approach may be applied to the reconstruction of muscle tissue.

O-Glycan-Dependent Interaction between MUC1 Glycopeptide and MY.1E12 Antibody by NMR, Molecular Dynamics and Docking Simulations.

Anti-mucin1 (MUC1) antibodies have been widely used for breast cancer diagnosis and treatment. This is based on the fact that MUC1 undergoes aberrant glycosylation upon cancer progression, and anti-MUC1 antibodies differentiate changes in glycan structure. MY.1E12 is a promising anti-MUC1 antibody with a distinct specificity toward MUC1 modified with an immature O-glycan (NeuAcα(2-3)Galß(1-3)GalNAc) on a specific Thr. However, the structural basis for the interaction between MY.1E12 and MUC1 remains unclear. The aim of this study is to elucidate the mode of interaction between MY.1E12 and MUC1 O-glycopeptide by NMR, molecular dynamics (MD) and docking simulations. NMR titration using MUC1 O-glycopeptides suggests that the epitope is located within the O-linked glycan and near the O-glycosylation site. MD simulations of MUC1 glycopeptide showed that the O-glycosylation significantly limits the flexibility of the peptide backbone and side chain of the O-glycosylated Thr. Docking simulations using modeled MY.1E12 Fv and MUC1 O-glycopeptide, suggest that VH mainly contributes to the recognition of the MUC1 peptide portion while VL mainly binds to the O-glycan part. The VH/VL-shared recognition mode of this antibody may be used as a template for the rational design and development of anti-glycopeptide antibodies.

3D Structural Insights into ß-Glucans and Their Binding Proteins.

ß(1,3)-glucans are a component of fungal and plant cell walls. The ß-glucan of pathogens is recognized as a non-self-component in the host defense system. Long ß-glucan chains are capable of forming a triple helix structure, and the tertiary structure may profoundly affect the interaction with ß-glucan-binding proteins. Although the atomic details of ß-glucan binding and signaling of cognate receptors remain mostly unclear, X-ray crystallography and NMR analyses have revealed some aspects of ß-glucan structure and interaction. Here, we will review three-dimensional (3D) structural characteristics of ß-glucans and the modes of interaction with ß-glucan-binding proteins.

3D Structures of IgA, IgM, and Components.

Immunoglobulin G (IgG) is currently the most studied immunoglobin class and is frequently used in antibody therapeutics in which its beneficial effector functions are exploited. IgG is composed of two heavy chains and two light chains, forming the basic antibody monomeric unit. In contrast, immunoglobulin A (IgA) and immunoglobulin M (IgM) are usually assembled into dimers or pentamers with the contribution of joining (J)-chains, which bind to the secretory component (SC) of the polymeric Ig receptor (pIgR) and are transported to the mucosal surface. IgA and IgM play a pivotal role in various immune responses, especially in mucosal immunity. Due to their structural complexity, 3D structural study of these molecules at atomic scale has been slow. With the emergence of cryo-EM and X-ray crystallographic techniques and the growing interest in the structure-function relationships of IgA and IgM, atomic-scale structural information on IgA-Fc and IgM-Fc has been accumulating. Here, we examine the 3D structures of IgA and IgM, including the J-chain and SC. Disulfide bridging and N-glycosylation on these molecules are also summarized. With the increasing information of structure-function relationships, IgA- and IgM-based monoclonal antibodies will be an effective option in the therapeutic field.

Ribitol in Solution Is an Equilibrium of Asymmetric Conformations.

Ribitol (C5H12O5), an acyclic sugar alcohol, is present on mammalian α-dystroglycan as a component of O-mannose glycan. In this study, we examine the conformation and dynamics of ribitol by database analysis, experiments, and computational methods. Database analysis reveals that the anti-conformation (180°) is populated at the C3-C4 dihedral angle, while the gauche conformation (±60°) is seen at the C2-C3 dihedral angle. Such conformational asymmetry was born out in a solid-state 13C-NMR spectrum of crystalline ribitol, where C1 and C5 signals are unequal. On the other hand, solution 13C-NMR has identical chemical shifts for C1 and C5. NMR 3J coupling constants and OH exchange rates suggest that ribitol is an equilibrium of conformations, under the influence of hydrogen bonds and/or steric hinderance. Molecular dynamics (MD) simulations allowed us to discuss such a chemically symmetric molecule, pinpointing the presence of asymmetric conformations evidenced by the presence of correlations between C2-C3 and C3-C4 dihedral angles. These findings provide a basis for understanding the dynamic structure of ribitol and the function of ribitol-binding enzymes.

Complementary Design for Pairing between Two Types of Nanoparticles Mediated by a Bispecific Antibody: Bottom-Up Formation of Porous Materials from Nanoparticles.

Recent advances in biotechnology have enabled the generation of antibodies with high affinity for the surfaces of specific inorganic materials. Herein, we report the synthesis of functional materials from multiple nanomaterials by using a small bispecific antibody recombinantly constructed from gold-binding and ZnO-binding antibody fragments. The bispecific antibody-mediated spontaneous linkage of gold and ZnO nanoparticles forms a binary gold-ZnO nanoparticle composite membrane. The relatively low melting point of the gold nanoparticles and the solubility of ZnO in dilute acidic solution then allowed for the bottom-up synthesis of a nanoporous gold membrane by means of a low-energy, low-environmental-load protocol. The nanoporous gold membrane showed high catalytic activity for the reduction of p-nitrophenol to p-aminophenol by sodium borohydride. Here, we show the potential utility of nanoparticle pairing mediated by bispecific antibodies for the bottom-up construction of nanostructured materials from multiple nanomaterials.

Phosphate-Catalyzed Succinimide Formation from Asp Residues: A Computational Study of the Mechanism.

Aspartic acid (Asp) residues in proteins and peptides are prone to the non-enzymatic reactions that give biologically uncommon l-ß-Asp, d-Asp, and d-ß-Asp residues via the cyclic succinimide intermediate (aminosuccinyl residue, Suc). These abnormal Asp residues are known to have relevance to aging and pathologies. Despite being non-enzymatic, the Suc formation is thought to require a catalyst under physiological conditions. In this study, we computationally investigated the mechanism of the Suc formation from Asp residues that were catalyzed by the dihydrogen phosphate ion, H2PO4-. We used Ac-l-Asp-NHMe (Ac = acetyl, NHMe = methylamino) as a model compound. The H2PO4- ion (as a catalyst) and two explicit water molecules (as solvent molecules stabilizing the negative charge) were included in the calculations. All of the calculations were performed by density functional theory with the B3LYP functional. We revealed a phosphate-catalyzed two-step mechanism (cyclization-dehydration) of the Suc formation, where the first step is predicted to be rate-determining. In both steps, the reaction involved a proton relay mediated by the H2PO4- ion. The calculated activation barrier for this mechanism (100.3 kJ mol-1) is in reasonable agreement with an experimental activation energy (107 kJ mol-1) for the Suc formation from an Asp-containing peptide in a phosphate buffer, supporting the catalytic mechanism of the H2PO4- ion that is revealed in this study.

Phosphate-Catalyzed Succinimide Formation from an NGR-Containing Cyclic Peptide: A Novel Mechanism for Deammoniation of the Tetrahedral Intermediate.

Spontaneous deamidation in the Asn-Gly-Arg (NGR) motif that yields an isoAsp-Gly-Arg (isoDGR) sequence has recently attracted considerable attention because of the possibility of application to dual tumor targeting. It is well known that Asn deamidation reactions in peptide chains occur via the five-membered ring succinimide intermediate. Recently, we computationally showed by the B3LYP density functional theory method, that inorganic phosphate and the Arg side chain can catalyze the NGR deamidation using a cyclic peptide, c[CH2CO⁻NGRC]⁻NH2. In this previous study, the tetrahedral intermediate of the succinimide formation was assumed to be readily protonated at the nitrogen originating from the Asn side chain by the solvent water before the release of an NH3 molecule. In the present study, we found a new mechanism for the decomposition of the tetrahedral intermediate that does not require the protonation by an external proton source. The computational method is the same as in the previous study. In the new mechanism, the release of an NH3 molecule occurs after a proton exchange between the peptide and the phosphate and conformational changes. The rate-determining step of the overall reaction course is the previously reported first step, i.e., the cyclization to form the tetrahedral intermediate.

Succinimide Formation from an NGR-Containing Cyclic Peptide: Computational Evidence for Catalytic Roles of Phosphate Buffer and the Arginine Side Chain.

The Asn-Gly-Arg (NGR) motif and its deamidation product isoAsp-Gly-Arg (isoDGR) have recently attracted considerable attention as tumor-targeting ligands. Because an NGR-containing peptide and the corresponding isoDGR-containing peptide target different receptors, the spontaneous NGR deamidation can be used in dual targeting strategies. It is well known that the Asn deamidation proceeds via a succinimide derivative. In the present study, we computationally investigated the mechanism of succinimide formation from a cyclic peptide, c[CH2CO-NGRC]-NH2, which has recently been shown to undergo rapid deamidation in a phosphate buffer. An H2PO4- ion was explicitly included in the calculations. We employed the density functional theory using the B3LYP functional. While geometry optimizations were performed in the gas phase, hydration Gibbs energies were calculated by the SM8 (solvation model 8) continuum model. We have found a pathway leading to the five-membered ring tetrahedral intermediate in which both the H2PO4- ion and the Arg side chain act as catalyst. This intermediate, once protonated at the NH2 group on the five-membered ring, was shown to easily undergo NH3 elimination leading to the succinimide formation. This study is the first to propose a possible catalytic role for the Arg side chain in the NGR deamidation.

Racemization of the Succinimide Intermediate Formed in Proteins and Peptides: A Computational Study of the Mechanism Catalyzed by Dihydrogen Phosphate Ion.

In proteins and peptides, d-aspartic acid (d-Asp) and d-ß-Asp residues can be spontaneously formed via racemization of the succinimide intermediate formed from l-Asp and l-asparagine (l-Asn) residues. These biologically uncommon amino acid residues are known to have relevance to aging and pathologies. Although nonenzymatic, the succinimide racemization will not occur without a catalyst at room or biological temperature. In the present study, we computationally investigated the mechanism of succinimide racemization catalyzed by dihydrogen phosphate ion, H2PO4-, by B3LYP/6-31+G(d,p) density functional theory calculations, using a model compound in which an aminosuccinyl (Asu) residue is capped with acetyl (Ace) and NCH3 (Nme) groups on the N- and C-termini, respectively (Ace-Asu-Nme). It was shown that an H2PO4- ion can catalyze the enolization of the Hα-Cα-C=O portion of the Asu residue by acting as a proton-transfer mediator. The resulting complex between the enol form and H2PO4- corresponds to a very flat intermediate region on the potential energy surface lying between the initial reactant complex and its mirror-image geometry. The calculated activation barrier (18.8 kcal·mol-1 after corrections for the zero-point energy and the Gibbs energy of hydration) for the enolization was consistent with the experimental activation energies of Asp racemization.

A Computational Study of the Mechanism of Succinimide Formation in the Asn-His Sequence: Intramolecular Catalysis by the His Side Chain.

The rates of deamidation reactions of asparagine (Asn) residues which occur spontaneously and nonenzymatically in peptides and proteins via the succinimide intermediate are known to be strongly dependent on the nature of the following residue on the carboxyl side (Xxx). The formation of the succinimide intermediate is by far the fastest when Xxx is glycine (Gly), the smallest amino acid residue, while extremely slow when Xxx is bulky such as isoleucine (Ile) and valine (Val). In this respect, it is very interesting to note that the succinimide formation is definitely accelerated when Xxx is histidine (His) despite its large size. In this paper, we computationally show that, in an Asn-His sequence, the His side-chain imidazole group (in the neutral Nε-protonated form) can specifically catalyze the formation of the tetrahedral intermediate in the succinimide formation by mediating a proton transfer. The calculations were performed for Ace-Asn-His-Nme (Ace = acetyl, Nme = methylamino) as a model compound by the density functional theory with the B3LYP functional and the 6-31+G(d,p) basis set. We also show that the tetrahedral intermediate, once protonated at the NH2 group, easily releases an ammonia molecule to give the succinimide species.

Acetic acid-catalyzed formation of N-phenylphthalimide from phthalanilic acid: a computational study of the mechanism.

In glacial acetic acid, phthalanilic acid and its monosubstituents are known to be converted to the corresponding phthalimides in relatively good yields. In this study, we computationally investigated the experimentally proposed two-step (addition-elimination or cyclization-dehydration) mechanism at the second-order Møller-Plesset perturbation (MP2) level of theory for the unsubstituted phthalanilic acid, with an explicit acetic acid molecule included in the calculations. In the first step, a gem-diol tetrahedral intermediate is formed by the nucleophilic attack of the amide nitrogen. The second step is dehydration of the intermediate to give N-phenylphthalimide. In agreement with experimental findings, the second step has been shown to be rate-determining. Most importantly, both of the steps are catalyzed by an acetic acid molecule, which acts both as proton donor and acceptor. The present findings, along with those from our previous studies, suggest that acetic acid and other carboxylic acids (in their undissociated forms) can catalyze intramolecular nucleophilic attacks by amide nitrogens and breakdown of the resulting tetrahedral intermediates, acting simultaneously as proton donor and acceptor. In other words, double proton transfers involving a carboxylic acid molecule can be part of an extensive bond reorganization process from cyclic hydrogen-bonded complexes.

Glycolic acid-catalyzed deamidation of asparagine residues in degrading PLGA matrices: a computational study.

Poly(lactic-co-glycolic acid) (PLGA) is a strong candidate for being a drug carrier in drug delivery systems because of its biocompatibility and biodegradability. However, in degrading PLGA matrices, the encapsulated peptide and protein drugs can undergo various degradation reactions, including deamidation at asparagine (Asn) residues to give a succinimide species, which may affect their potency and/or safety. Here, we show computationally that glycolic acid (GA) in its undissociated form, which can exist in high concentration in degrading PLGA matrices, can catalyze the succinimide formation from Asn residues by acting as a proton-transfer mediator. A two-step mechanism was studied by quantum-chemical calculations using Ace-Asn-Nme (Ace = acetyl, Nme = NHCH3) as a model compound. The first step is cyclization (intramolecular addition) to form a tetrahedral intermediate, and the second step is elimination of ammonia from the intermediate. Both steps involve an extensive bond reorganization mediated by a GA molecule, and the first step was predicted to be rate-determining. The present findings are expected to be useful in the design of more effective and safe PLGA devices.

Acetic acid can catalyze succinimide formation from aspartic acid residues by a concerted bond reorganization mechanism: a computational study.

Succinimide formation from aspartic acid (Asp) residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe) as a model compound, we propose the possibility that acetic acid (AA), which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition) to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds) occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism.

Roles of intramolecular and intermolecular hydrogen bonding in a three-water-assisted mechanism of succinimide formation from aspartic acid residues.

Aspartic acid (Asp) residues in peptides and proteins are prone to isomerization to the ß-form and racemization via a five-membered succinimide intermediate. These nonenzymatic reactions have relevance to aging and age-related diseases. In this paper, we report a three water molecule-assisted, six-step mechanism for the formation of succinimide from Asp residues found by density functional theory calculations. The first two steps constitute a stepwise iminolization of the C-terminal amide group. This iminolization involves a quintuple proton transfer along intramolecular and intermolecular hydrogen bonds formed by the C-terminal amide group, the side-chain carboxyl group, and the three water molecules. After a conformational change (which breaks the intramolecular hydrogen bond involving the iminol nitrogen) and a reorganization of water molecules, the iminol nitrogen nucleophilically attacks the carboxyl carbon of the Asp side chain to form a five-membered ring. This cyclization is accompanied by a triple proton transfer involving two water molecules, so that a gem-diol tetrahedral intermediate is formed. The last step is dehydration of the gem-diol group catalyzed by one water molecule, and this is the rate-determining step. The calculated overall activation barrier (26.7 kcal mol(-1)) agrees well with an experimental activation energy.

Solution NMR Analysis of O-Glycopeptide-Antibody Interaction.

O-Linked glycans potentially play a functional role in cellular recognition events. Recent structural analyses suggest that O-glycosylation can be a specific signal for a lectin receptor which recognizes both the O-glycan and the adjacent polypeptide region. Further, certain antibodies specifically bind to the O-glycosylated peptide. There is growing interest in the mechanism by which O-glycans on proteins are specifically recognized by lectins and antibodies. The recognition system may be common to many O-glycosylated proteins; however, there is limited 3D structural information on the dual recognition of glycan and protein. This chapter describes a solution NMR analysis of the interaction between MUC1 O-glycopeptide and anti-MUC1 antibody MY.1E12.