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Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing 15N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a 13C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling.

Ubiquitin (Ub) molecules can be enzymatically connected through a specific isopeptide linkage, thereby mediating various cellular processes by binding to Ub-interacting proteins through their hydrophobic surfaces. The Lys48-linked Ub chains, which serve as tags for proteasomal degradation, undergo conformational interconversions between open and closed states, in which the hydrophobic surfaces are exposed and shielded, respectively. Here, we provide a quantitative view of such dynamic processes of Lys48-linked triUb and tetraUb in solution. The native and cyclic forms of Ub chains are prepared with isotope labeling by in vitro enzymatic reactions. Our comparative NMR analyses using monomeric Ub and cyclic diUb as reference molecules enabled the quantification of populations of the open and closed states for each Ub unit of the native Ub chains. The data indicate that the most distal Ub unit in the Ub chains is the most apt to expose its hydrophobic surface, suggesting its preferential involvement in interactions with the Ub-recognizing proteins. We also demonstrate that a mutational modification of the distal end of the Ub chain can remotely affect the solvent exposure of the hydrophobic surfaces of the other Ub units, suggesting that Ub chains could be unique design frameworks for the creation of allosterically controllable multidomain proteins.

Guillain&ndash, Barr&eacute, syndrome, an autoimmune neuropathy characterized by acute limb weakness, is often preceded by Campylobacter jejuni infection. Molecular mimicry exists between the bacterial lipo-oligosaccharide and human ganglioside. Such C. jejuni infection induces production of immunoglobulin G1 (IgG1) autoantibodies against GM1 and causes complement-mediated motor nerve injury. For elucidating the molecular mechanisms linking autoantigen recognition and complement activation, we characterized the dynamic interactions of anti-GM1 IgG autoantibodies on ganglioside-incorporated membranes. Using high-speed atomic force microscopy, we found that the IgG molecules assemble into a hexameric ring structure on the membranes depending on their specific interactions with GM1. Complement component C1q was specifically recruited onto these IgG rings. The ring formation was inhibited by an IgG-binding domain of staphylococcal protein A bound at the cleft between the CH2 and CH3 domains. These data indicate that the IgG assembly is mediated through Fc&ndash, Fc interactions, which are promoted under on-membrane conditions due to restricted translational diffusion of IgG molecules. Reduction and alkylation of the hinge disulfide impaired IgG ring formation, presumably because of an increase in conformational entropic penalty. Our findings provide mechanistic insights into the molecular processes involved in Guillain&ndash, syndrome and, more generally, into antigen-dependent interplay between antibodies and complement components on membranes.

Fcγ receptor (FcγR) promotes various immune responses through interactions with the Fc portion of immunoglobulin G (IgG). FcγRIIIb is a glycosylphosphatidylinositol-linked protein expressed on neutrophils and triggers degranulation and opsonic phagocytosis. The extracellular region of FcγR is composed of two Ig-fold domains and can be cleaved as a soluble form (sFcγRIIIb), which is also reactive with complement receptor type 3. Although structure and Fc interaction of sFcγRIIIb have been characterized by X-ray crystallography, little has been known about its structure in solution. We herein report the backbone NMR assignments of human sFcγRIIIb to gain basic understanding of functional IgG-FcγRIII interactions of immunological and biopharmaceutical interest regarding the structural investigation.

To understand a biological system, it is important to observe structures of biomolecules in the solution where the system is functionalized. Small-Angle X-ray Scattering coupled with Size Exclusion Chromatography (SEC-SAXS) is one of techniques to selectively observe the target molecules in the multi-component system. However, this technique is believed to be available only with a synchrotron-based SAXS instrument due to requirement of high beam intensity and, therefore, the limitation of the beam time was obstacle to satisfy demands from many bio-researchers. We newly developed Laboratory-based Size exclusion chromatography SAXS System (La-SSS) by utilizing a latest laboratory-based SAXS instrument and finely optimization of the balance between flow rate, cell volume, irradiation time and so on. La-SSS succeeded not only decoupling of target protein(s) from non-specific aggregates but also measurement of each concerned component in a multi-component system. In addition, an option: “stopping mode”, which is designed for improving statistics of SAXS profile, realized a high S/N data acquisition for the most interesting protein in a multi-component system. Furthermore, by utilizing a column having small bed volume, the small-scale SEC-SAXS study makes available. Through optimization of instrumental parameters and environments, La-SSS is highly applicable for experimental requirements from various biological samples. It is strongly expected that a La-SSS concept must be a normal option for laboratory-based SAXS in the near future.

Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, FcγRIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with FcγRIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering.

Eukaryotic proteasomes harbor heteroheptameric &alpha, rings, each composed of seven different but homologous subunits &alpha, 1&ndash, &alpha, 7, which are correctly assembled via interactions with assembly chaperones. The human proteasome &alpha, 7 subunit is reportedly spontaneously assembled into a homotetradecameric double ring, which can be disassembled into single rings via interaction with monomeric &alpha, 6. We comprehensively characterized the oligomeric state of human proteasome &alpha, subunits and demonstrated that only the &alpha, 7 subunit exhibits this unique, self-assembling property and that not only &alpha, 6 but also &alpha, 4 can disrupt the &alpha, 7 double ring. We also demonstrated that mutationally monomerized &alpha, 7 subunits can interact with the intrinsically monomeric &alpha, 4 and &alpha, 6 subunits, thereby forming heterotetradecameric complexes with a double-ring structure. The results of this study provide additional insights into the mechanisms underlying the assembly and disassembly of proteasomal subunits, thereby offering clues for the design and creation of circularly assembled hetero-oligomers based on homo-oligomeric structural frameworks.

Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used to analyze population-averaged NMR quantities. Essentially, to apply NMR successfully, both the type of experiment and equation to fit the data must be carefully and specifically chosen for the protein-ligand interaction under analysis. In this review, we first explain the exchange regimes and kinetic models of protein-ligand interactions, and then describe the NMR methods that quantitatively analyze these specific interactions.

Immunoglobulin G (IgG) is a major serum glycoprotein that exerts the role of antibody in the immune system. This multifunctional glycoprotein couples antigen recognition with a variety of effector functions promoted via interactions with various IgG-binding proteins. Given its versatile functionality, IgG has recently been used for therapeutic interventions. Evidence indicates that the carbohydrate moieties of IgG glycoproteins critically affect their antibody functions, particularly the effector functions mediated by the interactions with Fcγ receptors (FcγRs). N-glycans at specific positions of FcγR also contribute both positively and negatively to the interactions with IgG. The integration of multilateral biophysical approaches, including X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, has provided structural insights into the mechanisms underlying the glycofunctions of this interacting system. The N-glycans of IgG and FcγR mediate their interactions by either strengthening or weakening the affinity on the basis of their glycoforms. Moreover, the N-glycosylation of IgG-Fc is a prerequisite to maintain the integrity of the quaternary structure of the sites interacting with the effector molecules and can also control functionally relevant local conformations. The biopharmaceutical significance of these glycan functions is discussed from a structural point of view.

Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing with such complicated systems, given that the target molecules can be isotopically labeled. Methods of metabolic isotope labeling in recombinant glycoproteins have been developed recently using a variety of eukaryotic production vehicles, including mammalian, yeast, insect, and plant cells, each of which has a distinct N-glycan diversification pathway. Yeast genetic engineering has enabled the overexpression of homogeneous high-mannose-type oligosaccharides with 13C labeling for NMR characterization of their conformational dynamics. The utility of stable isotope-assisted NMR spectroscopy has also been demonstrated using the Fc fragment of immunoglobulin G (IgG) as a model glycoprotein, providing useful information regarding intramolecular carbohydrate–protein interactions. Transverse relaxation optimization of intact IgG with a molecular mass of 150 kDa has been achieved by tailored deuteration of selected amino acid residues using a mammalian expression system. This offers a useful probe for the characterization of molecular interaction networks in multimolecular crowded systems typified by serum. Perspectives regarding the development of techniques for tailoring glycoform designs and isotope labeling of recombinant glycoproteins are also discussed.

Glycophobia in structural biology is strongly associated with the unpredictable, heterogeneous nature of protein glycosylation and the complex, flexible structures of the glycoprotein glycans. Moreover, glycoproteins cannot be produced by conventional bacterial expression systems. Nuclear magnetic resonance (NMR) spectroscopy assisted by other analytical and preparative techniques can now successfully address these issues. Recombinant glycoproteins can be expressed with stable isotope labeling using a variety of eukaryotic production vehicles. Glycoforms of glycoproteins can be remodeled by genetic engineering of the production vehicles as well as in vitro enzymatic reactions. Stable-isotope-assisted NMR techniques have provided detailed information regarding conformational dynamics and interactions of the carbohydrate chains in solution, giving insights into the functional mechanisms of glycoprotein glycans.

Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system.

Target-specific monoclonal antibodies can be routinely acquired, but the sequences of naturally acquired antibodies are not always affinity-matured and methods that increase antigen affinity are desirable. Most biophysical studies have focused on the complementary determining region (CDR), which directly contacts the antigen; however, it remains difficult to increase the affinity as much as desired. While strategies to alter the CDR to increase antibody affinity are abundant, those that target non-CDR regions are scarce. Here we describe a new method, designated fluctuation editing, which identifies potential mutation sites and engineers a high-affinity antibody based on conformational fluctuations observed by NMR relaxation dispersion. Our data show that relaxation dispersion detects important fluctuating residues that are not located in the CDR and that increase antigen–antibody affinity by point mutation. The affinity-increased mutants are shown to fluctuate less in their free form and to form a more packed structure in their antigen-bound form.

Major histocompatibility complex (MHC) molecules are loaded with a wide variety of self- and non-self-peptides in their binding grooves and present these to T cell receptors (TCRs) in order to activate the adaptive immune system. A large number of crystal structures of different MHC alleles with different bound peptides have been determined, and they have been found to be quite similar to one another regardless of the bound peptide sequence. The structures do not change markedly even when forming complexes with TCRs. Nonetheless, the degree of TCR activation does differ markedly depending on the peptide presented by the MHC. Recent structural studies in solution rather than as crystals have suggested that the conformational dynamics of MHC molecules may be responsible for the MHC stability differences. Furthermore, it was shown that the conformational dynamics of MHC molecules is important for peptide loading and presentation to TCR. Here, we describe the static and dynamic structures of MHC molecules and appropriate methods to analyze them. We focus particularly on nuclear magnetic resonance (NMR), one of the most powerful tools to study dynamic properties of proteins. The number of such studies in the literature is limited, but in this review, we show that NMR is valuable for elucidating the structural dynamics of MHC molecules.

A recently developed integrative approach combining varied types of experimental data has been successfully applied to three-dimensional modelling of larger biomacromolecular complexes. Deuteration-assisted small-angle neutron scattering (SANS) plays a unique role in this approach by making it possible to observe selected components in the complex. It enables integrative modelling of biomolecular complexes based on building-block structures typically provided by X-ray crystallography. In this integrative approach, it is important to be aware of the flexible properties of the individual building blocks. Here we examine the ability of SANS to detect a subtle conformational change of a multidomain protein using the Fc portion of human immunoglobulin G (IgG) interacting with a soluble form of the low-affinity Fcγ receptor IIIb (sFcγRIIIb) as a model system. The IgG-Fc glycoprotein was subjected to SANS in the absence and presence of 75%-deuterated sFcγRIIIb, which was matched out in D2O solution. This inverse contrast-matching technique enabled selective observation of SANS from IgG-Fc, thereby detecting its subtle structural deformation induced by the receptor binding. The SANS data were successfully interpreted by considering previously reported crystallographic data and an equilibrium between free and sFcγRIIIb-bound forms. Our SANS data thus demonstrate the applicability of SANS in the integrative approach dealing with biomacromolecular complexes composed of weakly associated building blocks with conformational plasticity., Highlights • IgG-Fc glycoprotein was structurally characterized by small-angle neutron scattering. • Fc was selectively observed under equilibrium between free and receptor-bound forms. • Receptor-induced conformational change of Fc was successfully detected.

We studied the interaction between GroES and a single-ring mutant (SR1) of GroEL by the NMR titration of 15N-labeled GroES with SR1 at three different temperatures (20, 25 and 30°C) in the presence of 3 mM ADP in 100 mM KCl and 10 mM MgCl2 at pH 7.5. We used SR1 instead of wild-type double-ring GroEL to precisely control the stoichiometry of the GroES binding to be 1:1 ([SR1]:[GroES]). Native heptameric GroES was very flexible, showing well resolved cross peaks of the residues in a mobile loop segment (residue 17–34) and at the top of a roof hairpin (Asn51) in the heteronuclear single quantum coherence spectra. The binding of SR1 to GroES caused the cross peaks to disappear simultaneously, and hence it occurred in a single-step cooperative manner with significant immobilization of the whole GroES structure. The binding was thus entropic with a positive entropy change (219 J/mol/K) and a positive enthalpy change (35 kJ/mol), and the binding constant was estimated at 1.9×105 M−1 at 25°C. The NMR titration in 3 mM ATP also indicated that the binding constant between GroES and SR1 increased more than tenfold as compared with the binding constant in 3 mM ADP. These results will be discussed in relation to the structure and mechanisms of the chaperonin GroEL/GroES complex.

The Fc portion of immunoglobulin G (IgG) is a horseshoe-shaped homodimer, which interacts with various effector proteins, including Fc&gamma, receptors (Fc&gamma, Rs). These interactions are critically dependent on the pair of N-glycans packed between the two CH2 domains. Fucosylation of these N-glycans negatively affects human IgG1-Fc&gamma, RIIIa interaction. The IgG1-Fc crystal structures mostly exhibit asymmetric quaternary conformations with divergent orientations of CH2 with respect to CH3. We aimed to provide dynamic views of IgG1-Fc by performing long-timescale molecular dynamics (MD) simulations, which were experimentally validated by small-angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our simulation results indicated that the dynamic conformational ensembles of Fc encompass most of the previously reported crystal structures determined in both free and complex forms, although the major Fc conformers in solution exhibited almost symmetric, stouter quaternary structures, unlike the crystal structures. Furthermore, the MD simulations suggested that the N-glycans restrict the motional freedom of CH2 and endow quaternary-structure plasticity through multiple intramolecular interaction networks. Moreover, the fucosylation of these N-glycans restricts the conformational freedom of the proximal tyrosine residue of functional importance, thereby precluding its interaction with Fc&gamma, RIIIa. The dynamic views of Fc will provide opportunities to control the IgG interactions for developing therapeutic antibodies.

Exploration of the conformational spaces of flexible oligosaccharides is essential to gain deeper insights into their functional mechanisms. Here we characterised dynamic conformation of a high-mannose-type dodecasaccharide with a terminal glucose residue, a critical determinant recognised by molecular chaperones. The dodecasaccharide was prepared by our developed chemoenzymatic technique, which uses 13 C labelling and lanthanide tagging to detect conformation-dependent paramagnetic effects by NMR spectroscopy. The NMR-validated molecular dynamics simulation produced the dynamic conformational ensemble of the dodecasaccharide. This determined its spatial distribution as well as the glycosidic linkage conformation of the terminal glucose determinant. Moreover, comparison of our results with previously reported crystallographic data indicates that the chaperone binding to its target oligosaccharides involves an induced-fit mechanism.

Antibody-dependent cellular cytotoxicity (ADCC) is promoted through interaction between the Fc region of immunoglobulin G1 (IgG1) and Fcγ receptor IIIa (FcγRIIIa), depending on N-glycosylation of these glycoproteins. In particular, core fucosylation of IgG1-Fc N-glycans negatively affects this interaction and thereby compromises ADCC activity. To address the mechanisms of this effect, we performed replica-exchange molecular dynamics simulations based on crystallographic analysis of a soluble form of FcγRIIIa (sFcγRIIIa) in complex with IgG1-Fc. Our simulation highlights increased conformational fluctuation of the N-glycan at Asn162 of sFcγRIIIa upon fucosylation of IgG1-Fc, consistent with crystallographic data giving no interpretable electron density for this N-glycan, except for the innermost part. The fucose residue disrupts optimum intermolecular carbohydrate-carbohydrate interactions, rendering this sFcγRIIIa glycan distal from the Fc glycan. Moreover, our simulation demonstrates that core fucosylation of IgG1-Fc affects conformational dynamics and rearrangements of surrounding amino acid residues, typified by Tyr296 of IgG1-Fc, which was more extensively involved in the interaction with sFcγRIIIa without Fc core fucosylation. Our findings offer a structural foundation for designing and developing therapeutic antibodies with improved ADCC activity.