Solution NMR Experiments Research Articles

A salt bridge of the C-terminal carboxyl group regulates PHPT1 substrate affinity and catalytic activity.

PHPT1 is a histidine phosphatase that modulates signaling in eukaryotes through its catalytic activity. Here, we present an analysis of the structure and dynamics of PHPT1 through a combination of solution NMR, molecular dynamics, and biochemical experiments. We identify a salt bridge formed between the R78 guanidinium moiety and the C-terminal carboxyl group on Y125 that is critical for ligand binding. Disruption of the salt bridge by appending a glycine residue at the C-terminus (G126) leads to a decrease in catalytic activity and binding affinity for the pseudo substrate, para-nitrophenylphosphate (pNPP), as well as the active site inhibitor, phenylphosphonic acid (PPA). We show through NMR chemical shift, 15N relaxation measurements, and analysis of molecular dynamics trajectories, that removal of this salt bridge results in an active site that is altered both structurally and dynamically thereby significantly impacting enzymatic function and confirming the importance of this electrostatic interaction.

Insights into the photoswitch based on 5-deazaFMN and LOV2 from Avena sativa: a combined absorption and NMR spectroscopy study.

The LOV2 domain from Avena sativa (As) is a blue light receptor that undergoes adduct formation with the native flavin mononucleotide (FMN) cofactor [Salomon et al., Biochemistry, 2000, 39, 9401]. We report the photochemical changes of AsLOV2 through cofactor exchange with the FMN analogue 5-deazaFMN. Absorption spectroscopy shows that upon illumination a thermodynamically stable adduct is formed. We were able to confirm the structure of the adduct by introducing 13C-labelled 5-deazaFMN isotopologues in solution NMR experiments. Dark-adapted state recovery can be photo-induced, providing a photoswitch that is easy to manipulate. The robust photocycle is repeatable without significant loss. Based on the data presented we propose the system as an alternative to wild-type AsLOV2 for applications in optogenetics.

Controlling the Redox Catalytic Activity of a Cyclic Selenide Fused to 18-Crown-6 by the Conformational Transition Induced by Coordination to an Alkali Metal Ion.

trans-3,4-Dihydroxyselenolane (DHS), a water-soluble cyclic selenide, exhibits selenoenzyme-like unique redox activities through reversible oxidation to the corresponding selenoxide. Previously, we demonstrated that DHS can be applied as an antioxidant against lipid peroxidation and a radioprotector by means of adequate modifications of the two hydroxy (OH) groups. Herein, we synthesized new DHS derivatives with a crown-ether ring fused to the OH groups (DHS-crown-n (n = 4 to 7), 1-4) and investigated their behaviors of complex formation with various alkali metal salts. According to the X-ray structure analysis, it was found that the two oxygen atoms of DHS change the directions from diaxial to diequatorial by complexation. The similar conformational transition was also observed in solution NMR experiments. The 1H NMR titration in CD3OD further confirmed that DHS-crown-6 (3) forms stable 1:1 complexes with KI, RbCl and CsCl, while it forms a 2:1 complex with KBPh4. The results suggested that the 1:1 complex (3·MX) exchanges the metal ion with metal-free 3 through the formation of the 2:1 complex. The redox catalytic activity of 3 was evaluated using a selenoenzyme model reaction between H2O2 and dithiothreitol. The activity was significantly reduced in the presence of KCl due to the complex formation. Thus, the redox catalytic activity of DHS could be controlled by the conformational transition induced by coordination to an alkali metal ion.

Supramolecular Host–Guest Assemblies of [M6Cl14]2–, M = Mo, W, Clusters with γ-Cyclodextrin for the Development of CLUSPOMs

Host–guest assemblies open up opportunities for developing novel functional CLUSPOM multicomponent systems based on transition metal clusters (CLUS), polyoxometalates (POMs) and macrocyclic organic ligands. In water–ethanol solution γ-cyclodextrin (γ-CD) interacts with halide metal clusters [M6Cl14]2– (M = Mo, W) to form sandwich-type structures. The supramolecular association between the clusters and CDs, however, remains weak in solution, and the interactions are not strong enough to prevent the hydrolysis of the inorganic guest. Although analysis of the resulting crystal structures reveals inclusion complexation, 1H NMR experiments in solution show no specific affinity between the two components. The luminescent properties of the host–guest compounds in comparison with the initial cluster complexes are also studied to evaluate the influence of CD.

Structural Insights into the Interaction between Bacillus subtilis SepF Assembly and FtsZ by Solid-State NMR Spectroscopy.

In many species of Gram-positive bacteria, SepF participated in the membrane tethering of FtsZ Z-ring during bacteria division. However, atomic-level details of interaction between SepF and FtsZ in an assembled state are lacking. Here, by combining solid-state NMR (SSNMR) with biochemical analyses, the interaction of Bacillus subtilis SepF and the C-terminal domain (CTD) of FtsZ was investigated. We obtained near complete chemical shift assignments of SepF and determined the structural model of the SepF monomer. Interaction with FtsZ-CTD caused further packing of SepF rings, and SSNMR experiments revealed the affected residues locating at α1, α2, β3, and β4 of SepF. Solution NMR experiments of dimeric SepF constructed by point mutation strategy proved a prerequisite role of α-α interface formation in SepF for FtsZ binding. Overall, our results provide structural insights into the mechanisms of SepF-FtsZ interaction for better understanding the function of SepF in bacteria.

Resolving Chemical and Spatial Heterogeneities at Complex Electrochemical Interfaces in Li-Ion Batteries

The high specific capacities of Ni-rich transition-metal oxides have garnered immense interest for improving the energy density of Li-ion batteries (LIBs). Despite the potential of these materials, Ni-rich cathodes suffer from interfacial instabilities that lead to crystallographic rearrangement of the active material surface as well as the formation of a cathode electrolyte interphase (CEI) layer on the composite surface during electrochemical cycling. While changes in crystallographic structure can be detected with diffraction-based methods, probing the chemistry of the disordered, heterogeneous CEI layer is challenging. In this work, we use a combination of ex situ solid-state nuclear magnetic resonance (SSNMR) spectroscopy and X-ray photoemission electron microscopy (XPEEM) to provide chemical and spatial information, on the nanometer length scale, on the CEI deposited on LiNi0.8Mn0.1Co0.1O2 (NMC811) composite cathode films. XPEEM elemental maps offer insight into the lateral arrangement of the electrolyte decomposition products that comprise the CEI and paramagnetic interactions (assessed with electron paramagnetic resonance (EPR) and relaxation measurements) in 13C SSNMR provide information on the radial arrangement of the CEI from the NMC811 particles outward. Using this approach, we find that LiF, Li2CO3, and carboxy-containing structures are directly appended to NMC811 active particles, whereas soluble species detected during in situ 1H and 19F solution NMR experiments (e.g., alkyl carbonates, HF, and vinyl compounds) are randomly deposited on the composite surface. We show that the combined approach of ex situ SSNMR and XPEEM, in conjunction with in situ solution NMR, allows spatially resolved, molecular-level characterization of paramagnetic surfaces and new insights into electrolyte oxidation mechanisms in porous electrode films.

Aggregation and the Intrinsic Structural Disorder of Dipeptide Repeat Peptides of C9orf72-Related Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Characterized by NMR.

Dipeptide repeats (DPRs) are known to play important roles in C9ORF72-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies on DPRs have reported on the kinetics of aggregation, toxicity, and low-resolution morphology of the aggregates of these peptides. While the dipeptide hexa-repeats of Gly-Pro [(GP)6] have been shown to be nonaggregating, Gly-Ala [(GA)6] and Gly-Arg [(GR)6] exhibited the formation of neurotoxic aggregates. However, structural studies of these DPRs have been elusive. In this study, we explored the feasibility of a high-resolution monitoring of a real-time aggregation of these peptides in a solution by using NMR experiments. Although (GP)6 is disordered and nonaggregating, the existence of cis and trans conformations was observed from NMR spectra. It was remarkable that the (GR)6 exhibited the formation of multiple conformations, whereas the hydrophobic and low-soluble (GA)6 aggregated fast in a temperature-dependent manner. These results demonstrate the feasibility of monitoring the minor conformational changes from highly disordered peptides, aggregation kinetics, and the formation of small molecular weight aggregates by solution NMR experiments. The ability to detect cis and trans local isomerizations in (GP)6 is noteworthy and could be valuable to study intrinsically disordered proteins/peptides by NMR. The early detection of minor conformational changes could be valuable in better understanding the mechanistic insights into the formation of toxic intermediates and the development of approaches to inhibit them and, potentially, aid in the development of compounds to treat the devastating C9ORF72-related ALS and FTD diseases.

Potential Use of Flavonoids Baicalin, Baicalein and Quercetin as Drug Absorption Enhancer by Mitigating Epithelial Tight Junction

The tight junction (TJ) is the apical-most intercellular junction complex, serving as a biological barrier of intercellular spaces between epithelial cells. The TJ's integrity is thought to be maintained by a key protein–protein interaction between C-terminal motifs of claudins (CLDs) and the postsynaptic density 95 (PSD-95)/discs large/zonula occludens 1 (ZO-1; PDZ) domains of ZO-1. Weak but direct interaction of baicalin, baicalein, and hesperetin—which are known pharmacologically active flavonoids—with ZO-1(PDZ1) have been assessed by solution NMR experiments. We observed TJ-mitigating activity of these flavonoids against Madin-Darby canine kidney (MDCK) II cells with the downregulation of subcellular localization of CLD-2 at TJs. In addition, we also found that quercetin, another physiologically relevant flavonoid, showed down regulation of TJ integrity, although quercetin did not bind ZO-1(PDZ1). We propose that inhibition of protein-protein interaction mediated by PDZ domains of TJ scaffolding proteins is one of the potential mechanisms for TJ-mitigation. We further confirmed that 1.2 times increase of paracellular efflux of FITC-labeled insulin of baicalin and baicalein on Caco-2 cell monolayers. In addition, a marked decrease of TEER by quercetin on Caco-2 cell monolayers was observed. These results suggested some of flavonoids are useful as a drug absorption enhancer.

NMR unveils an N-terminal interaction interface on acetylated-α-synuclein monomers for recruitment to fibrils

Amyloid fibril formation of α-synuclein (αS) is associated with multiple neurodegenerative diseases, including Parkinson's disease (PD). Growing evidence suggests that progression of PD is linked to cell-to-cell propagation of αS fibrils, which leads to seeding of endogenous intrinsically disordered monomer via templated elongation and secondary nucleation. A molecular understanding of the seeding mechanism and driving interactions is crucial to inhibit progression of amyloid formation. Here, using relaxation-based solution NMR experiments designed to probe large complexes, we probe weak interactions of intrinsically disordered acetylated-αS (Ac-αS) monomers with seeding-competent Ac-αS fibrils and seeding-incompetent off-pathway oligomers to identify Ac-αS monomer residues at the binding interface. Under conditions that favor fibril elongation, we determine that the first 11 N-terminal residues on the monomer form a common binding site for both fibrils and off-pathway oligomers. Additionally, the presence of off-pathway oligomers within a fibril seeding environment suppresses seeded amyloid formation, as observed through thioflavin-T fluorescence experiments. This highlights that off-pathway αS oligomers can act as an auto-inhibitor against αS fibril elongation. Based on these data taken together with previous results, we propose a model in which Ac-αS monomer recruitment to the fibril is driven by interactions between the intrinsically disordered monomer N terminus and the intrinsically disordered flanking regions (IDR) on the fibril surface. We suggest that this monomer recruitment may play a role in the elongation of amyloid fibrils and highlight the potential of the IDRs of the fibril as important therapeutic targets against seeded amyloid formation.

Luminescent cyclometalated platinum(ii) complexes with acyclic diaminocarbene ligands: structural, photophysical and biological properties.

Four new cyclometalated Pt(ii) complexes bearing acyclic diaminocarbene (ADC) ligands, [Pt(C^N)Cl{C(NHXyl)(NHR)}] [C^N = 2,6-difluorophenylpyridine (dfppy), phenylquinoline (pq); R = Pr 3a, 4a, CH2Ph 3b, 4b], were prepared by the nucleophilic attack on the isocyanide [Pt(C^N)Cl(CNXyl)] (C^N = dfppy 1, pq 2) by the corresponding amine RNH2 (R = Pr, CH2Ph). Complexes 3 show in their 1H NMR spectra in CDCl3 a notable concentration dependence, with a clear variation of the δH (NHXyl) signal, suggesting an assembling process implying donor-acceptor NHXylCl bonding, also supported by 1D-PGSE (Pulse Field Gradient Spin Echo) and 2D-DOSY (Diffusion Ordered Spectroscopy) NMR experiments in solution and X-ray diffraction studies. The intermolecular interactions in compounds 3a and 3b were studied by using Hirshfeld surface analysis and Non-Covalent Interaction (NCI) methods on their X-ray structures. Their photophysical properties were investigated by absorption and emission spectroscopies and also by TD-DFT calculations performed on 3a and 4b. These complexes show green (3) or orange (4) phosphorescence, attributed to a mixed 3IL/3MLCT excited state. The carbene ligand does not affect the emission maxima but it produces an increase of the quantum yields in relation to the isocyanide in the precursors. In fluid solutions, the emission is not concentration-dependent, but the complexes may show aggregation induced emission as detailed for complexes 3a and 4a. In addition, cytotoxicity studies in the human cell lines A549 (lung carcinoma) and HeLa (cervix carcinoma) showed good activity for these complexes and 3a, 3b and 4a exhibit a strong effect on DNA electrophoretic mobility. To the best of our knowledge, compounds 3 and 4 represent the first examples of cycloplatinated complexes bearing acyclic diamino carbenes with antiproliferative properties.

Contrasting the conformational effects of α-O-GalNAc and α-O-Man glycan protein modifications and their impact on the mucin-like region of alpha-dystroglycan.

We have carried out a comparative study of the conformational impact of modifications to threonine residues of either α-O-Man or α-O-GalNAc in the context of a sequence from the mucin-like region of α-dystroglycan. Both such modifications can coexist in this domain of the glycoprotein. Solution NMR experiments and molecular dynamics calculations were employed. Comparing the results for an unmodified peptide Ac- PPTTTTKKP-NH2 sequence from α-dystroglycan, and glycoconjugates with either modification on the Ts, we find that the impact of the α-O-Man modification on the peptide scaffold is quite limited, while that of the α-O-GalNAc is more profound. The results for the α-O-GalNAc glycoconjugate are consistent with what has been seen earlier in other systems. Further examination of the NMR-based structure and the MD results suggest a more extensive network of hydrogen bond interactions within the α-O-GalNAc-threonine residue than has been previously appreciated, which influences the properties of the protein backbone. The conformational effects are relevant to the mechanical properties of α-dystroglycan.

Fragment-derived modulators of an industrial β-glucosidase.

A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme–substrate complex.

Calcium and hydroxyapatite binding site of human vitronectin provides insights to abnormal deposit formation

The human blood protein vitronectin (Vn) is a major component of the abnormal deposits associated with age-related macular degeneration, Alzheimer's disease, and many other age-related disorders. Its accumulation with lipids and hydroxyapatite (HAP) has been demonstrated, but the precise mechanism for deposit formation remains unknown. Using a combination of solution and solid-state NMR experiments, cosedimentation assays, differential scanning fluorimetry (DSF), and binding energy calculations, we demonstrate that Vn is capable of binding both soluble ionic calcium and crystalline HAP, with high affinity and chemical specificity. Calcium ions bind preferentially at an external site, at the top of the hemopexin-like (HX) domain, with a group of four Asp carboxylate groups. The same external site is also implicated in HAP binding. Moreover, Vn acquires thermal stability upon association with either calcium ions or crystalline HAP. The data point to a mechanism whereby Vn plays an active role in orchestrating calcified deposit formation. They provide a platform for understanding the pathogenesis of macular degeneration and other related degenerative disorders, and the normal functions of Vn, especially those related to bone resorption.

Unsaturated Fatty Acid-Induced Conformational Transitions and Aggregation of the Repeat Domain of Tau.

Background: The intrinsically disordered, amyloidogenic protein Tau associates with diverse classes of molecules, including proteins, nucleic acids, and lipids. Mounting evidence suggests that fatty acid molecules could play a role in the dysfunction of this protein, however, their interaction with Tau remains poorly characterized. Methods: In a bid to elucidate the association of Tau with unsaturated fatty acids at the sub-molecular level, we carried out a variety of solution NMR experiments in combination with circular dichroism and fluorescence measurements. Our study shows that Tau4RD, the highly basic four-repeat domain of Tau, associates strongly with arachidonic and oleic acid assemblies in a high lipid/protein ratio, perturbing their supramolecular states and itself undergoing time-dependent structural adaptation. The structural signatures of Tau4RD/fatty acid aggregates appear similar for arachidonic acid and oleic acid, however, they are distinct from those of another prototypical intrinsically disordered protein, α-synuclein, when bound to these lipids, revealing protein-specific conformational adaptations. Both fatty acid molecules are found to invariably promote the self-aggregation of Tau4RD and of α-synuclein. Conclusions: This study describes the reciprocal influence that Tau4RD and fatty acids exert on their conformational states, contributing to our understanding of fundamental aspects of Tau/lipid co-assembly.

Structure and allosteric activity of a single-disulfide conopeptide from Conus zonatus at human α3β4 and α7 nicotinic acetylcholine receptors

Conopeptides are neurotoxic peptides in the venom of marine cone snails and have broad therapeutic potential for managing pain and other conditions. Here, we identified the single-disulfide peptides Czon1107 and Cca1669 from the venoms of Conus zonatus and Conus caracteristicus, respectively. We observed that Czon1107 strongly inhibits the human α3β4 (IC50 15.7 ± 3.0 μm) and α7 (IC50 77.1 ± 0.05 μm) nicotinic acetylcholine receptor (nAChR) subtypes, but the activity of Cca1669 remains to be identified. Czon1107 acted at a site distinct from the orthosteric receptor site. Solution NMR experiments revealed that Czon1107 exists in equilibrium between conformational states that are the result of a key Ser4-Pro5cis-trans isomerization. Moreover, we found that the X-Pro amide bonds in the inter-cysteine loop are rigidly constrained to cis conformations. Structure-activity experiments of Czon1107 and its variants at positions P5 and P7 revealed that the conformation around the X-Pro bonds (cis-trans) plays an important role in receptor subtype selectivity. The cis conformation at the Cys6-Pro7 peptide bond was essential for α3β4 nAChR subtype allosteric selectivity. In summary, we have identified a unique single-disulfide conopeptide with a noncompetitive, potentially allosteric inhibitory mechanism at the nAChRs. The small size and rigidity of the Czon1107 peptide could provide a scaffold for rational drug design strategies for allosteric nAChR modulation. This new paradigm in the "conotoxinomic" structure-function space provides an impetus to screen venom from other Conus species for similar, short bioactive peptides that allosterically modulate ligand-gated receptor function.

Surface coordination chemistry of germanium nanocrystals synthesized by microwave-assisted reduction in oleylamine

As surface ligands play a critical role in the colloidal stability and optoelectronic properties of semiconductor nanocrystals, we used solution NMR experiments to investigate the surface coordination chemistry of Ge nanocrystals synthesized by a microwave-assisted reduction of GeI2 in oleylamine. The as-synthesized Ge nanocrystals are coordinated to a fraction of strongly bound oleylamide ligands (with covalent X-type Ge-NHR bonds) and a fraction of more weakly bound (or physisorbed) oleylamine, which readily exchanges with free oleylamine in solution. The fraction of strongly bound oleylamide ligands increases with increasing synthesis temperature, which also correlates with better colloidal stability. Thiol and carboxylic acid ligands bind to the Ge nanocrystal surface only upon heating, suggesting a high kinetic barrier to surface binding. These incoming ligands do not displace native oleylamide ligands but instead appear to coordinate to open surface sites, confirming that the as-prepared nanocrystals are not fully passivated. These findings will allow for a better understanding of the surface chemistry of main group nanocrystals and the conditions necessary for ligand exchange to ultimately maximize their functionality.

Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics.

CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.

Origin of conformational dynamics in a globular protein

Protein structures are dynamic, undergoing motions that can play a vital role in function. However, the link between primary sequence and conformational dynamics remains poorly understood. Here, we studied how conformational dynamics can arise in a globular protein by evaluating the impact of individual core-residue substitutions in DANCER-3, a streptococcal protein G domain β1 variant that we previously designed to undergo a specific mode of conformational exchange that has never been observed in the wild-type protein. Using a combination of solution NMR experiments and molecular dynamics simulations, we demonstrate that only two mutations are necessary to create this conformational exchange, and that these mutations work synergistically, with one destabilizing the native structure and the other allowing two new conformational states to be accessed on the energy landscape. Overall, our results show how dynamics can appear in a stable globular fold, a critical step in the molecular evolution of dynamics-linked functions.

Drug-carrier binding and enzymatic carrier digestion in amorphous solid dispersions containing proteins as carrier

In order to further explain the ability of gelatin 50PS and bovine serum albumin (BSA) to generate supersaturation of a series of poorly soluble drugs (carbamazepine, cinnarizine, diazepam, itraconazole, nifedipine, indomethacin, darunavir (ethanolate), ritonavir, fenofibrate, griseofulvin, ketoconazole, naproxen, phenylbutazone and phenytoin), drug-polymer binding was investigated using solution NMR and equilibrium dialysis experiments. Binding characteristics of the biopolymers were compared to those of PVP, PVPVA and HPMC. Since both biopolymers are prone to enzymatic digestion, we evaluated the influence of proteolytic enzymes like pepsin and pancreatin on the dissolution properties of poorly soluble compounds when formulated as amorphous solid dispersions with gelatin 50PS and BSA. Evidence is being presented that supports the importance of drug-polymer binding in inducing and stabilizing supersaturation of poorly soluble drugs and enhancing dissolution from ASDs. In fact, BSA displayed drug binding with nearly all tested model drugs while in case of gelatin 50PS binding was observed for 5 out of 12 drugs. Addition of pepsin or pancreatin during dissolution of the biopolymer-containing ASDs leads to a drop in the concentration of the drug pointing to enzymatic digestion of the gelatin and BSA. However, after digestion, these formulations still outperformed their crystalline counterparts.

Structure and Function of Molecular Chaperones that Govern Immune Peptide Loading.

Major histocompatibility class I (MHC-I) molecules bind peptides derived from cellular synthesis and display them at the cell surface for recognition by receptors on T lymphocytes (TCR) or natural killer (NK) cells. Such recognition provides a crucial step in autoimmunity, identification of bacterial and viral pathogens, and anti-tumor responses. Understanding the mechanism by which such antigenic peptides in the ER are loaded and exchanged for higher affinity peptides onto MHC molecules has recently been clarified by cryo-EM and X-ray studies of the multimolecular peptide loading complex (PLC) and a unimolecular tapasin-like chaperone designated TAPBPR. Insights from these structural studies and complementary solution NMR experiments provide a basis for understanding mechanisms related to immune antigen presentation.