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Structure characterization and classification is frequently based on local environment information of all or selected atomic sites in the crystal structure.
We report the novel crystal structure and characterization of symmetrical, homodimeric humanized heavy-chain-only antibodies or dimers (HC2s). HC2s were found to be significantly coexpressed and secreted along with mAbs from transient CHO HC/LC cotransfection, resulting in an unacceptable mAb developability attribute. Expression of full-length HC2s in the absence of LC followed by purification resulted in HC2s with high purity and thermal stability similar to conventional mAbs. The V H and C H 1 portion of the heavy chain (or Fd) was also efficiently expressed and yielded a stable, covalent, and reducible dimer (Fd2). Mutagenesis of all heavy chain cysteines involved in disulfide bond formation revealed that Fd2 intermolecular disulfide formation was similar to Fabs and elucidated requirements for Fd2 folding and expression. For one HC2, we solved the crystal structure of the Fd2 domain to 2.9 Å, revealing a highly symmetrical homodimer that is structurally similar to Fabs and is mediated by conserved (C H 1) and variable (V H ) contacts with all CDRs positioned outward for target binding. Interfacial dimer contacts revealed by the crystal structure were mutated for two HC2s and were found to dramatically affect HC2 formation while maintaining mAb bioactivity, offering a potential means to modulate novel HC2 formation through engineering. These findings indicate that human heavy-chain dimers can be secreted efficiently in the absence of light chains, may show good physicochemical properties and stability, are structurally similar to Fabs, offer insights into their mechanism of formation, and may be amenable as a novel therapeutic modality.
HLA class I proteins, a critical component in adaptive immunity, bind and present intracellular Ags to CD8+ T cells. The extreme polymorphism of HLA genes and associated peptide binding specificities leads to challenges in various endeavors, including neoantigen vaccine development, disease association studies, and HLA typing. Supertype classification, defined by clustering functionally similar HLA alleles, has proven helpful in reducing the complexity of distinguishing alleles. However, determining supertypes via experiments is impractical, and current in silico classification methods exhibit limitations in stability and functional relevance. In this study, by incorporating three-dimensional structures we present a method for classifying HLA class I molecules with improved breadth, accuracy, stability, and flexibility. Critical for these advances is our finding that structural similarity highly correlates with peptide binding specificity. The new classification should be broadly useful in peptide-based vaccine development and HLA–disease association studies.
Abstract Estimating the accuracy of quaternary structural models of protein complexes and assemblies (EMA) is important for predicting quaternary structures and applying them to studying protein function and interaction. The pairwise similarity between structural models is proven useful for estimating the quality of protein tertiary structural models, but it has been rarely applied to predicting the quality of quaternary structural models. Moreover, the pairwise similarity approach often fails when many structural models are of low quality and similar to each other. To address the gap, we developed a hybrid method (MULTICOM_qa) combining a pairwise similarity score (PSS) and an interface contact probability score (ICPS) based on the deep learning inter‐chain contact prediction for estimating protein complex model accuracy. It blindly participated in the 15th Critical Assessment of Techniques for Protein Structure Prediction (CASP15) in 2022 and performed very well in estimating the global structure accuracy of assembly models. The average per‐target correlation coefficient between the model quality scores predicted by MULTICOM_qa and the true quality scores of the models of CASP15 assembly targets is 0.66. The average per‐target ranking loss in using the predicted quality scores to rank the models is 0.14. It was able to select good models for most targets. Moreover, several key factors (i.e., target difficulty, model sampling difficulty, skewness of model quality, and similarity between good/bad models) for EMA are identified and analyzed. The results demonstrate that combining the multi‐model method (PSS) with the complementary single‐model method (ICPS) is a promising approach to EMA.
Peptide and protein drug molecules fold into higher order structures (HOS) in formulation and these folded structures are often critical for drug efficacy and safety. Generic or biosimilar drug products (DPs) need to show similar HOS to the reference product. The solution NMR spectroscopy is a non-invasive, chemically and structurally specific analytical method that is ideal for characterizing protein therapeutics in formulation. However, only limited NMR studies have been performed directly on marketed DPs and questions remain on how to quantitively define similarity. Here, NMR spectra were collected on marketed peptide and protein DPs, including calcitonin-salmon, liraglutide, teriparatide, exenatide, insulin glargine and rituximab. The 1D 1 H spectral pattern readily revealed protein HOS heterogeneity, exchange and oligomerization in the different formulations. Principal component analysis (PCA) applied to two rituximab DPs showed consistent results with the previously demonstrated similarity metrics of Mahalanobis distance (D M ) of 3.3. The 2D 1 H- 13 C HSQC spectral comparison of insulin glargine DPs provided similarity metrics for chemical shift difference (Δδ) and methyl peak profile, i.e., 4 ppb for 1 H, 15 ppb for 13 C and 98% peaks with equivalent peak height. Finally, 2D 1 H- 15 N sofast HMQC was demonstrated as a sensitive method for comparison of small protein HOS. The application of NMR procedures and chemometric analysis on therapeutic proteins offer quantitative similarity assessments of DPs with practically achievable similarity metrics.
Optical and 3.3-mm data for the Seyfert galaxy NGC 1275 (3C 84) are compared. Similarities exist between the variations at the two wavelengths with regard to amplitude, time scale, and interval between events. The question is raised whether these similarities may be the result of a common triggering mechanism.
Flavonoids are potent antioxidants that play a role in defense against pathogens, UV-radiation, and the detoxification of reactive oxygen species. Dihydroflavonol 4-reductase (DFR) and flavanone 4-reductase (FNR) reduce dihydroflavonols and flavanones, respectively, using NAD(P)H to produce flavan-(3)-4-(di)ols in flavonoid biosynthesis. Anthocyanidin reductase (ANR) reduces anthocyanidins to flavan-3-ols. In addition to their sequences, the 3D structures of recombinant DFR, FNR and ANR from sorghum and switchgrass showed a high level of similarity. The catalytic mechanism, substrate-specificity and key residues of three reductases were deduced from crystal structures, site-directed mutagenesis, molecular docking, kinetics, and thermodynamic ana-lyses. Although DFR displayed its highest activity against dihydroflavonols, it also showed activity against flavanones and anthocyanidins. It was inhibited by the flavonol quercetin and high concentrations of dihydroflavonols/flavonones. SbFNR1 and SbFNR2 did not show any activity against dihydroflavonols. However, SbFNR1 displayed activity against flavanones and ANR activity against two anthocyanidins, cyanidin and pelargonidin. Therefore, SbFNR1 and SbFNR2 could be specific ANR isozymes without delphinidin activity. Sorghum has high concentrations of 3-deoxyanthocyanidins in vivo, supporting the observed high activity of SbDFR against flavonols. Mining of expression data indicated substantial induction of these three reductase genes in both switchgrass and sorghum in response to biotic stress. Key signature sequences for proper DFR/ANR classification are proposed and could form the basis for future metabolic engineering of flavonoid metabolism.
QUESTS provides a strategy to quantify the uncertainty and the information content (entropy) of datasets of atomistic simulations. The code implements the calculation of a descriptor, compares information contents of datasets, verifies whether given structures are contained in specific datasets, and more. This code base enables better design of training sets for machine learning force fields as well as understanding some thermodynamic properties of materials.
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Activated protein C is a trypsin-like protease with anticoagulant and cytoprotective properties that is generated by thrombin from the zymogen precursor protein C in a reaction greatly accelerated by the cofactor thrombomodulin. The molecular details of this activation remain elusive due to the lack of structural information. We now fill this gap by providing information on the overall structural organization of these proteins using single molecule Förster resonance energy transfer and small angle X-ray scattering. Under physiological conditions, both zymogen and protease adopt a conformation with all domains vertically aligned along an axis 76 Å long and maximal particle size of 120 Å. This conformation is stabilized by binding of Ca 2+ to the Gla domain and is affected minimally by interaction with thrombin. Hence, the zymogen protein C likely interacts with the thrombin-thrombomodulin complex through a rigid body association that produces a protease with essentially the same structural architecture. This scenario stands in contrast to an analogous reaction in the coagulation cascade where conversion of the zymogen prothrombin to the protease meizothrombin by the prothrombinase complex is linked to a large conformational transition of the entire protein. The presence of rigid EGF domains in protein C as opposed to kringles in prothrombin likely accounts for the different conformational plasticity of the two zymogens. The new structural features reported here for protein C have general relevance to vitamin K-dependent clotting factors containing EGF domains, such as factors VII, IX, and X.
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Abstract not provided.
The phononic dispersion relations calculated by solving an eigenvalue problem while applying Bloch-Floquet boundary conditions are applied to three varieties of phononic pseudocrystals interposers useful for suppressing the transmission of structure-born vibration.
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Fabrication of scale model of Apollo service module and lunar module cone extension, fairing between command and service modules, and modified command module base plate and sting
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