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NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.

Gaussia Luciferase (GLuc) is a renowned reporter protein that can catalyze the oxidation of coelenterazine (CTZ) and emit a bright light signal. GLuc comprises two consecutive repeats that form the enzyme body and a central putative catalytic cavity. However, deleting the C-terminal repeat only limited reduces the activity (over 30% residual luminescence intensity detectable), despite being a key part of the cavity. How does the remaining GLuc (tGLuc) catalyze CTZ? To address this question, we built a structural model of tGLuc by removing the C-terminal repeat from the resolved structure of intact GLuc, and verified that the cavity-forming component in GLuc remains stable and provides an open-mouth cavity in tGLuc during 500 ns MD simulations in water. Docking simulation and a followed umbrella sampling analysis further revealed that the cavity on tGLuc has a high affinity for CTZ, with a binding energy of up to -114 kJ/mol. Moreover, R76, a validated activity-critical amino acid residue, resides in the cavity and forms a stable hydrogen bond with CTZ. Then, we constructed a cluster model to examine the CTZ oxidation pathway in the cavity using Density Functional Theory (DFT) calculations. The result showed that the pathway consists of four elementary reactions, with the highest Gibbs energy barrier being 65.4 kJ/mol. Both intramolecular electron transfer and the convergence of S1/S0 potential energy surfaces occurred in the last elementary reaction, which was regarded as the reported Chemically-Initiated-Electron-Exchange-Luminescence (CIEEL) reaction. Geometry and wavefunction analysis on the pathway indicated that R76 plays a vital role in CTZ oxidation, which first anchors the environmental oxygen molecule and induces it to form a singlet biradical state, facilitating its attack on CTZ. Subsequently, R76 and the adjacent Q88, positioned near R76 through the tGLuc refolding process, stabilize the transition states and facilitate the emergence of radical electrons on CTZ at the onset of the CIEEL reaction, which contributes to the subsequent intramolecular electron transfer and the production of excited amide product. This study provides a comprehensive explanation of tGLuc's catalytic mechanism. However, it is important to note that these findings are specific to tGLuc and may not extend to other CTZ-based luciferases, particularly those lacking arginine in their catalytic cavities, which likely operate via distinct mechanisms. Author summary: The luciferase-catalyzed bioluminescence has garnered widespread application across various fields. Among luciferases, Gaussia luciferase (GLuc) is distinguished by its small size and intense luminescence, making it an ideal luminescent probe. Despite advances in GLuc's application development, its catalytic luminescence mechanism remains poorly understood. A major challenge arises from the structural analysis, which reveals GLuc as a monomeric enzyme with a single catalytic chamber. Surprisingly, even after deleting critical C-terminal sequences essential for chamber formation, the truncated enzyme retains considerable catalytic activity. In this study, we utilized molecular dynamics simulations to investigate how the remaining GLuc sequence refolds to regenerate the catalytic chamber. Furthermore, through density functional theory calculations and a cluster model, we mapped the complete reaction potential energy surface for CTZ oxidation within the reformed chamber, identifying R76 as the key catalytic residue and elucidating its role in reducing the reaction barrier to facilitate catalysis. Prior mutagenesis research has confirmed the critical role of R76 in catalytic activity, corroborating our computational results. [ABSTRACT FROM AUTHOR]

Author(s): Ryunosuke Saiki [sup.1] , Yukihide Momozawa [sup.2] , Yasuhito Nannya [sup.1] , Masahiro M. Nakagawa [sup.1] [sup.3] , Yotaro Ochi [sup.1] , Tetsuichi Yoshizato [sup.1] , Chikashi Terao [sup.4] [...], Clonal hematopoiesis (CH) in apparently healthy individuals is implicated in the development of hematological malignancies (HM) and cardiovascular diseases. Previous studies of CH analyzed either single-nucleotide variants and indels (SNVs/indels) or copy number alterations (CNAs), but not both. Here, using a combination of targeted sequencing of 23 CH-related genes and array-based CNA detection of blood-derived DNA, we have delineated the landscape of CH-related SNVs/indels and CNAs in 11,234 individuals without HM from the BioBank Japan cohort, including 672 individuals with subsequent HM development, and studied the effects of these somatic alterations on mortality from HM and cardiovascular disease, as well as on hematological and cardiovascular phenotypes. The total number of both types of CH-related lesions and their clone size positively correlated with blood count abnormalities and mortality from HM. CH-related SNVs/indels and CNAs exhibited statistically significant co-occurrence in the same individuals. In particular, co-occurrence of SNVs/indels and CNAs affecting DNMT3A, TET2, JAK2 and TP53 resulted in biallelic alterations of these genes and was associated with higher HM mortality. Co-occurrence of SNVs/indels and CNAs also modulated risks for cardiovascular mortality. These findings highlight the importance of detecting both SNVs/indels and CNAs in the evaluation of CH. Analysis of single-nucleotide variants and copy number alterations gives a more complete picture of clonal hematopoiesis and its impact on hematological malignancy and cardiovascular disease.

Objective: To evaluate the effects of primary anti-dengue virus envelop protein domain 3 (DENV-ED3) antibodies on secondary heterotypic anti-DENV ED3 antibody responses and the status of anti-DENV antibody responses against multivalent DENV ED3s in mice. Methods: Four different DENV-ED3s were purified and their biophysical characteristics were confirmed. Swiss albino mice aged 3-4 weeks were immunized with four different DENV-ED3s and the anti-ED3 IgG responses were determined by ELISA. Results: Firstly, the primary 1ED3-2ED3-3ED3 cross-reactive anti-DENV1 ED3 response boosted the secondary anti-2ED3 and anti-3ED3 antibody responses. In contrast, primary anti-2ED3 and anti-3ED3 antibodies neither had cross-recognition of 1ED3, nor had any effect on secondary anti-1ED3 response. Besides, the strict serospecificity of the anti-4ED3 sera did not affect other secondary anti-DENV ED3 responses. Secondly, 1ED3, 2ED3, and 3ED3 were co-dominantly immunogenic in trivalent ED3 formulations. However, the poorly immunogenic 4ED3 became almost non-immunogenic when injected after or together with 2ED3 and 3ED3, but showed slightly increased immunogenicity when injected with 1ED3, suggesting an adjuvanticity of 1ED3 on 4ED3's immunogenicity. Conclusions: Although DENV1~4 ED3s share similar sequence homologies and structures, their immune induction potentials differ significantly in terms of immune magnitude, sero-specificity, and sero-cross-reactivity. Such intrinsic features of DENV1~4 ED3s may lead to 'antigen interference', limiting both the understanding of dengue etiology and the success of dengue vaccine development, which needs to neutralize all four DENV serotypes equivalently. [ABSTRACT FROM AUTHOR]