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Abstract: Introduction: Z-disk proteins play an essential role in stabilizing the contractile muscle apparatus. Genetic variants in these proteins are associated with cardiomyopathies, a group of genetic cardiac diseases linked to sudden cardiac death. Several studies have identified pathogenic variants in two prominent Z-disk proteins, alpha-actinin (ACTN2) and filamin C (FLNC). Notably, a 2014 study identified the ACTN2 variant M228T in a family of 11 patients with cardiomyopathy. Similarly, a recent investigation uncovered the FLNC variant M82K in two cardiomyopathy families. Both variants reside within the actin-binding domain of their respective Z-disk proteins, which is crucial for organizing and binding actin-thin filaments. However, the mechanisms by which these variants alter protein structure and function remain undefined. This study investigated the structural alterations and functional consequences associated with the ACTN2-M228T and FLNC-M82K variants. Methods: To explore the structural modifications in the ACTN2-M228T and FLNC-M82K mutant proteins, structural modelling approaches were employed. Additionally, recombinant proteins were expressed in a bacterial system and subsequently purified. A comprehensive array of biophysical techniques was utilized to detect alterations in protein structure. Functional assessments were performed using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) that incorporated the ACTN2-M228T variant. Results: Structural modeling predictions revealed that both ACTN2-M228T and FLNC-M82K variants share a common mechanism that adversely impact the regulatory function of the actin-binding domain. This hypothesis was further evaluated using actin-binding assays. The mutant proteins exhibited increased aggregation identified through mass photometry and size exclusion chromatography coupled with either multi-angle light scattering or small-angle X-ray scattering (SAXS). Moreover, both variants demonstrated reduced solubility and structural stability based on solubility and enzymatic digestion assays. The thermal stability of mutant proteins was also compromised, as assessed through SAXS and differential scanning fluorimetry. To correlate the identified structural alterations with disease mechanisms, the functional implications of ACTN2-M228T variant were assessed using an iPSC-CM model. Mutant cardiomyocytes showed increased ACTN2 aggregate formation and upregulation of hypertrophy, fibrosis, and autophagy markers. Furthermore, significant degradation of ACTN2 was observed through biochemical fractionation assays. ACTN2 destabilization was further evaluated using proteasome and protease inhibitors targeting the ubiquitin-proteosome system and the autophagy-lysosomal pathway, to elucidate their potential roles in the protein degradation process. Conclusion: Collectively, this study provides valuable insights into the effects of Z-disk variants on protein structure and function. The integration of structural and functional approaches represents a crucial step towards understanding disease pathways for cardiomyopathy-linked Z-disk variants.

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Abstract: Multi-crystal processing of X-ray diffraction data has become highly automated to keep pace with the current high-throughput capabilities afforded by beamlines. A significant challenge, however, is the automated clustering of such data based on subtle differences such as ligand binding or conformational shifts. Intensity-based hierarchical clustering has been shown to be a viable method of identifying such subtle structural differences, but the interpretation of the resulting dendrograms is difficult to automate. Using isomorphous crystals of bovine, porcine and human insulin, the existing clustering methods in the multi-crystal processing software xia2.multiplex were validated and their limits were tested. It was determined that weighting the pairwise correlation coefficient calculations with the intensity uncertainties was required for accurate calculation of the pairwise correlation coefficient matrix (correlation clustering) and dimension optimization was required when expressing this matrix as a set of coordinates representing data sets (cosine-angle clustering). Finally, the introduction of the OPTICS spatial density-based clustering algorithm into DIALS allowed the automatic output of species-pure clusters of bovine, porcine and human insulin data sets.

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Abstract: Multifunctionality, processivity, and thermostability are critical for the cost-effective enzymatic saccharification of non-food plant biomass polymers such as β-glucans, celluloses, and xylans to generate biofuels and other valuable products. We present molecular insights into a processive multifunctional endo-1,3-1,4-β-D-glucanase (Tt_End5A) from the hyperthermophilic bacterium Thermogutta terrifontis. Tt_End5A demonstrated activities against a broad spectrum of β-polysaccharides, including barley glucan, lichenan, carboxymethyl cellulose, regenerated amorphous cellulose (RAC), Avicel, xylan, laminarin, mannan, curdlan, xanthan, and various chromogenic substrates at pH 7 and temperatures ranging from 70-80°C. The enzyme exhibited a high level of processivity on RAC and retained over 90% activity at 80 °C for an extended period, indicating exceptional thermal stability. The 1.20 Å crystal structure of the Tt_End5A catalytic domain revealed an archetypal glycoside hydrolase family 5 (GH5) catalytic TIM-(β/α)8-barrel, supplemented with additional β-strands, elongated α-helices, and a rare cis-non-Pro (His481-cis-Ala482) peptide. A large central cleft was observed in the 3D structure, which is likely related to the enzyme's multifunctionality and processivity. The catalytic domain is preceded by a novel N-terminal multivalent carbohydrate-binding module (CBM) that enhances the enzymatic degradation of insoluble polysaccharides. Mutagenesis studies, ligand interaction analyses, and the structurally conserved positions of E329 and E448 in Tt_End5A suggest that these residues function as the proton donor and nucleophile in the catalytic mechanism. Owing to its multifunctionality and processivity, Tt_End5A can reduce the need for multiple saccharification enzymes to generate fermentable sugars from plant biomass for bioethanol production. Additionally, it holds promise for applications in the pharmaceutical, feed, and food industries.

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Abstract: The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the ‘arms race’ with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics.

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Abstract: A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities of homogenous, well diffracting microcrystals. Efficient high-throughput screening of batch-grown microcrystals and the determination of ground-state structures from different conditions is thus of considerable value in the early stages of a project. Here, a highly sample-efficient methodology to measure serial crystallography data from microcrystals by raster scanning within standard in situ 96-well crystallization plates is described. Structures were determined from very small quantities of microcrystal suspension and the results were compared with those from other sample-delivery methods. The analysis of a two-dimensional batch crystallization screen using this method is also described as a useful guide for further optimization and the selection of appropriate conditions for scaling up microcrystallization.