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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: N-utilization substance A (NusA) is a regulatory factor with pleiotropic functions in gene expression in bacteria. Archaea encode two conserved small proteins, NusA1 and NusA2, with domains orthologous to the two RNA binding K Homology (KH) domains of NusA. Here, we report the crystal structures of NusA2 from Sulfolobus acidocaldarius and Saccharolobus solfataricus obtained at 3.1 Å and 1.68 Å, respectively. NusA2 comprises an N-terminal zinc finger followed by two KH-like domains lacking the GXXG signature. Despite the loss of the GXXG motif, NusA2 binds single-stranded RNA. Mutations in the zinc finger domain compromise the structural integrity of NusA2 at high temperatures and molecular dynamics simulations indicate that zinc binding provides an energy barrier preventing the domain from reaching unfolded states. A structure-guided phylogenetic analysis of the KH-like domains supports the notion that the NusA2 clade is ancestral to the ribosomal protein eS7 in eukaryotes, implying a potential role of NusA2 in translation.

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Abstract: The identification of multiple simultaneous orientations of small molecule inhibitors binding to a protein target is a common challenge. It has recently been reported that the conformational heterogeneity of ligands is widely underreported in the Protein Data Bank, which is likely to impede optimal exploitation to improve affinity of these ligands. Significantly less is even known about multiple binding orientations for fragments (<300 Da), although this information would be essential for subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here, we use recently reported fragment hits for the SARS-CoV-2 non-structural protein 1 (nsp1) N-terminal domain to propose a general procedure for unambiguously identifying binding orientations of 2-dimensional fragments containing either sulphur or chloro substituents within the wavelength range of most tunable beamlines. By measuring datasets at two energies, using a tunable beamline operating in vacuum and optimised for data collection at very low X-ray energies, we show that the anomalous signal can be used to identify multiple orientations in small fragments containing sulphur and/or chloro substituents or to verify recently reported conformations. Although in this specific case we identified the positions of sulphur and chlorine in fragments bound to their protein target, we are confident that this work can be further expanded to additional atoms or ions which often occur in fragments. Finally, our improvements in the understanding of binding orientations will also serve to improve the rational optimisation of SARS-CoV-2 nsp1 fragment hits.

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Abstract: Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation.

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Abstract: The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin–binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca2+ in nonmuscle cells. Here we report the mechanism of Ca2+-mediated regulation of Entamoeba histolytica α-actinin-2 (EhActn2) with features expected for the common ancestor of Entamoeba and higher eukaryotic α-actinins. Crystal structures of Ca2+-free and Ca2+-bound EhActn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the EhActn2 CaMD for Ca2+, binding of which can only be regulated in the presence of physiological concentrations of Mg2+. Ca2+ binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin–binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that EhActn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery.