Journal of structural biology最新文献

The mevalonate pathway provides isoprenoid building blocks required for the biosynthesis of more complex downstream products, including cholesterol, as well as for the posttranslational prenylation of membrane-associated proteins. Farnesyl pyrophosphate synthase (FPPS) is a key regulatory enzyme in this pathway and an established drug target for bone-resorption disorders, with more recent interest in its inhibition as a potential anticancer strategy. In addition to classical active-site inhibitors such as nitrogen-containing bisphosphonates, several chemically distinct small molecules inhibit FPPS via an allosteric site involved in a product-mediated feedback regulation. Here, we report the discovery of a previously unrecognized ligand-binding site in FPPS. Crystallographic analysis reveals that several bisphosphonate compounds, previously thought to bind to the allosteric site under metal-free conditions, instead bind to a distinct cryptic pocket. Located adjacent to the known allosteric site, this pocket is absent in the native enzyme conformation. Its formation is driven by a conformational rearrangement of the C-terminal helix, which alternates between opening the allosteric pocket and the cryptic pocket in a mutually exclusive manner. Molecular dynamics simulations indicate that the cryptic pocket does not open spontaneously from the native state on the simulated timescale and likely requires ligand binding. Once induced, the open conformation is stabilized by residues Phe239 and Ile348. Together, these findings expand the known conformational landscape of FPPS and identify a new ligandable site that may be relevant for future chemical biology and drug discovery efforts.

The Zika virus protease, composed of the cofactor region from NS2B and the N-terminal region of NS3, plays a critical role in viral polyprotein maturation and represents an attractive therapeutic target. However, developing small-molecule inhibitors for its highly hydrophilic active site remains challenging, highlighting the importance of pursuing allosteric inhibition strategies. In this study, we engineered an NS2B-NS3 protease containing an 18-residue NS2B sequence linked to the N-terminal region of NS3 via a glycine-rich linker. We determined its crystal structure and obtained the solution NMR spectrum with backbone resonance assigned. This new construct was used in fragment screening and two new fragments were identified. This design excludes the C-terminal part of NS2B cofactor region, whose conformation is influenced by substrate or inhibitor binding, making the construct particularly valuable for screening and characterizing allosteric inhibitors.

Single amino acid substitutions in the ATP-binding domain of ACVRL1, a key receptor in the bone morphogenetic protein (BMP) signaling pathway, are frequently classified as variants of uncertain significance (VUS), complicating molecular diagnosis for pulmonary arterial hypertension (PAH) and Hereditary Hemorrhagic Telangiectasia (HHT). Since aberrant ATP binding disrupts downstream SMAD1/5/8 phosphorylation, we employed molecular dynamics (MD) simulations to quantitatively assess the functional impact of these variants. We first validated our approach on 20 known pathogenic/likely pathogenic variants within 5Å of the ATP-binding site, finding that 18 (90%) caused significant alterations in binding affinity (|d| ≥ 0.8, p < 0.001). We then applied this protocol to all known VUS, conflicting, and unclassified variants within the same region, reclassifying 20 of 32 (63%) as likely pathogenic. Comprehensive in silico mutagenesis of all possible substitutions at ATP-binding pocket positions, combined with InterVar classification under HHT phenotype, enabled reclassification of 9 of 12 (75%) VUS as likely pathogenic. Finally, we demonstrated the applicability of this approach in two PAH patients with HHT carrying ACVRL1 VUS. This work establishes MD simulation of ATP-binding affinity as an effective and scalable tool for the functional interpretation of kinase variants, with broad potential for application across other disease-associated kinases.

Micro-computed tomography and semantic segmentation provide insights into the structural hierarchy of biomineralized tissues, as exemplified by stony coral skeletons. Non-destructive imaging reveals mechanistic aspects of skeletal growth, including variations in porosity, density, and skeletal thickness across species and in the context of disease. Recent developments in semantic segmentation based on convolutional neural network models are poised to transform the streamlined analysis of large 3D tomography datasets, surpassing the performance of traditional segmentation methods. In this work, a series of U-Net deep learning models were trained and applied on exemplary micro-CT datasets from stony corals pertaining to Montastraea cavernosa and Porites astreoides species for the segmentation of pores and skeleton. The models were statistically evaluated, revealing that Attention U-Net was the top performer with respect to computational efficiency, accuracy, and generalizability, followed by U-Net++ and standard U-Net. Our analysis highlights accuracy limitations of U-Net-based deep learning segmentations that can result in false-positive or false-negative classifications. The segmented 3D models were utilized to perform porosity, bulk density, and thickness analyses of each dataset, revealing quantitative differences between the two species, as well as between healthy and stony coral tissue loss disease afflicted M. cavernosa coral skeletons. This work provides a framework for streamlined training and deployment of deep learning models for semantic segmentation of calcified tissues that inform our understanding of skeletogenesis and growth patterns across species and pathogenesis contexts.

The terrestrial isopod Armadillidium vulgare possesses a hierarchically organized tergite cuticle mineralized with calcium carbonate, comprising crystalline calcite and amorphous calcium carbonate (ACC). In this study, we assessed the effects of dietary calcium carbonate polymorphs (calcite, aragonite) and a non-carbonate control (quartz) on cuticle mineralization and layer-specific microstructural organization. Isopods were reared under controlled-feeding conditions using calcite, aragonite, or quartz, and their cuticles were analyzed using scanning electron microscopy (SEM), Raman spectroscopy, and synchrotron X-ray diffraction (XRD). SEM observations indicated that diets containing calcite or aragonite promoted marked thickening and development of mineralized lamellar structures within the exo- and endocuticles, whereas quartz-fed individuals exhibited significantly reduced cuticle mineralization. Raman spectroscopy revealed that the endocuticle consistently contained calcite-type ACC, irrespective of whether calcite or aragonite was provided as the dietary carbonate source. In contrast, the exocuticle exhibited unique characteristics of a more structurally ordered carbonate phase, consistent with a transitional state between ACC and crystalline calcite. Synchrotron XRD analyses of bulk cuticle specimens detected only calcite reflections across all feeding conditions, with no evidence of aragonite even in aragonite-fed isopods, indicating a selective crystallization process. These findings demonstrate that A. vulgare establishes a predominantly calcite-based carbonate system within its cuticle-comprising calcite and calcite-type ACC-largely independent of the external calcium carbonate polymorph source. Polymorph-independent ACC stabilization, layer-dependent carbonate ordering, and exclusive calcite crystallization provide insights into biomineralization mechanisms in terrestrial isopods and establish a framework for future research on carbonate phase evolution in cuticle architecture and function.