Atopic dermatitis (AD) is characterized by a T-helper cell type 2 (Th2) inflammatory response leading to skin damage with erythema and edema. Comparative fecal sample analysis has uncovered a strong correlation between AD and Faecalibacterium prausnitzii strain A2-165, specifically associated with butyrate production. Therefore, understanding the functional mechanisms of crucial enzymes in the butyrate pathway, such as 3-hydroxybutyryl-CoA dehydrogenase of A2-165 (A2HBD), is imperative. Here, we have successfully elucidated the three-dimensional structure of A2HBD in complex with acetoacetyl-CoA and NAD⁺ at a resolution of 2.2Å using the PAL-11C beamline (third generation). Additionally, X-ray data of A2HBD in complex with acetoacetyl-CoA at a resolution of 1.9 Å were collected at PAL-XFEL (fourth generation) utilizing Serial Femtosecond Crystallography (SFX). The monomeric structure of A2HBD consists of two domains, N-terminal and C-terminal, with cofactor binding occurring at the N-terminal domain, while the C-terminal domain facilitates dimerization. Our findings elucidate the binding mode of NAD⁺ to A2HBD. Upon acetoacetyl-CoA binding, the crystal structure revealed a significant conformational change in the Clamp-roof domain (root-mean-square deviation of 2.202 Å). Notably, residue R143 plays a critical role in capturing the adenine phosphate ring, underlining its significance in substrate recognition and catalytic activity. The binding mode of acetoacetyl-CoA was also clarified, indicating its lower stability compared to NAD⁺. Furthermore, the conformational change of hydrophobic residues near the catalytic cavity upon substrate binding resulted in cavity shrinkage from an open to closed conformation. This study confirms the conformational changes of catalytic triads involved in the catalytic reaction and presents a proposed mechanism for substrate reduction based on structural observations.

Fasciola hepatica remains a global health and economic concern, and treatment still relies heavily on triclabendazole. At the parasite–host interface, F. hepatica calcium-binding proteins (FhCaBPs) have a unique EF-hand/DLC-like domain fusion found only in trematodes. This makes it a parasite-specific target for small compounds and vaccinations. To enable novel therapeutic strategies, we report the first elevated-resolution structure of a full-length FhCaBP4. The apo structure was determined at 1.93 Å resolution, revealing a homodimer architecture that integrates an N-terminal, calmodulin-like, EF-hand pair with a C-terminal dynein light chain (DLC)-like domain. Structure-guided in silico mutagenesis identified a flexible, 16-residue β4–β5 loop (LTGSYWMKFSHEPFMS) with an FSHEPF core that demonstrates greater energetic variability than its FhCaBP2 counterpart, likely explaining the distinct ligand-binding profiles of these paralogs. Molecular dynamics simulations and AlphaFold3 modeling suggest that EF-hand 2 acts as the primary calcium-binding site, with calcium coordination inducing partial rigidification and modest expansion of the protein structure. Microscale thermophoresis confirmed calcium as the major ligand, while calmodulin antagonists bound with lower affinity and praziquantel demonstrated no interaction. Thermal shift assays revealed calcium-dependent stabilization and a merger of biphasic unfolding transitions. These results suggest that FhCaBP4 functions as a calcium-responsive signaling hub, with an allosterically coupled EF-hand–DLC interface that could serve as a structurally tractable platform for drug targeting in trematodes.

The exosome multiprotein complex plays a critical role in RNA processing and degradation. This system governs the regulation of mRNA quality, degradation in the cytoplasm, the processing of short noncoding RNA, and the breakdown of RNA fragments. We determined two crystal structures of exosome components from Thermoplasma acidophilum (Taci): one with a resolution of 2.3 Å that reveals the central components (TaciRrp41 and TaciRrp42), and another with a resolution of 3.5 Å that displays the whole exosome (TaciRrp41, TaciRrp42, and TaciRrp4). The fundamental exosome structure revealed the presence of a heterodimeric complex consisting of TaciRrp41 and TaciRrp42. The structure comprises nine subunits, with TaciRrp41 and TaciRrp42 arranged in a circular configuration, while TaciRrp4 is located at the apex. The RNA degradation capabilities of the TaciRrp4:41:42 complex were verified by RNA degradation assays, consistent with prior findings in other archaeal exosomes. The resemblance between archaeal exosomes and bacterial PNPase suggests a common mechanism for RNA degradation. Despite sharing comparable topologies, the surface charge distributions of TaciRrp4 and other archaea structures are surprisingly distinct. Different RNA breakdown substrates may be responsible for this variation. These newfound structural findings enhance our comprehension of RNA processing and degradation in biological systems.

Methionine sulfoxide reductase A (MsrA) protects proteins from oxidative damage through cysteine-based redox cycling, but how mammalian MsrA is structurally regulated during oxidation and reductive recycling has remained unclear. We report three crystal structures of mouse MsrA (mMsrA) that capture distinct redox states - reduced (3.0 Å), BME-trapped intermediate-like (BTIL; 1.6 Å), and oxidized (1.9 Å) - and reveal an unrecognized layer of regulation centered on Cys107. The BTIL structure visualizes catalytic Cys72 as a mixed disulfide with β-mercaptoethanol (BME) alongside a sulfenic acid at Cys107, a configuration not previously observed. The oxidized structure resolves the canonical Cys72-Cys218 disulfide together with an unexpected Cys107-Cys227 pairing that locks a C-terminal loop into a cap over the active site. LC-MS/MS mapping, MD simulations, activity assays, and thioredoxin (Trx)-trapping experiments converge on a model in which Cys72 drives catalysis, while Cys107-mediated disulfide rearrangement and Cys227-directed Trx entry gate substrate access and enzymatic regeneration - defining a noncanonical oxidized state that expands the mechanistic repertoire of mammalian MsrA under oxidative stress.