Structural basis for the function of the N-terminal domain of the ATPase CopA from Bacillus subtilis.

The solution structure of the N-terminal region (151 amino acids) of a copper ATPase, CopA, from Bacillus subtilis, is reported here. It consists of two domains, CopAa and CopAb, linked by two amino acids. It is found that the two domains, which had already been separately characterized, interact one to the other through a hydrogen bond network and a few hydrophobic interactions, forming a single rigid body. The two metal binding sites are far from one another, and the short link between the domains prevents them from interacting. This and the surface electrostatic potential suggest that each domain receives copper from the copper chaperone, CopZ, independently and transfers it to the membrane binding site of CopA. The affinity constants of silver(I) and copper(I) are similar for the two sites as monitored by NMR. Because the present construct "domain-short link-domain" is shared also by the last two domains of the eukaryotic copper ATPases and several residues at the interface between the two domains are conserved, the conclusions of the present study have general validity for the understanding of the function of copper ATPases

Solution Structure of Cox11, a Novel Type of β-Immunoglobulin-like Fold Involved in CuB Site Formation of Cytochrome c Oxidase

Cytochrome c oxidase assembly process involves many accessory proteins including Cox11, which is a copper-binding protein required for Cu incorporation into the Cu(B) site of cytochrome c oxidase. In a genome wide search, a number of Cox11 homologs are found in all of the eukaryotes with complete genomes and in several Gram-negative bacteria. All of them possess a highly homologous soluble domain and contain an N-terminal fragment that anchors the protein to the membrane. An anchor-free construct of 164 amino acids was obtained from Sinorhizobium meliloti, and the first structure of this class of proteins is reported here. The apoform has an immunoglobulin-like fold with a novel type of beta-strand organization. The copper binding motif composed of two highly conserved cysteines is located on one side of the beta-barrel structure. The apoprotein is monomeric in the presence of dithiothreitol, whereas it dimerizes in the absence of the reductant. When copper(I) binds, NMR and extended x-ray absorption fine structure (EXAFS) data indicate a dimeric protein state with two thiolates bridging two copper(I) ions. The present results advance the knowledge on the poorly understood molecular aspects of cytochrome c oxidase assembly

Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins

Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochemical basis for explaining the pathological phenotypes of patients with mutations in BOLA3. DOI: http://dx.doi.org/10.7554/eLife.16673.00

The FDA-Approved Antiviral Raltegravir Inhibits Fascin1-Dependent Invasion of Colorectal Tumor Cells In Vitro and In Vivo

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Serrated adenocarcinoma (SAC) has been recently recognized by the WHO as a histological CRC with bad prognosis. Consistent with previous evidence, our group identified Fascin1 as a protein directly related to the invasiveness of tumor cells, overexpressed and positively correlated with worse survival in various carcinomas, including SAC. Therefore, Fascin1 has emerged as an ideal target for cancer treatment. In the present study, virtual screening has been carried out from a library of 9591 compounds, thus identifying the FDA-approved anti-retroviral raltegravir (RAL) as a potential Fascin1 blocker. In vitro and in vivo results show that RAL exhibits Fascin1-binding activity and Fascin1-dependent anti-invasive and anti-metastatic properties against CRC cells both in vitro and in vivo

Ultrasound-based techniques in diabetic bone disease: State of the art and future perspectives

Bone fragility is increasingly recognized as an important complication of diabetes mellitus (DM), and both type 1 (T1DM) and type 2 (T2DM) diabetes are associated with a higher risk of fracture. The causes of bone fragility in diabetic patients are not yet fully understood; probably they are linked to low bone mineral density (BMD), poor bone quality due to the alterations in bone remodelling, microarchitecture and composition of the bone matrix. Quantitative ultrasound (QUS) is a validated, low-cost and free ionizing radiation alternative to DXA measurement of BMD for the assessment of fracture risk. The results obtained by using QUS in T1DM and T2DM have been summarized and reported in this review. QUS technique presents some benefits but also some limits. These limits could be overcome by radiofrequency echographic multispectrometry (REMS) that is a non-ionizing technology recently introduced for the assessment of bone status that can also calculate parameters related to bone quality and strength. Therefore, REMS may represent a promising approach to evaluate bone status and fragility fracture risk in DM subjects

Bone Fracture in Rett Syndrome: Mechanisms and Prevention Strategies

The present study aimed to evaluate the burden and management of fragility fractures in subjects with Rett syndrome. We searched all relevant medical literature from 1 January 1986 to 30 June 2023 for studies under the search term “Rett syndrome and fracture”. The fracture frequency ranges from a minimum of 13.9% to a maximum of 36.1%. The majority of such fractures occur in lower limb bones and are associated with low bone mineral density. Anticonvulsant use, joint contractures, immobilization, low physical activity, poor nutrition, the genotype, and lower calcium and vitamin D intakes all significantly impair skeletal maturation and bone mass accrual in Rett syndrome patients, making them more susceptible to fragility fractures. This review summarizes the knowledge on risk factors for fragility fracture in patients with Rett syndrome and suggests a possible diagnostic and therapeutic care pathway for improving low bone mineral density and reducing the risk of fragility fractures. The optimization of physical activity, along with adequate nutrition and the intake of calcium and vitamin D supplements, should be recommended. In addition, subjects with Rett syndrome and a history of fracture should consider using bisphosphonates

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with H-1 detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of N-15 and C-13 nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu+ (diamagnetic) or Cu2+ (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to H-1-H-1 distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a Gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable