The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA؉ ATPase superfamily. A feature common to AAA؉ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5-8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain.Poliovirus is the prototype member of the Picornaviridae family. The 7.5-kb positive sense RNA genome encodes both capsid and noncapsid proteins that are necessary for virus replication. Translation of the viral genome into a single polyprotein yields both functionally distinct precursors and final products that are required for productive viral replication via an orchestrated series of co-and post-translational cleavage events catalyzed by viral proteinases. Replication of the viral RNA occurs in the cytoplasm, localized on the surfaces of newly formed membranous structures that develop after infection. Viral and host proteins involved in viral RNA replication form a poorly characterized, nuclease-resistant replication complex associated with the remodeled membrane structures. Numerous studies have demonstrated that viral protein 2C and its precursor 2BC play key roles in viral RNA replication, yet their actual biochemical functions in this complex reaction remain undefined (1-3). Protein 2C has been shown to interact with other elements of the viral replication apparatus, including 3AB (4), 3C proteinase (5), and the cloverleaf structure at the 5Ј-end of the viral genome (6). More recently it was shown that reticulon-3, a cellular protein involved in membrane trafficking and endoplasmic reticulum structure, binds polioviral as well as other picornaviral 2C proteins, and this plays an essential albeit undefined role...
Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc 1 Man 7-9 GlcNAc 2 present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc 1 Man 7-9 GlcNAc 2 , determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (K b ) and changes in enthalpy of binding (⌬H b°) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19 ؋ 10 4 M ؊1 at 279 K is obtained when glucose occurs in ␣-1,3 linkage to Man␣Me as in Glc␣1-3Man␣Me. The binding constant increases by 25-fold from di-to trisaccharide and doubles from tri-to tetrasaccharide, demonstrating that the entire Glc␣1-3Man␣1-2Man␣1-2Man␣Me structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis á vis legume lectins, which have the same fold as this chaperone, are discussed. Calreticulin (CRT),1 along with calnexin, serves as a molecular chaperone in the endoplasmic reticulum (ER) of eukaryotic cells. Although calreticulin is a soluble, luminal protein, calnexin is a type I membrane protein (1, 2). Segments of these proteins share amino acid identity ranging from 42 to 78% (3). Calreticulin is a highly conserved ubiquitous protein (M r 46,000) and has been implicated in Ca 2ϩ storage and intracellular Ca 2ϩ signaling in the sarcoplasmic and endoplasmic reticula (4, 5). CRT has been divided into three regions: the N-terminal, the C-terminal, and the central P-domain, which consists of short sequence motifs repeated three times in tandem. The N-terminal domain is highly conserved among CRTs from different species and potentially mediates interactions between CRT and the ER folding catalysts, protein disulfide isomerase and ERp57 (6, 7). Recent studies show that the P-domain, previously thought to be involved in oligosaccharide binding, interacts directly with ERp57 (8 -10). The C-domain is characterized by a high content of acidic residues (4, 11), which is consistent with the location of a low affinity (K d ϭ ϳ1-2 mM), high capacity (ϳ25-50 mol) calcium-binding site (12) and contains the ER retrieval sequence.The ER plays an essential role in the folding and maturation of newly synthesized proteins in the secretory pathway. ER quality control operates at various levels; one of the most comm...
Recent improvements in direct electron detectors, microscope technology and software provided the stimulus for a `quantum leap' in the application of cryo-electron microscopy in structural biology, and many national and international centres have since been created in order to exploit this. Here, a new facility for cryo-electron microscopy focused on single-particle reconstruction of biological macromolecules that has been commissioned at the European Synchrotron Radiation Facility (ESRF) is presented. The facility is operated by a consortium of institutes co-located on the European Photon and Neutron Campus and is managed in a similar fashion to a synchrotron X-ray beamline. It has been open to the ESRF structural biology user community since November 2017 and will remain open during the 2019 ESRF–EBS shutdown.
Fatty acid β‐oxidation (FAO) and oxidative phosphorylation (OXPHOS) are mitochondrial redox processes that generate ATP. The biogenesis of the respiratory Complex I, a 1 MDa multiprotein complex that is responsible for initiating OXPHOS, is mediated by assembly factors including the mitochondrial complex I assembly (MCIA) complex. However, the organisation and the role of the MCIA complex are still unclear. Here we show that ECSIT functions as the bridging node of the MCIA core complex. Furthermore, cryo‐electron microscopy together with biochemical and biophysical experiments reveal that the C‐terminal domain of ECSIT directly binds to the vestigial dehydrogenase domain of the FAO enzyme ACAD9 and induces its deflavination, switching ACAD9 from its role in FAO to an MCIA factor. These findings provide the structural basis for the MCIA complex architecture and suggest a unique molecular mechanism for coordinating the regulation of the FAO and OXPHOS pathways to ensure an efficient energy production.
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