A new type of flexible CP12 protein in the marine diatom <i>Thalassiosira pseudonana</i>

International audienceBackground: CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background. Methods: A combination of biochemical, bioinformatics and biophysical methods including electrospray ionizationmass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12. Results: Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions. Conclusions: These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle

RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae

RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning—possibly mediated by intrinsically disordered protein segments—is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication

Inherent Structural Disorder and Dimerisation of Murine Norovirus NS1-2 Protein

Human noroviruses are highly infectious viruses that cause the majority of acute, non-bacterial epidemic gastroenteritis cases worldwide. The first open reading frame of the norovirus RNA genome encodes for a polyprotein that is cleaved by the viral protease into six non-structural proteins. The first non-structural protein, NS1-2, lacks any significant sequence similarity to other viral or cellular proteins and limited information is available about the function and biophysical characteristics of this protein. Bioinformatic analyses identified an inherently disordered region (residues 1–142) in the highly divergent N-terminal region of the norovirus NS1-2 protein. Expression and purification of the NS1-2 protein of Murine norovirus confirmed these predictions by identifying several features typical of an inherently disordered protein. These were a biased amino acid composition with enrichment in the disorder promoting residues serine and proline, a lack of predicted secondary structure, a hydrophilic nature, an aberrant electrophoretic migration, an increased Stokes radius similar to that predicted for a protein from the pre-molten globule family, a high sensitivity to thermolysin proteolysis and a circular dichroism spectrum typical of an inherently disordered protein. The purification of the NS1-2 protein also identified the presence of an NS1-2 dimer in Escherichia coli and transfected HEK293T cells. Inherent disorder provides significant advantages including structural flexibility and the ability to bind to numerous targets allowing a single protein to have multiple functions. These advantages combined with the potential functional advantages of multimerisation suggest a multi-functional role for the NS1-2 protein

Common Features at the Start of the Neurodegeneration Cascade

A single-molecule study reveals that neurotoxic proteins share common structural features that may trigger neurodegeneration, thus identifying new targets for therapy and diagnosis

AlphaFold, small-angle X-ray scattering and ensemble modelling: a winning combination for intrinsically disordered proteins

Artificial intelligence has revolutionized many societal and scientific domains, and structural biology benefits from one of its most spectacular breakthroughs. The deep learning algorithm AlphaFold can predict with unparalleled accuracy the threedimensional structure of folded proteins based solely on their sequence. In 1972, Christian Anfinsen was awarded the Nobel Prize in Chemistry for his groundbreaking work on ribonuclease which demonstrated that all the protein's folding information is encoded within its sequence. This ignited a surge of experimental and computational investigations into protein folding to unravel what was at the time referred to as the 'second half of the genetic code' and understand the mechanisms governing the protein's pathway towards its final three-dimensional conformation. Now, half a century after Anfinsen's seminal article (Anfinsen, 1973), it appears that a long-awaited milestone has been reached and that one can now correlate a onedimensional string of amino acids to an accurate three-dimensional structure. However, challenges still persist. The fundamental nature of artificial intelligence lies in its initial training using data that have accumulated over many years, the Protein Data Bank (PDB) in the case of AlphaFold. The PDB suffers from inherent biases: certain families of structures are under-represented due to the limitations of the methods used to solve the protein structures, in particular X-ray crystallization and cryo-electron microscopy (cryoEM). This is typically the case of proteins that exhibit a high conformational flexibility and cannot be described by a single three-dimensional structure. In particular, intrinsically disordered proteins (IDPs) or regions (IDRs) of proteins can adopt a tremendous number of conformations and this high dynamic is inherent to their function. Because of their flexibility, these polypeptides are often invisible in the structures solved by X-ray crystallization or cryoEM. Their flexibility may even preclude protein crystallization or lead to poorly diffracting crystals. In addition, a short disordered segment can undergo an induced folding upon binding to a partner, but may adopt different folds depending on the binding partner and the interface of interaction, further complicating accurate predictions. Therefore, AlphaFold could not train properly on disordered structures, which lack structural homologues in the PDB. Furthermore, AlphaFold uses multiple sequence alignments, while IDRs are very difficult to align because they are poorly conserved. For all these reasons AlphaFold cannot predict for now the 3D structure of long flexible disordered regions with accuracy and provides low confidence scores on these predictions. AlphaFold's creators even suggest that the low confidence scores can be used as a method to predict protein disorder. Therefore, the single static representation of the structure of proteins containing IDRs provided by AlphaFold cannot convey all their structural and functional properties. A very interesting strategy to circumvent this difficulty is to integrate AlphaFold predictions with ensemble molecular modelling and experimentally derived constraints, as proposed by Brookes et al. (2023) in their paper entitled AlphaFold-predicted protein structures and small-angle X-ray scattering: insights from an extended examination of selected data in the Small-Angle Scattering Biological Data Bank. Small-angle X-ray scattering (SAXS) is a particularly suitable experimental method for providing such constraints. SAXS data contain all the 3D information on the various conformations adopted by a macromolecule in solution. By calculating the SAXS profile from atomic coordinates and comparing it with experimental SAXS data, it becomes possible to select an ensemble of conformations that collectively contribute to the observed scatterin

Orchestration of algal metabolism by protein disorder

International audienceIntrinsically disordered proteins (IDPs) are proteins that provide many functional advantages in a large number of metabolic and signalling pathways. Because of their high flexibility that endows them with pressure-, heat- and acid-resistance, IDPs are valuable metabolic regulators that help algae to cope with extreme conditions of pH, temperature, pressure and light. They have, however, been overlooked in these organisms. In this review, we present some well-known algal IDPs, including the conditionally disordered CP12, a protein involved in the regulation of CO2 assimilation, as probably the best known example, whose disorder content is strongly dependent on the redox conditions, and the essential pyrenoid component 1 that serves as a scaffold for ribulose-1, 5-bisphosphate carboxylase/oxygenase. We also describe how some enzymes are regulated by protein regions, called intrinsically disordered regions (IDRs), such as ribulose-1, 5-bisphosphate carboxylase/oxygenase activase, the A2B2 form of glyceraldehyde-3-phosphate dehydrogenase and the adenylate kinase. Several molecular chaperones, which are crucial for cell proteostasis, also display significant disorder propensities such as the algal heat shock proteins HSP33, HSP70 and HSP90. This review confirms the wide distribution of IDPs in algae but highlights that further studies are needed to uncover their full role in orchestrating algal metabolism