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

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

A Trajectory of Discovery: Metabolic Regulation by the Conditionally Disordered Chloroplast Protein, CP12

International audienceThe chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO2 assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have

Absence of residual structure in the intrinsically disordered regulatory protein CP12 in its reduced state

International audienceThe redox switch protein CP12 is a key player of the regulation of the Benson–Calvin cycle. Its oxidation state is controlled by the formation/dissociation of two intramolecular disulphide bridges during the day/night cycle. CP12 was known to be globally intrinsically disordered on a large scale in its reduced state, while being partly ordered in the oxidised state. By combining Nuclear Magnetic Resonance and Small Angle X-ray Scattering experiments, we showed that, contrary to secondary structure or disorder predictions, reduced CP12 is fully disordered, with no transient or local residual structure likely to be precursor of the structures identified in the oxidised active state and/or in the bound state with GAPDH or PRK. These results highlight the diversity of the mechanisms of regulation of conditionally disordered redox switches, and question the stability of oxidised CP12 scaffold

Assessing Protein Disorder and Induced Folding

Intrinsically disordered proteins (IDPs) defy the structure–function paradigm as they fulfill essential biological functions while lacking well-defined secondary and tertiary structures. Conformational and spectroscopic analyses showed that IDPs do not constitute a uniform family, and can be divided into subfamilies as a function of their residual structure content. Residual intramolecular interactions are thought to facilitate binding to a partner and then induced folding. Comprehensive information about experimental approaches to investigate structural disorder and induced folding is still scarce. We herein provide hints to readily recognize features typical of intrinsic disorder and review the principal techniques to assess structural disorder and induced folding. We describe their theoretical principles and discuss their respective advantages and limitations. Finally, we point out the necessity of using different approaches and show how information can be broadened by the use of multiples techniques. Proteins 2006. © 2005 Wiley-Liss, Inc

Activation of the LicT transcriptional antiterminator involves a domain swing/lock mechanism provoking massive structural changes.

The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in β-glucoside metabolism. It consists of an N-terminal RNA-binding domain (co-antiterminator (CAT)) and two phosphorylatable phosphotransferase system regulation domains (PRD1 and PRD2). In the activated state, each PRD forms a dimeric unit with the phosphorylation sites totally buried at the dimer interface. Here we present the 1.95 Å resolution structure of the inactive LicT PRDs as well as the molecular solution structure of the full-length protein deduced from small angle x-ray scattering. Comparison of native (inactive) and mutant (constitutively active) PRD crystal structures shows massive tertiary and quaternary rearrangements of the entire regulatory domain. In the inactive state, a wide swing movement of PRD2 results in dimer opening and brings the phosphorylation sites to the protein surface. This movement is accompanied by additional structural rearrangements of both the PRD1-PRD1 ′ interface and the CAT-PRD1 linker. Small angle x-ray scattering experiments indicate that the amplitude of the PRD2 swing might even be wider in solution than in the crystals. Our results suggest that PRD2 is highly mobile in the native protein, whereas it is locked upon activation by phosphorylation

Cryptic Disorder Out of Disorder: Encounter between Conditionally Disordered CP12 and Glyceraldehyde-3-Phosphate Dehydrogenase

International audienc

The C-terminal domain of the measles virus nucleoprotein is intrinsically disordered and folds upon binding to the C-terminal moiety of the phosphoprotein.

The nucleoprotein of measles virus consists of an N-terminal moiety, N(CORE), resistant to proteolysis and a C-terminal moiety, N(TAIL), hypersensitive to proteolysis and not visible as a distinct domain by electron microscopy. We report the bacterial expression, purification, and characterization of measles virus N(TAIL). Using nuclear magnetic resonance, circular dichroism, gel filtration, dynamic light scattering, and small angle x-ray scattering, we show that N(TAIL) is not structured in solution. Its sequence and spectroscopic and hydrodynamic properties indicate that N(TAIL) belongs to the premolten globule subfamily within the class of intrinsically disordered proteins. The same epitopes are exposed in N(TAIL) and within the nucleoprotein, which rules out dramatic conformational changes in the isolated N(TAIL) domain compared with the full-length nucleoprotein. Most unstructured proteins undergo some degree of folding upon binding to their partners, a process termed "induced folding." We show that N(TAIL) is able to bind its physiological partner, the phosphoprotein, and that it undergoes such an unstructured-to-structured transition upon binding to the C-terminal moiety of the phosphoprotein. The presence of flexible regions at the surface of the viral nucleocapsid would enable plastic interactions with several partners, whereas the gain of structure arising from induced folding would lead to modulation of these interactions. These results contribute to the study of the emerging field of natively unfolded proteins