ABSTRACT
Recent advances in structural biophysics and integrative modelling methods now allow us to decipher the structures of large macromolecular assemblies. Understanding the dynamics and mechanisms involved in their biological function requires rigorous integration of all available data. We have developed a complete modelling pipeline that includes analyses to extract biologically significant information by consistently combining automated and interactive human-guided steps. We illustrate this idea with two examples. First, we describe the ryanodine receptor, an ion channel that controls ion flux across the cell membrane through transitions between open and closed states. The conformational changes associated with the transitions are small compared to the considerable system size of the receptor; it is challenging to consistently track these states with the available cryo-EM structures. The second example involves homologous recombination, in which long filaments of a recombinase protein and DNA catalyse the exchange of homologous DNA strands to reliably repair DNA double-strand breaks. The nucleoprotein filament reaction intermediates in this process are short-lived and heterogeneous, making their structures particularly elusive. The pipeline we describe, which incorporates experimental and theoretical knowledge combined with state-of-the-art interactive and immersive modelling tools, can help overcome these challenges. In both examples, we point to new insights into biological processes that arise from such interdisciplinary approaches.
ABSTRACT
Poly(ornithine-co-citrulline)s are ureido-based polymers, which were shown to exhibit tunable upper critical solution temperature (UCST) behavior, a property that can be exploited to develop thermoresponsive nanoparticles for controlled drug delivery systems. To gain insight into the driving forces that govern the formation and dissolution processes of poly(ornithine-co-citrulline) nanoparticles, a molecular dynamics (MD) simulation study has been carried out using MARTINI-based protein coarse-grained models. Multi-microsecond simulations at temperatures ranging from 280 to 370 K show that the fully reparametrized version 3.0 of MARTINI force field is able to capture the dependence on temperature of poly(ornithine-co-citrulline) aggregation and dissolution, while version 2.2 could not account for it. Furthermore, the phase separation observed in these simulations allowed us to extrapolate a phase diagram based on the Flory-Huggins theory of polymer solution, which could help in future rational design of drug delivery nanoparticles based on poly(amino acid)s.
ABSTRACT
Scaffolding proteins play important roles in supporting the plasma membrane (sarcolemma) of muscle cells. Among them, dystrophin strengthens the sarcolemma through protein-lipid interactions, and its absence due to gene mutations leads to the severe Duchenne muscular dystrophy. Most of the dystrophin protein consists of a central domain made of 24 spectrin-like coiled-coil repeats (R). Using small angle neutron scattering (SANS) and the contrast variation technique, we specifically probed the structure of the three first consecutive repeats 1-3 (R1-3), a part of dystrophin known to physiologically interact with membrane lipids. R1-3 free in solution was compared to its structure adopted in the presence of phospholipid-based bicelles. SANS data for the protein/lipid complexes were obtained with contrast-matched bicelles under various phospholipid compositions to probe the role of electrostatic interactions. When bound to anionic bicelles, large modifications of the protein three-dimensional structure were detected, as revealed by a significant increase of the protein gyration radius from 42 ± 1 to 60 ± 4 Å. R1-3/anionic bicelle complexes were further analyzed by coarse-grained molecular dynamics simulations. From these studies, we report an all-atom model of R1-3 that highlights the opening of the R1 coiled-coil repeat when bound to the membrane lipids. This model is totally in agreement with SANS and click chemistry/mass spectrometry data. We conclude that the sarcolemma membrane anchoring that occurs during the contraction/elongation process of muscles could be ensured by this coiled-coil opening. Therefore, understanding these structural changes may help in the design of rationalized shortened dystrophins for gene therapy. Finally, our strategy opens up new possibilities for structure determination of peripheral and integral membrane proteins not compatible with different high-resolution structural methods.
Subject(s)
Dystrophin/chemistry , Dystrophin/metabolism , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Humans , Micelles , Molecular Dynamics Simulation , Protein Binding , Protein Conformation, alpha-HelicalABSTRACT
Dystrophin, encoded by the DMD gene, is critical for maintaining plasma membrane integrity during muscle contraction events. Mutations in the DMD gene disrupting the reading frame prevent dystrophin production and result in severe Duchenne muscular dystrophy (DMD); in-frame internal deletions allow production of partly functional internally deleted dystrophin and result in less severe Becker muscular dystrophy (BMD). Many known BMD deletions occur in dystrophin's central domain, generally considered to be a monotonous rod-shaped domain based on the knowledge of spectrin family proteins. However, the effects caused by these deletions, ranging from asymptomatic to severe BMD, argue against the central domain serving only as a featureless scaffold. We undertook structural studies combining small-angle X-ray scattering and molecular modeling in an effort to uncover the structure of the central domain, as dystrophin has been refractory to characterization. We show that this domain appears to be a tortuous and complex filament that is profoundly disorganized by the most severe BMD deletion (loss of exons 45-47). Despite the preservation of large parts of the binding site for neuronal nitric oxide synthase (nNOS) in this deletion, computational approaches failed to recreate the association of dystrophin with nNOS. This observation is in agreement with a strong decrease of nNOS immunolocalization in muscle biopsies, a parameter related to the severity of BMD phenotypes. The structural description of the whole dystrophin central domain we present here is a first necessary step to improve the design of microdystrophin constructs toward the goal of a successful gene therapy for DMD.
Subject(s)
Dystrophin/chemistry , Dystrophin/genetics , Gene Deletion , Muscular Dystrophy, Duchenne/genetics , Binding Sites , Exons , Humans , Molecular Docking Simulation , Muscular Dystrophy, Duchenne/enzymology , Nitric Oxide Synthase Type I/metabolism , Protein Domains , Reading Frames , Scattering, Small Angle , Solutions , X-Ray DiffractionABSTRACT
Duchenne muscular dystrophy is a lethal genetic defect that is associated with the absence of dystrophin protein. Lack of dystrophin protein completely abolishes muscular nitric-oxide synthase (NOS) function as a regulator of blood flow during muscle contraction. In normal muscles, nNOS function is ensured by its localization at the sarcolemma through an interaction of its PDZ domain with dystrophin spectrin-like repeats R16 and R17. Early studies suggested that repeat R17 is the primary site of interaction but ignored the involved nNOS residues, and the R17 binding site has not been described at an atomic level. In this study, we characterized the specific amino acids involved in the binding site of nNOS-PDZ with dystrophin R16-17 using combined experimental biochemical and structural in silico approaches. First, 32 alanine-scanning mutagenesis variants of dystrophin R16-17 indicated the regions where mutagenesis modified the affinity of the dystrophin interaction with the nNOS-PDZ. Second, using small angle x-ray scattering-based models of dystrophin R16-17 and molecular docking methods, we generated atomic models of the dystrophin R16-17·nNOS-PDZ complex that correlated well with the alanine scanning identified regions of dystrophin. The structural regions constituting the dystrophin interaction surface involve the A/B loop and the N-terminal end of helix B of repeat R16 and the N-terminal end of helix A' and a small fraction of helix B' and a large part of the helix C' of repeat R17. The interaction surface of nNOS-PDZ involves its main ß-sheet and its specific C-terminal ß-finger.
Subject(s)
Dystrophin/chemistry , Nitric Oxide Synthase Type I/chemistry , Alanine/chemistry , Binding Sites , Biotinylation , Dystrophin-Associated Proteins/chemistry , Exons , Humans , Molecular Dynamics Simulation , Muscle, Skeletal/enzymology , Mutagenesis , Mutation , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Scattering, Radiation , X-RaysABSTRACT
During protein synthesis, many translating ribosomes are bound together with an mRNA molecule to form polysomes (or polyribosomes). While the spatial organization of bacterial polysomes has been well studied in vitro, little is known about how they cluster when cellular conditions are highly constrained. To better understand this, we used electron tomography, template matching, and three-dimensional modeling to analyze the supramolecular network of ribosomes after induction of translational pauses. In Escherichia coli, we overexpressed an mRNA carrying a polyproline motif known to induce pausing during translation. When working with a strain lacking transfer-messenger RNA, the principle actor in the "trans-translation" rescuing system, the cells survived the hijacking of the translation machinery but this resulted in a sharp modification of the ribosomal network. The results of our experiments demonstrate that single ribosomes are replaced with large amounts of compacted polysomes. These polysomes are highly organized, principally forming hairpins and dimers of hairpins that stack together. We propose that these spatial arrangements help maintain translation efficiency when the rescue systems are absent or overwhelmed.
Subject(s)
Escherichia coli/chemistry , Escherichia coli/metabolism , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Polyribosomes/chemistry , Polyribosomes/metabolism , Electron Microscope Tomography , Imaging, Three-Dimensional , Peptides/metabolism , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Dystrophin is a large skeletal muscle protein located at the internal face of the plasma membrane and interacting with membrane phospholipids and a number of cytosolic proteins. Binding of neuronal nitric oxide synthase (nNOS) to dystrophin appears to be crucial for exercise-induced increases in blood supply in muscle cells. By contrast, utrophin, the developmental homologous protein of dystrophin, does not display nNOS interaction. Recent in vitro and in vivo experiments showed that the dystrophin region involved in nNOS binding is located in spectrin-like repeats R16 and R17 of its filamentous central domain. Using homology modeling and atomistic molecular dynamics simulation, we compared the structural organization and surface potentials of dystrophin, utrophin, and chimeric fragments, thus revisiting the dystrophin-nNOS binding region. Our simulation results are in good agreement with experimental data. They provide a three-dimensional representation of the repeats and give insight into the molecular organization of the regions involved in dystrophin-nNOS interaction. This study also further elucidates the physical properties crucial for this interaction, particularly the presence of a large hydrophobic patch. These results will be helpful to improving our understanding of the phenotypic features of patients bearing mutations in the nNOS-binding region of dystrophin.
Subject(s)
Dystrophin/chemistry , Dystrophin/metabolism , Nitric Oxide Synthase Type I/chemistry , Nitric Oxide Synthase Type I/metabolism , Amino Acid Motifs , Dystrophin/genetics , Humans , Molecular Dynamics Simulation , Nitric Oxide Synthase Type I/genetics , Protein Binding , Protein Structure, SecondaryABSTRACT
A finely tuned balance of translation, storage and decay of mRNAs (mRNAs) is important for the regulation of gene expression. In eukaryotic cells, this takes place in dynamic cytoplasmic RNA-protein granules termed Processing bodies (P-bodies). In this study, by using immunoelectron tomography, 3D modeling and template matching, we analyze the size and the organization of the polysomes in the vicinity of human P-bodies. Our results show the presence of several polysomes that are compatible with a translational activity around P-bodies. Therefore, movement of mRNAs between polysomes and P-bodies can take place when the two compartments are in close contact. The presence of initiation factors in the proximity of P-bodies also suggests that translation of mRNAs can resume at the periphery of these granules.