Regulation of T Cell Receptor Activation by Dynamic Membrane Binding of the CD3ɛ Cytoplasmic Tyrosine-Based Motif

SummaryMany immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3ɛ cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3ɛ residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3ɛ ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation

An Unusual Topological Structure of the HIV-1 Rev Response Element

SummaryNuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an “A”-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ∼55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the “A” and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA

Outcome of the First wwPDB Hybrid / Integrative Methods Task Force Workshop

Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models

Conjoined use of EM and NMR in RNA structure refinement.

More than 40% of the RNA structures have been determined using nuclear magnetic resonance (NMR) technique. NMR mainly provides local structural information of protons and works most effectively on relatively small biomacromolecules. Hence structural characterization of large RNAs can be difficult for NMR alone. Electron microscopy (EM) provides global shape information of macromolecules at nanometer resolution, which should be complementary to NMR for RNA structure determination. Here we developed a new energy term in Xplor-NIH against the density map obtained by EM. We conjointly used NMR and map restraints for the structure refinement of three RNA systems—U2/U6 small-nuclear RNA, genome-packing motif (Ψ(CD))2 from Moloney murine leukemia virus, and ribosome-binding element from turnip crinkle virus. In all three systems, we showed that the incorporation of a map restraint, either experimental or generated from known PDB structure, greatly improves structural precision and accuracy. Importantly, our method does not rely on an initial model assembled from RNA duplexes, and allows full torsional freedom for each nucleotide in the torsion angle simulated annealing refinement. As increasing number of macromolecules can be characterized by both NMR and EM, the marriage between the two techniques would enable better characterization of RNA three-dimensional structures

A Practical Implicit Membrane Potential for NMR Structure Calculations of Membrane Proteins

AbstractThe highly anisotropic environment of the lipid bilayer membrane imposes significant constraints on the structures and functions of membrane proteins. However, NMR structure calculations typically use a simple repulsive potential that neglects the effects of solvation and electrostatics, because explicit atomic representation of the solvent and lipid molecules is computationally expensive and impractical for routine NMR-restrained calculations that start from completely extended polypeptide templates. Here, we describe the extension of a previously described implicit solvation potential, eefxPot, to include a membrane model for NMR-restrained calculations of membrane protein structures in XPLOR-NIH. The key components of eefxPot are an energy term for solvation free energy that works together with other nonbonded energy functions, a dedicated force field for conformational and nonbonded protein interaction parameters, and a membrane function that modulates the solvation free energy and dielectric screening as a function of the atomic distance from the membrane center, relative to the membrane thickness. Initial results obtained for membrane proteins with structures determined experimentally in lipid bilayer membranes show that eefxPot affords significant improvements in structural quality, accuracy, and precision. Calculations with eefxPot are straightforward to implement and can be used to both fold and refine structures, as well as to run unrestrained molecular-dynamics simulations. The potential is entirely compatible with the full range of experimental restraints measured by various techniques. Overall, it provides a useful and practical way to calculate membrane protein structures in a physically realistic environment

A practical implicit solvent potential for NMR structure calculation

The benefits of protein structure refinement in water are well documented. However, performing structure refinement with explicit atomic representation of the solvent molecules is computationally expensive and impractical for NMR-restrained structure calculations that start from completely extended polypeptide templates. Here we describe a new implicit solvation potential, EEFx (Effective Energy Function for XPLOR-NIH), for NMR-restrained structure calculations of proteins in XPLOR-NIH. The key components of EEFx are an energy term for solvation energy that works together with other nonbonded energy functions, and a dedicated force field for conformational and nonbonded protein interaction parameters. The initial results obtained with EEFx show that significant improvements in structural quality can be obtained. EEFx is computationally efficient and can be used both to fold and refine structures. Overall, EEFx improves the quality of protein conformation and nonbonded atomic interactions. Moreover, such benefits are accompanied by enhanced structural precision and enhanced structural accuracy, reflected in improved agreement with the cross-validated dipolar coupling data. Finally, implementation of EEFx calculations is straightforward and computationally efficient. Overall, EEFx provides a useful method for the practical calculation of experimental protein structures in a physically realistic environment

xplor-nih script

<p>xplor-nih script for RNA structure refinement </p

Structure refinement of ribosome-binding element from turnip crinkle virus genome.

<p>(A) Superposition of 20 structures, obtained by refining against NMR, secondary structure, and knowledge-based restraints. The RMS deviation for the bundle is 13.24±0.56 Å. (B) When incorporating of SAXS and P-P envelope distance restraints, the structural convergence is improved to 8.20±0.44 Å. (C) With the incorporation of additional map restraints, the RMS deviation is further lowed to 2.35±0.05 Å, for the 20-structure bundle. (D) By allowing only translation movement for the RNA duplexes but not the rotational movement, refined against both NMR and map restraints, the convergence of the calculated structures can be improved to 0.76±0.03 Å. The structure for generating the density map (the first model of PDB structure 2KRL) is colored red. The RMS difference between the structure calculated and the reference structure is 2.65±0.27 Å and 0.94±0.04 Å for (C) and (D), respectively.</p