Bente Vestergaard

A cryo-electron microscopic study of ribosome-bound termination factor RF2

Protein synthesis takes place on the ribosome, where genetic information carried by messenger RNA is translated into a sequence of amino acids. This process is terminated when a stop codon moves into the ribosomal decoding centre (DC) and is recognized by a class-1 release factor (RF). RFs have a conserved GGQ amino-acid motif, which is crucial for peptide release and is believed to interact directly with the peptidyl-transferase centre (PTC) of the 50S ribosomal subunit. Another conserved motif of RFs (SPF in RF2) has been proposed to interact directly with stop codons in the DC of the 30S subunit. The distance between the DC and PTC is ∼73 Å. However, in the X-ray structure of RF2, SPF and GGQ are only 23 Å apart, indicating that they cannot be at DC and PTC simultaneously. Here we show that RF2 is in an open conformation when bound to the ribosome, allowing GGQ to reach the PTC while still allowing SPF–stop-codon interaction. The results indicate new interpretations of accuracy in termination, and have implications for how the presence of a stop codon in the DC is signalled to PTC.

Structure of the Escherichia coli ribosomal termination complex with release factor 2

Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein–transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the ‘SPF’ loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.

Bacterial Polypeptide Release Factor RF2 Is Structurally Distinct from Eukaryotic eRF1

Bacterial release factor RF2 promotes termination of protein synthesis, specifically recognizing stop codons UAA or UGA. The crystal structure of Escherichia coli RF2 has been determined to a resolution of 1.8 A. RF2 is structurally distinct from its eukaryotic counterpart eRF1. The tripeptide SPF motif, thought to confer RF2 stop codon specificity, and the universally conserved GGQ motif, proposed to be involved with the peptidyl transferase center, are exposed in loops only 23 A apart, and the structure suggests that stop signal recognition is more complex than generally believed.

Low-resolution structure of a vesicle disrupting α-synuclein oligomer that accumulates during fibrillation

One of the major hallmarks of Parkinson disease is aggregation of the protein α-synuclein (αSN). Aggregate cytotoxicity has been linked to an oligomeric species formed at early stages in the aggregation process. Here we follow the fibrillation process of αSN in solution over time using small angle X-ray scattering and resolve four major coexisting species in the fibrillation process, namely monomer, dimer, fibril and an oligomer. By ab initio modeling to fit the data, we obtain a low-resolution structure of a symmetrical and slender αSN fibril in solution, consisting of a repeating unit with a maximal distance of 900 Å and a diameter of ∼180 Å. The same approach shows the oligomer to be shaped like a wreath, with a central channel and with dimensions corresponding to the width of the fibril. The structure, accumulation and decay of this oligomer is consistent with an on-pathway role for the oligomer in the fibrillation process. We propose an oligomer-driven αSN fibril formation mechanism, where the fibril is built from the oligomers. The wreath-shaped structure of the oligomer highlights its potential cytotoxicity by simple membrane permeabilization. This is confirmed by the ability of the purified oligomer to disrupt liposomes. Our results provide the first structural description in solution of a potentially cytotoxic oligomer, which accumulates during the fibrillation of αSN.

BioXTAS RAW, a software program for high- throughput automated small-angle X-ray scattering data reduction and preliminary analysis

A fully open source software program for automated two-dimensional and one-dimensional data reduction and preliminary analysis of isotropic small-angle X-ray scattering (SAXS) data is presented. The program is freely distributed, following the open-source philosophy, and does not rely on any commercial software packages. BioXTAS RAW is a fully automated program that, via an online feature, reads raw two-dimensional SAXS detector output files and processes and plots data as the data files are created during measurement sessions. The software handles all steps in the data reduction. This includes mask creation, radial averaging, error bar calculation, artifact removal, normalization and q calibration. Further data reduction such as background subtraction and absolute intensity scaling is fast and easy via the graphical user interface. BioXTAS RAW also provides preliminary analysis of one-dimensional data in terms of the indirect Fourier transform using the objective Bayesian approach to obtain the pair-distance distribution function, PDDF, and is thereby a free and open-source alternative to existing PDDF estimation software. Apart from the TIFF input format, the program also accepts ASCII-format input files and is currently compatible with one-dimensional data files from SAXS beamlines at a number of synchrotron facilities. BioXTAS RAW is written in Python with C++ extensions.

A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-d-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)2 (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.

Ionotropic glutamate-like receptor δ2 binds d-serine and glycine

The orphan glutamate-like receptor GluRδ2 is predominantly expressed in Purkinje cells of the central nervous system. The classification of GluRδ2 to the ionotropic glutamate receptor family is based on sequence similarities, because GluRδ2 does not form functional homomeric glutamate-gated ion channels in transfected cells. Studies in GluRδ2−/− knockout mice as well as in mice with naturally occurring mutations in the GluRδ2 gene have demonstrated an essential role of GluRδ2 in cerebellar long-term depression, motor learning, motor coordination, and synaptogenesis. However, the lack of a known agonist has hampered investigations on the function of GluRδ2. In this study, the ligand-binding core of GluRδ2 (GluRδ2–S1S2) was found to bind neutral amino acids such as d-serine and glycine, as demonstrated by isothermal titration calorimetry. Direct evidence for binding of d-serine and structural rearrangements in the binding cleft of GluRδ2–S1S2 is provided by x-ray structures of GluRδ2–S1S2 in its apo form and in complex with d-serine. Functionally, d-serine and glycine were shown to inactivate spontaneous ion-channel conductance in GluRδ2 containing the lurcher mutation (EC50 values, 182 and 507 μM, respectively). These data demonstrate that the GluRδ2 ligand-binding core is capable of binding ligands and that cleft closure of the ligand-binding core can induce conformational changes that alter ion permeation.

Crystal structure of the kainate receptor GluR5 ligand-binding core in complex with (S)-glutamate

The X‐ray structure of the ligand‐binding core of the kainate receptor GluR5 (GluR5‐S1S2) in complex with (S)‐glutamate was determined to 1.95 Å resolution. The overall GluR5‐S1S2 structure comprises two domains and is similar to the related AMPA receptor GluR2‐S1S2J. (S)‐glutamate binds as in GluR2‐S1S2J. Distinct features are observed for Ser741, which stabilizes a highly coordinated network of water molecules and forms an interdomain bridge. The GluR5 complex exhibits a high degree of domain closure (26°) relative to apo GluR2‐S1S2J. In addition, GluR5‐S1S2 forms a novel dimer interface with a different arrangement of the two protomers compared to GluR2‐S1S2J.

High-throughput small angle X-ray scattering from proteins in solution using a microfluidic front-end.

This manuscript presents, for the first time, the method of automated structural analysis of biomolecules in solution on a microfluidic chip. A polymer-based micrototal analysis system for high-throughput Small-Angle X-ray Scattering (SAXS) data collection from biological macromolecules has been developed. The bioXTAS chip features an integrated X-ray transparent 200 nL sample chamber and diffusion-based mixing of protein and buffer solutions. Software for fully automated fluidic control, data acquisition, and data analysis has been developed. The proof-of concept is based on data using bovine serum albumin as the model system. It confirms the quality of SAXS data generated from small sample volumes and furthermore validates the on-chip mixing capabilities. SAXS data on the gradual unfolding of BSA induced by an anionic surfactant exemplifies how the bioXTAS chip can be used to follow and identify structural changes and proves the feasibility of high-throughput structural analysis in solution. In total, this shows that the bioXTAS chip has the potential for becoming a powerful tool for automated high-throughput structural analysis of macromolecular systems.

A new small-angle X-ray scattering set-up on the crystallography beamline I711 at MAX-lab.

A small-angle X-ray scattering (SAXS) set-up has recently been developed at beamline I711 at the MAX II storage ring in Lund (Sweden). An overview of the required modifications is presented here together with a number of application examples. The accessible q range in a SAXS experiment is 0.009-0.3 A(-1) for the standard set-up but depends on the sample-to-detector distance, detector offset, beamstop size and wavelength. The SAXS camera has been designed to have a low background and has three collinear slit sets for collimating the incident beam. The standard beam size is about 0.37 mm x 0.37 mm (full width at half-maximum) at the sample position, with a flux of 4 x 10(10) photons s(-1) and lambda = 1.1 A. The vacuum is of the order of 0.05 mbar in the unbroken beam path from the first slits until the exit window in front of the detector. A large sample chamber with a number of lead-throughs allows different sample environments to be mounted. This station is used for measurements on weakly scattering proteins in solutions and also for colloids, polymers and other nanoscale structures. A special application supported by the beamline is the effort to establish a micro-fluidic sample environment for structural analysis of samples that are only available in limited quantities. Overall, this work demonstrates how a cost-effective SAXS station can be constructed on a multipurpose beamline.