Publications by authors named "Adam Round"

59 Publications

Triangular Self-Assembling Coiled-Coil Protein Origami.

ACS Chem Biol 2021 02 21;16(2):310-315. Epub 2021 Jan 21.

Department of Synthetic Biology and Immunology, National Institute of Chemistry, 1000 Ljubljana, Slovenia.

Coiled-coil protein origami (CCPO) polyhedra are designed self-assembling nanostructures constructed from coiled coil (CC)-forming modules connected into a single chain. For testing new CCPO building modules, simpler polyhedra could be used that should maintain most features relevant to larger scaffolds. We show the design and characterization of nanoscale single-chain triangles, composed of six concatenated parallel CC dimer-forming segments connected by flexible linker peptides. The polypeptides self-assembled in bacteria in agreement with the design, and the shape of the polypeptides was confirmed with small-angle X-ray scattering. Fusion with split-fluorescent protein domains was used as a functional assay in bacteria, based on the discrimination between the correctly folded and misfolded nanoscale triangles comprising correct, mismatched, or truncated modules. This strategy was used to evaluate the optimal size of linkers between CC segments which comprised eight amino acid residues.
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http://dx.doi.org/10.1021/acschembio.0c00812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901019PMC
February 2021

Segmented flow generator for serial crystallography at the European X-ray free electron laser.

Nat Commun 2020 09 9;11(1):4511. Epub 2020 Sep 9.

School of Molecular Sciences, Arizona State University, Tempe, AZ, 85287-1604, USA.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.
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http://dx.doi.org/10.1038/s41467-020-18156-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481229PMC
September 2020

K vs. Na Effects on the Self-Assembly of Guanosine 5'-Monophosphate: A Solution SAXS Structural Study.

Nanomaterials (Basel) 2020 Mar 28;10(4). Epub 2020 Mar 28.

Department of Life and Environmental Sciences, Università Politecnica delle Marche, 60131 Ancona, Italy.

The hierarchical process of guanosine (G) self-assembly, leading in aqueous solution and in the presence of metal cations to the formation of G-quadruplexes, represents an intriguing topic both for the biological correlation with telomerase activity and for the nano-technological applications, as demonstrated by the current measured in a quadruplex wire 100 nm long. Similar to G-rich DNA sequences and G-oligonucleotides, the guanosine 5'-monophosphate (GMP) self-aggregates in water to form quadruplexes. However, due to the absence of a covalent axial backbone, this system can be very useful to understand the chemical-physical conditions that govern the guanosine supramolecular aggregation. We have then investigated by in-solution Synchrotron Small Angle X-ray Scattering technique the role of different cations in promoting the quadruplex formation as a function of concentration and temperature. Results show how potassium, with its peculiar biological traits, favours the G-quadruplex elongation process in respect to other cations (Na + , but also NH 4 + and Li + ), determining the longest particles in solution. Moreover, the formation and the elongation of G-quadruplexes have been demonstrated to be controlled by both GMP concentration and excess cation content, even if they specifically contribute to these processes in different ways. The occurrence of condensed liquid crystalline phases was also detected, proving that excess cations play also unspecific effects on the effective charges on the G-quadruplex surface.
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http://dx.doi.org/10.3390/nano10040629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7221663PMC
March 2020

E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E2.

J Biol Chem 2019 07 3;294(30):11404-11419. Epub 2019 Jun 3.

Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping, Sweden

The E3 ubiquitin-protein ligase TRIM21, of the RING-containing tripartite motif (TRIM) protein family, is a major autoantigen in autoimmune diseases and a modulator of innate immune signaling. Together with ubiquitin-conjugating enzyme E2 E1 (UBE2E1), TRIM21 acts both as an E3 ligase and as a substrate in autoubiquitination. We here report a 2.82-Å crystal structure of the human TRIM21 RING domain in complex with the human E2-conjugating UBE2E1 enzyme, in which a ubiquitin-targeted TRIM21 substrate lysine was captured in the UBE2E1 active site. The structure revealed that the direction of lysine entry is similar to that described for human proliferating cell nuclear antigen (PCNA), a small ubiquitin-like modifier (SUMO)-targeted substrate, and thus differs from the canonical SUMO-targeted substrate entry. In agreement, we found that critical UBE2E1 residues involved in the capture of the TRIM21 substrate lysine are conserved in ubiquitin-conjugating E2s, whereas residues critical for SUMOylation are not conserved. We noted that coordination of the acceptor lysine leads to remodeling of amino acid side-chain interactions between the UBE2E1 active site and the E2-E3 direct interface, including the so-called "linchpin" residue conserved in RING E3s and required for ubiquitination. The findings of our work support the notion that substrate lysine activation of an E2-E3-connecting allosteric path may trigger catalytic activity and contribute to the understanding of specific lysine targeting by ubiquitin-conjugating E2s.
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http://dx.doi.org/10.1074/jbc.RA119.008485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663867PMC
July 2019

Small-angle neutron and X-ray scattering analysis of the supramolecular organization of rhodopsin in photoreceptor membrane.

Biochim Biophys Acta Biomembr 2019 10 30;1861(10):183000. Epub 2019 May 30.

Department of Molecular Physiology, Biological Faculty Lomonosov Moscow State University, Leninskie Gory1, Moscow 119991, Russia; Emanuel Institute of Biochemical Physics Russian Academy of Sciences, Kosygin st.4, Moscow 119334, Russia; Joint Institute for Nuclear Research, Joliot-Curie, 6 Dubna, Moscow region 141980, Russia.

The supramolecular organization of the visual pigment rhodopsin in the photoreceptor membrane remains contentious. Specifically, whether this G protein-coupled receptor functions as a monomer or dimer remains unknown, as does the presence or absence of ordered packing of rhodopsin molecules in the photoreceptor membrane. Completely opposite opinions have been expressed on both issues. Herein, using small-angle neutron and X-ray scattering approaches, we performed a comparative analysis of the structural characteristics of the photoreceptor membrane samples in buffer, both in the outer segment of photoreceptor cells, and in the free photoreceptor disks. The average distance between the centers of two neighboring rhodopsin molecules was found to be ~5.8 nm in both cases. The results indicate an unusually high packing density of rhodopsin molecules in the photoreceptor membrane, but molecules appear to be randomly distributed in the membrane without any regular ordering.
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http://dx.doi.org/10.1016/j.bbamem.2019.05.022DOI Listing
October 2019

The Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography instrument of the European XFEL: initial installation.

J Synchrotron Radiat 2019 May 12;26(Pt 3):660-676. Epub 2019 Apr 12.

European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany.

The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation - in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described.
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http://dx.doi.org/10.1107/S1600577519003308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510195PMC
May 2019

Megahertz serial crystallography.

Authors:
Max O Wiedorn Dominik Oberthür Richard Bean Robin Schubert Nadine Werner Brian Abbey Martin Aepfelbacher Luigi Adriano Aschkan Allahgholi Nasser Al-Qudami Jakob Andreasson Steve Aplin Salah Awel Kartik Ayyer Saša Bajt Imrich Barák Sadia Bari Johan Bielecki Sabine Botha Djelloul Boukhelef Wolfgang Brehm Sandor Brockhauser Igor Cheviakov Matthew A Coleman Francisco Cruz-Mazo Cyril Danilevski Connie Darmanin R Bruce Doak Martin Domaracky Katerina Dörner Yang Du Hans Fangohr Holger Fleckenstein Matthias Frank Petra Fromme Alfonso M Gañán-Calvo Yaroslav Gevorkov Klaus Giewekemeyer Helen Mary Ginn Heinz Graafsma Rita Graceffa Dominic Greiffenberg Lars Gumprecht Peter Göttlicher Janos Hajdu Steffen Hauf Michael Heymann Susannah Holmes Daniel A Horke Mark S Hunter Siegfried Imlau Alexander Kaukher Yoonhee Kim Alexander Klyuev Juraj Knoška Bostjan Kobe Manuela Kuhn Christopher Kupitz Jochen Küpper Janine Mia Lahey-Rudolph Torsten Laurus Karoline Le Cong Romain Letrun P Lourdu Xavier Luis Maia Filipe R N C Maia Valerio Mariani Marc Messerschmidt Markus Metz Davide Mezza Thomas Michelat Grant Mills Diana C F Monteiro Andrew Morgan Kerstin Mühlig Anna Munke Astrid Münnich Julia Nette Keith A Nugent Theresa Nuguid Allen M Orville Suraj Pandey Gisel Pena Pablo Villanueva-Perez Jennifer Poehlsen Gianpietro Previtali Lars Redecke Winnie Maria Riekehr Holger Rohde Adam Round Tatiana Safenreiter Iosifina Sarrou Tokushi Sato Marius Schmidt Bernd Schmitt Robert Schönherr Joachim Schulz Jonas A Sellberg M Marvin Seibert Carolin Seuring Megan L Shelby Robert L Shoeman Marcin Sikorski Alessandro Silenzi Claudiu A Stan Xintian Shi Stephan Stern Jola Sztuk-Dambietz Janusz Szuba Aleksandra Tolstikova Martin Trebbin Ulrich Trunk Patrik Vagovic Thomas Ve Britta Weinhausen Thomas A White Krzysztof Wrona Chen Xu Oleksandr Yefanov Nadia Zatsepin Jiaguo Zhang Markus Perbandt Adrian P Mancuso Christian Betzel Henry Chapman Anton Barty

Nat Commun 2018 10 2;9(1):4025. Epub 2018 Oct 2.

Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607, Hamburg, Germany.

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.
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http://dx.doi.org/10.1038/s41467-018-06156-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168542PMC
October 2018

Insc:LGN tetramers promote asymmetric divisions of mammary stem cells.

Nat Commun 2018 03 9;9(1):1025. Epub 2018 Mar 9.

Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139, Milano, Italy.

Asymmetric cell divisions balance stem cell proliferation and differentiation to sustain tissue morphogenesis and homeostasis. During asymmetric divisions, fate determinants and niche contacts segregate unequally between daughters, but little is known on how this is achieved mechanistically. In Drosophila neuroblasts and murine mammary stem cells, the association of the spindle orientation protein LGN with the stem cell adaptor Inscuteable has been connected to asymmetry. Here we report the crystal structure of Drosophila LGN in complex with the asymmetric domain of Inscuteable, which reveals a tetrameric arrangement of intertwined molecules. We show that Insc:LGN tetramers constitute stable cores of Par3-Insc-LGN-Gαi complexes, which cannot be dissociated by NuMA. In mammary stem cells, the asymmetric domain of Insc bound to LGN:Gαi suffices to drive asymmetric fate, and reverts aberrant symmetric divisions induced by p53 loss. We suggest a novel role for the Insc-bound pool of LGN acting independently of microtubule motors to promote asymmetric fate specification.
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http://dx.doi.org/10.1038/s41467-018-03343-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844954PMC
March 2018

How to Analyze and Present SAS Data for Publication.

Adv Exp Med Biol 2017 ;1009:47-64

SPB/SFX instrument, European XFEL GmbH, Holzkoppel 4, 22869, Schenefeld, Germany.

SAS is a powerful technique to investigate oligomeric state and domain organization of macromolecules, e.g. proteins and nucleic acids, under physiological, functional and even time resolved conditions. However, reconstructing three dimensional structures from SAS data is inherently ambiguous, as no information about orientation and phase is available. In addition experimental artifacts such as radiation damage, concentration effects and incorrect background subtraction can hinder the interpretation of even lead to wrong results. In this chapter, explanations on how to analyze data and how to assess and minimize the influence of experimental artifacts on the data. Furthermore, guidelines on how to present the resulting data and models to demonstrate the data supports the conclusion being made and that it is not biased by artifacts, will be given.
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http://dx.doi.org/10.1007/978-981-10-6038-0_4DOI Listing
June 2018

Architecture of TAF11/TAF13/TBP complex suggests novel regulation properties of general transcription factor TFIID.

Elife 2017 11 7;6. Epub 2017 Nov 7.

BrisSynBio Centre, The School of Biochemistry, Faculty of Biomedical Sciences, University of Bristol, Bristol, United Kingdom.

General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-interaction domain (CTID) in TAF13, which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.
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http://dx.doi.org/10.7554/eLife.30395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690282PMC
November 2017

Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo.

Nat Biotechnol 2017 Nov 16;35(11):1094-1101. Epub 2017 Oct 16.

Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia.

Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized >20 single-chain protein cages in three shapes-tetrahedron, four-sided pyramid, and triangular prism-with the largest containing >700 amino-acid residues and measuring 11 nm in diameter. Their stability and folding kinetics were similar to those of natural proteins. Solution small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophysical analysis confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape.
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http://dx.doi.org/10.1038/nbt.3994DOI Listing
November 2017

Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

Sci Rep 2017 06 6;7(1):2848. Epub 2017 Jun 6.

Department of Structural & Computational Biology, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Vienna Biocenter Campus 5, A-1030, Vienna, Austria.

Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.
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http://dx.doi.org/10.1038/s41598-017-03220-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460201PMC
June 2017

Coupling High Throughput Microfluidics and Small-Angle X-ray Scattering to Study Protein Crystallization from Solution.

Anal Chem 2017 02 8;89(4):2282-2287. Epub 2017 Feb 8.

Laboratoire de Génie Chimique, Université de Toulouse, CNRS, INPT, UPS , 4 allée Emile Monso, 31432 Toulouse, France.

In this work, we propose the combination of small-angle X-ray scattering (SAXS) and high throughput, droplet based microfluidics as a powerful tool to investigate macromolecular interactions, directly related to protein solubility. For this purpose, a robust and low cost microfluidic platform was fabricated for achieving the mixing of proteins, crystallization reagents, and buffer in nanoliter volumes and the subsequent generation of nanodroplets by means of a two phase flow. The protein samples are compartmentalized inside droplets, each one acting as an isolated microreactor. Hence their physicochemical conditions (concentration, pH, etc.) can be finely tuned without cross-contamination, allowing the screening of a huge number of saturation conditions with a small amount of biological material. The droplet flow is synchronized with synchrotron radiation SAXS measurements to probe protein interactions while minimizing radiation damage. To this end, the experimental setup was tested with rasburicase (known to be very sensitive to denaturation), proving the structural stability of the protein in the droplets and the absence of radiation damage. Subsequently weak interaction variations as a function of protein saturation was studied for the model protein lysozime. The second virial coefficients (A2) were determined from the X-ray structure factors extrapolated to the origin. A2 obtained values were found to be in good agreement with data previously reported in literature but using only a few milligrams of protein. The experimental results presented here highlight the interest and convenience of using this methodology as a promising and potential candidate for studying protein interactions for the construction of phase diagrams.
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http://dx.doi.org/10.1021/acs.analchem.6b03492DOI Listing
February 2017

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline.

J Vis Exp 2017 01 5(119). Epub 2017 Jan 5.

Groupe de Microscopie Electronique et Méthodes, Institut de Biologie Structurale;

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.
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http://dx.doi.org/10.3791/54861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409194PMC
January 2017

SAXS Structural Studies of Dps from Deinococcus radiodurans Highlights the Conformation of the Mobile N-Terminal Extensions.

J Mol Biol 2017 03 11;429(5):667-687. Epub 2017 Jan 11.

ITQB-NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal. Electronic address:

The radiation-resistant bacterium Deinococcus radiodurans contains two DNA-binding proteins from starved cells (Dps): Dps1 (DR2263) and Dps2 (DRB0092). These are suggested to play a role in DNA interaction and manganese and iron storage. The proteins assemble as a conserved dodecameric structure with structurally uncharacterised N-terminal extensions. In the case of DrDps1, these extensions have been proposed to be involved in DNA interactions, while in DrDps2, their function has yet to be established. The reported data reveal the relative position of the N-terminal extensions to the dodecameric sphere in solution for both Dps. The low-resolution small angle X-ray scattering (SAXS) results show that the N-terminal extensions protrude from the spherical shell of both proteins. The SAXS envelope of a truncated form of DrDps1 without the N-terminal extensions appears as a dodecameric sphere, contrasting strongly with the protrusions observed in the full-length models. The effect of iron incorporation into DrDps2 was investigated by static and stopped-flow SAXS measurements, revealing dynamic structural changes upon iron binding and core formation, as reflected by a quick alteration of its radius of gyration. The truncated and full-length versions of DrDps were also compared on the basis of their interaction with DNA to analyse functional roles of the N-terminal extensions. DrDps1 N-terminal protrusions appear to be directly involved with DNA, whilst those from DrDps2 are indirectly associated with DNA binding. Furthermore, detection of DrDps2 in the D. radiodurans membrane fraction suggests that the N-terminus of the protein interacts with the membrane.
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http://dx.doi.org/10.1016/j.jmb.2017.01.008DOI Listing
March 2017

Development of tools to automate quantitative analysis of radiation damage in SAXS experiments.

J Synchrotron Radiat 2017 01 1;24(Pt 1):63-72. Epub 2017 Jan 1.

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

Biological small-angle X-ray scattering (SAXS) is an increasingly popular technique used to obtain nanoscale structural information on macromolecules in solution. However, radiation damage to the samples limits the amount of useful data that can be collected from a single sample. In contrast to the extensive analytical resources available for macromolecular crystallography (MX), there are relatively few tools to quantitate radiation damage for SAXS, some of which require a significant level of manual characterization, with the potential of leading to conflicting results from different studies. Here, computational tools have been developed to automate and standardize radiation damage analysis for SAXS data. RADDOSE-3D, a dose calculation software utility originally written for MX experiments, has been extended to account for the cylindrical geometry of the capillary tube, the liquid composition of the sample and the attenuation of the beam by the capillary material to allow doses to be calculated for many SAXS experiments. Furthermore, a library has been written to visualize and explore the pairwise similarity of frames. The calculated dose for the frame at which three subsequent frames are determined to be dissimilar is defined as the radiation damage onset threshold (RDOT). Analysis of RDOTs has been used to compare the efficacy of radioprotectant compounds to extend the useful lifetime of SAXS samples. Comparison of the RDOTs shows that, for radioprotectant compounds at 5 and 10 mM concentration, glycerol is the most effective compound. However, at 1 and 2 mM concentrations, dithiothreitol (DTT) appears to be most effective. Our newly developed visualization library contains methods that highlight the unusual radiation damage results given by SAXS data collected using higher concentrations of DTT: these observations should pave the way to the development of more sophisticated frame merging strategies.
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http://dx.doi.org/10.1107/S1600577516015083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5182020PMC
January 2017

Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection.

Structure 2017 01 15;25(1):107-120. Epub 2016 Dec 15.

Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, UK. Electronic address:

The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signaling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1):myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of ∼135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.
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http://dx.doi.org/10.1016/j.str.2016.11.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5222588PMC
January 2017

Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

PLoS Pathog 2016 Dec 14;12(12):e1006079. Epub 2016 Dec 14.

Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, Austria.

Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83) structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.
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http://dx.doi.org/10.1371/journal.ppat.1006079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5156371PMC
December 2016

Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules.

BMC Struct Biol 2016 10 27;16(1):18. Epub 2016 Oct 27.

European Molecular Biology Laboratory, 71 Avenue des Martyrs, Grenoble, 38000, France.

Background: Synchrotron radiation facilities are pillars of modern structural biology. Small-Angle X-ray scattering performed at synchrotron sources is often used to characterize the shape of biological macromolecules. A major challenge with high-energy X-ray beam on such macromolecules is the perturbation of sample due to radiation damage.

Results: By employing atomic force microscopy, another common technique to determine the shape of biological macromolecules when deposited on flat substrates, we present a protocol to evaluate and characterize consequences of radiation damage. It requires the acquisition of images of irradiated samples at the single molecule level in a timely manner while using minimal amounts of protein. The protocol has been tested on two different molecular systems: a large globular tetremeric enzyme (β-Amylase) and a rod-shape plant virus (tobacco mosaic virus). Radiation damage on the globular enzyme leads to an apparent increase in molecular sizes whereas the effect on the long virus is a breakage into smaller pieces resulting in a decrease of the average long-axis radius.

Conclusions: These results show that radiation damage can appear in different forms and strongly support the need to check the effect of radiation damage at synchrotron sources using the presented protocol.
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http://dx.doi.org/10.1186/s12900-016-0068-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081678PMC
October 2016

Online ion-exchange chromatography for small-angle X-ray scattering.

Acta Crystallogr D Struct Biol 2016 10 15;72(Pt 10):1090-1099. Epub 2016 Sep 15.

EMBL Grenoble Outstation, 71 Avenue des Martyrs, 38042 Grenoble, France.

Biological small-angle X-ray scattering (BioSAXS) is a powerful technique to determine the solution structure, particle size, shape and surface-to-volume ratio of macromolecules. However, a drawback is that the sample needs to be monodisperse. To ensure this, size-exclusion chromatography (SEC) has been implemented on many BioSAXS beamlines. Here, the integration of ion-exchange chromatography (IEC) using both continuous linear and step gradients on a beamline is described. Background subtraction for continuous gradients by shifting a reference measurement and two different approaches for step gradients, which are based on interpolating between two background measurements, are discussed. The results presented here serve as a proof of principle for online IEC and subsequent data treatment.
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http://dx.doi.org/10.1107/S2059798316012833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053136PMC
October 2016

Structural Basis of Host Autophagy-related Protein 8 (ATG8) Binding by the Irish Potato Famine Pathogen Effector Protein PexRD54.

J Biol Chem 2016 09 25;291(38):20270-20282. Epub 2016 Jul 25.

From the Department of Biological Chemistry, John Innes Centre, and

Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLR-type effector PexRD54 binds potato ATG8 via its ATG8 family-interacting motif (AIM) and perturbs host-selective autophagy. However, the structural basis of this interaction remains unknown. Here, we define the crystal structure of PexRD54, which includes a modular architecture, including five tandem repeat domains, with the AIM sequence presented at the disordered C terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effector's AIM sequence, and we established a model of the full-length PexRD54-ATG8CL complex using small angle x-ray scattering. Structure-informed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease.
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http://dx.doi.org/10.1074/jbc.M116.744995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025708PMC
September 2016

Noncovalent PEGylation via Lectin-Glycopolymer Interactions.

Biomacromolecules 2016 08 19;17(8):2719-25. Epub 2016 Jul 19.

School of Chemistry, National University of Ireland Galway , University Road, Galway, Ireland.

PEGylation, the covalent modification of proteins with polyethylene glycol, is an abundantly used technique to improve the pharmacokinetics of therapeutic proteins. The drawback with this methodology is that the covalently attached PEG can impede the biological activity (e.g., reduced receptor-binding capacity). Protein therapeutics with "disposable" PEG modifiers have potential advantages over the current technology. Here, we show that a protein-polymer "Medusa complex" is formed by the combination of a hexavalent lectin with a glycopolymer. Using NMR spectroscopy, small-angle X-ray scattering (SAXS), size exclusion chromatography, and native gel electrophoresis it was demonstrated that the fucose-binding lectin RSL and a fucose-capped polyethylene glycol (Fuc-PEG) form a multimeric assembly. All of the experimental methods provided evidence of noncovalent PEGylation with a concomitant increase in molecular mass and hydrodynamic radius. The affinity of the protein-polymer complex was determined by ITC and competition experiments to be in the micromolar range, suggesting that such systems have potential biomedical applications.
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http://dx.doi.org/10.1021/acs.biomac.6b00766DOI Listing
August 2016

The Use of Small-Angle Scattering for the Characterization of Multi Subunit Complexes.

Authors:
Adam Round

Adv Exp Med Biol 2016 ;896:329-50

European Molecular Biology Laboratory, Grenoble, 71 avenue des Martyrs, 38042, France.

As the continuing trend in structural biology is to probe ever more complex systems, new methodologies are being developed plus existing techniques are being expanded and adapted, to keep up with the demands of the research community. To investigate multi subunit complexes (protein-DNA, protein-RNA or protein-protein complexes) no one technique holds a monopoly, as each technique yields independent information inaccessible to the other methods, but can be used together in a complementary way. Additionally as large conformational changes are not unlikely, investigation of the dynamics of these systems under physiological conditions is needed to fully understand their function. Investigations under physiological conditions in solution are becoming more standardized and with more dedicated, automated beamlines available these experiments are easy to access by the general research community. As such the need for explanations of how to plan and undertake these experiments is needed. In this chapter we will cover the requirements of these experiments as well and how to plan undertake and analyze the results of such experiments.
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http://dx.doi.org/10.1007/978-3-319-27216-0_21DOI Listing
September 2016

Domain Organization of Vaccinia Virus Helicase-Primase D5.

J Virol 2016 May 14;90(9):4604-4613. Epub 2016 Apr 14.

Institut de Biologie Structurale (IBS), CEA, CNRS, University Grenoble Alpes, Grenoble, France

Unlabelled: Poxviridae are viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in the Phycodnaviridae We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single- and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins.

Importance: Since the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase for replication, as do poxviruses. Here we provide for the first time information about the domain structure and DNA-binding activity of D5, the poxvirus helicase-primase. This result not only refines the current model of the poxvirus replication fork but also will lead in the long run to a structural basis for antiviral drug design.
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http://dx.doi.org/10.1128/JVI.00044-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836322PMC
May 2016

FANCM interacts with PCNA to promote replication traverse of DNA interstrand crosslinks.

Nucleic Acids Res 2016 Apr 28;44(7):3219-32. Epub 2016 Jan 28.

Rudolf Virchow Center for Experimental Biomedicine, Institute for Structural Biology, University of Würzburg, D-97080 Würzburg, Germany

FANCM is a highly conserved DNA remodeling enzyme that promotes the activation of the Fanconi anemia DNA repair pathway and facilitates replication traverse of DNA interstrand crosslinks. However, how FANCM interacts with the replication machinery to promote traverse remains unclear. Here, we show that FANCM and its archaeal homolog Hef from Thermoplasma acidophilum interact with proliferating cell nuclear antigen (PCNA), an essential co-factor for DNA polymerases in both replication and repair. The interaction is mediated through a conserved PIP-box; and in human FANCM, it is strongly stimulated by replication stress. A FANCM variant carrying a mutation in the PIP-box is defective in promoting replication traverse of interstrand crosslinks and is also inefficient in promoting FANCD2 monoubiquitination, a key step of the Fanconi anemia pathway. Our data reveal a conserved interaction mode between FANCM and PCNA during replication stress, and suggest that this interaction is essential for FANCM to aid replication machines to traverse DNA interstrand crosslinks prior to post-replication repair.
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http://dx.doi.org/10.1093/nar/gkw037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838364PMC
April 2016

The solution configurations of inactive and activated DntR have implications for the sliding dimer mechanism of LysR transcription factors.

Sci Rep 2016 Jan 28;6:19988. Epub 2016 Jan 28.

Structural Bioloy Group, European Synchrotron Radiation Facility (ESRF), CS 40220, 38043 Grenoble Cedex 9, France.

LysR Type Transcriptional Regulators (LTTRs) regulate basic metabolic pathways or virulence gene expression in prokaryotes. Evidence suggests that the activation of LTTRs involves a conformational change from an inactive compact apo- configuration that represses transcription to an active, expanded holo- form that promotes it. However, no LTTR has yet been observed to adopt both configurations. Here, we report the results of structural studies of various forms of the LTTR DntR. Crystal structures of apo-DntR and of a partially autoinducing mutant H169T-DntR suggest that active and inactive DntR maintain a compact homotetrameric configuration. However, Small Angle X-ray Scattering (SAXS) studies on solutions of apo-, H169T- and inducer-bound holo-DntR indicate a different behaviour, suggesting that while apo-DntR maintains a compact configuration in solution both H169T- and holo-DntR adopt an expanded conformation. Models of the SAXS-obtained solution conformations of apo- and holo-DntR homotetramers in complex with promoter-operator region DNA are consistent with previous observations of a shifting of LTTR DNA binding sites upon activation and a consequent relaxation in the bend of the promoter-operator region DNA. Our results thus provide clear evidence at the molecular level which strongly supports the 'sliding dimer' hypothesis concerning LTTR activation mechanisms.
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http://dx.doi.org/10.1038/srep19988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730206PMC
January 2016

Evolution of the Plant Reproduction Master Regulators LFY and the MADS Transcription Factors: The Role of Protein Structure in the Evolutionary Development of the Flower.

Front Plant Sci 2015 6;6:1193. Epub 2016 Jan 6.

CNRS, Laboratoire de Physiologie Cellulaire & Végétale, UMR 5168Grenoble, France; Laboratoire de Physiologie Cellulaire & Végétale, University of Grenoble AlpesGrenoble, France; Commissariat à l´Energie Atomique et aux Energies Alternatives, Direction des Sciences du Vivant, Laboratoire de Physiologie Cellulaire & Végétale, Institut de Recherches en Technologies et Sciences pour le VivantGrenoble, France; Laboratoire de Physiologie Cellulaire & Végétale, Institut National de la Recherche AgronomiqueGrenoble, France.

Understanding the evolutionary leap from non-flowering (gymnosperms) to flowering (angiosperms) plants and the origin and vast diversification of the floral form has been one of the focuses of plant evolutionary developmental biology. The evolving diversity and increasing complexity of organisms is often due to relatively small changes in genes that direct development. These "developmental control genes" and the transcription factors (TFs) they encode, are at the origin of most morphological changes. TFs such as LEAFY (LFY) and the MADS-domain TFs act as central regulators in key developmental processes of plant reproduction including the floral transition in angiosperms and the specification of the male and female organs in both gymnosperms and angiosperms. In addition to advances in genome wide profiling and forward and reverse genetic screening, structural techniques are becoming important tools in unraveling TF function by providing atomic and molecular level information that was lacking in purely genetic approaches. Here, we summarize previous structural work and present additional biophysical and biochemical studies of the key master regulators of plant reproduction - LEAFY and the MADS-domain TFs SEPALLATA3 and AGAMOUS. We discuss the impact of structural biology on our understanding of the complex evolutionary process leading to the development of the bisexual flower.
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http://dx.doi.org/10.3389/fpls.2015.01193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701952PMC
January 2016

Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of PB2 Domains.

Mol Cell 2016 Jan 17;61(1):125-37. Epub 2015 Dec 17.

European Molecular Biology Laboratory Grenoble Outstation and Unit of Virus Host-Cell Interactions, University Grenoble Alpes-CNRS-EMBL, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France. Electronic address:

Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)-containing domain translocates ∼90 Å to bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs.
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http://dx.doi.org/10.1016/j.molcel.2015.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712189PMC
January 2016

Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei.

J Biol Chem 2015 Dec 22;290(51):30498-513. Epub 2015 Oct 22.

From the Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India,

Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis, or sleeping sickness. The causative parasite, Trypanosoma, encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. The latter are acidic membrane-enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg(2+) and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from TbbVSP1 crystal structure is further confirmed by SAXS and TEM data. SAXS data suggest structural flexibility in EF hand domains indicative of conformational plasticity within TbbVSP1.
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http://dx.doi.org/10.1074/jbc.M115.674176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683271PMC
December 2015

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α.

J Am Chem Soc 2015 Dec 16;137(48):15122-34. Epub 2015 Oct 16.

Univ. Grenoble Alpes , Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.
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http://dx.doi.org/10.1021/jacs.5b07765DOI Listing
December 2015