Molecular Mechanisms of Membrane Deformation by I-BAR Domain Proteins

SummaryBackgroundGeneration of membrane curvature is critical for the formation of plasma membrane protrusions and invaginations and for shaping intracellular organelles. Among the central regulators of membrane dynamics are the BAR superfamily domains, which deform membranes into tubular structures. In contrast to the relatively well characterized BAR and F-BAR domains that promote the formation of plasma membrane invaginations, I-BAR domains induce plasma membrane protrusions through a poorly understood mechanism.ResultsWe show that I-BAR domains induce strong PI(4,5)P2 clustering upon membrane binding, bend the membrane through electrostatic interactions, and remain dynamically associated with the inner leaflet of membrane tubules. Thus, I-BAR domains induce the formation of dynamic membrane protrusions to the opposite direction than do BAR and F-BAR domains. Strikingly, comparison of different I-BAR domains revealed that they deform PI(4,5)P2-rich membranes through distinct mechanisms. IRSp53 and IRTKS I-BARs bind membranes mainly through electrostatic interactions, whereas MIM and ABBA I-BARs additionally insert an amphipathic helix into the membrane bilayer, resulting in larger tubule diameter in vitro and more efficient filopodia formation in vivo. Furthermore, FRAP analysis revealed that whereas the mammalian I-BAR domains display dynamic association with filopodia, the C. elegans I-BAR domain forms relatively stable structures inside the plasma membrane protrusions.ConclusionsThese data define I-BAR domain as a functional member of the BAR domain superfamily and unravel the mechanisms by which I-BAR domains deform membranes to induce filopodia in cells. Furthermore, our work reveals unexpected divergence in the mechanisms by which evolutionarily distinct groups of I-BAR domains interact with PI(4,5)P2-rich membranes

Cryo-EM in structural virology : dissecting the icosahedral capsid

Technological advances especially in the last two decades have made it feasible to study virus structures to atomic or near-atomic resolution by a combination of cryogenic electron microscopy (cryo-EM) and image reconstruction or by X-ray crystallography. Drawing from these data, intertwined concepts of “viral self” and structure-based viral lineages been put forward and refined during the last decade. In order to address these theories, I used cryo-EM, 3D image reconstruction, homology modelling and de novo atomic modelling of proteins to study five viruses with icosahedrally symmetric protein capsids. The capsid structures of bacteriophage Bam35, two African horse sickness virus serotypes (AHSV-4 and AHSV-7 tVP2), archaeal head-tailed virus HSTV-1, and Nora virus (NORAV) that infects Drosophila melanogaster were solved to subnanometer resolution. I examine the methodological advances in the field of cryoEM and image processing that have occurred over the time span of the original articles included in the thesis, giving an empirical perspective on the important changes required to reach atomic resolution in virus structural determination. These included improved imaging, data recording, automation, and software developments. Notably, grid preparation is the area where the next strides need to be made to improve reproducibility and throughput. The results contribute to the general field of structural virology as well as shedding light into more specific areas, such as biological membrane modulation, archaeal viruses and vaccine development. Moreover, the hosts of the viruses studied span all three domains of life (bacteria, archaea and eukaryotes). This unusually wide sampling of the viral universe, or virosphere, creates an excellent basis for testing the utilisation of capsid structure in structure-based virus classification as well as verification of structure-based viral lineages. The results allowed unambiguous structure-based classification of the five studied viruses into the four previously postulated virus lineages; the picornavirus-like lineage, the HK97-like tailed-phage lineage, the PRD1/adenovirus lineage and the icosahedral dsRNA virus lineage. Furthermore, the NORAV reconstruction at 2.7 Å resolution provided structural evidence suggesting that NORAV is a representative of a new virus family within the order Picornavirales; a result not achievable by genetic evidence alone and a benchmark example of structure-based virus classification.Virusten rakenteiden tarkka kuvantaminen on mahdollista käyttäen kryogeenista elektronimikroskopiaa (kryo-EM) tai vaihtoehtoisesti röntgenkristallografiaa. Alan teknologinen kehitys viimeisen kahden vuosikymmenen aikana on ollut merkittävää, ja saatujen tulosten perusteella on kehitetty hypoteeseja virusten pohjimmaisesta olemuksesta sekä virusten kuorirakenteisiin perustuvista sukulinjoista. Väitöskirjassani tarkastelen viiden viruksen rakennetta näiden teorioiden valossa. Käytin kryogeenista elektronimikroskopiaa, tiheyskartan rekonstruointia, proteiinirakenteita vertailevaa mallinnusta ja de novo proteiinirakenteen mallinnusta viiteen virioniin joiden kuorirakenteet noudattavat 20 -tahoista eli ikosahedraalista symmetriaa. Selvitimme yhtä nanometriä paremmalla erotuskyvyllä kuorirakenteen bakteerivirus Bam35:stä, kahdesta afrikkalaista hevosruttoa aiheuttavasta viruksesta (AHSV-4 ja AHSV-7 tVP2), arkeoneja infektoivasta viruksesta (HSTV-1) ja Drosophila melanogaster -kärpästä infektoivasta Nora -viruksesta (NORAV). Työssäni tarkastelen kryo-EM-kuvantamis- ja rekonstruktiomenetelmien kehitystä alkuperäisjulkaisujen kattamalta ajanjaksolta. Pitkälle ajanjaksolle levittäytyvien tutkimustulosten rinnakkaisvertailu avaa yleisnäkymän niihin kriittisiin kehitysaskeliin jotka ovat olleet tarpeen atomiresoluution saavuttamisessa kryo-EM:ään perustuvassa virusten rakennetutkimuksessa. Nämä kehitysaskeleet perustuvat parannuksiin kuvan muodostuksessa, kuvan tallennusmenetelmissä, automaatiossa ja ohjelmistoissa. Näytehilojen valmistaminen näyttäytyy tärkeänä tulevaisuuden kehitysalueena. Saavutetut tutkimustulokset edistävät yleisemmällä tasolla rakennevirologian tietämystämme, valottaen lisäksi kapea-alaisempia kysymyksiä liittyen mm. biologisten kalvorakenteiden vuorovaikutuksiin, arkeonien viruksiin ja rokotuskehitykseen. Tutkittujen virusten isäntäsolut kattavat kaikki kolme eliökunnan päähaaraa (bakteerit, arkeonit ja aitotumalliset). Tämä virusten maailman, virosfäärin, laaja-alainen kattavuus tarjoaa erinomaiset lähtökohdat rakenteeseen perustuvan virusluokittelun käytännön kokeiluun sekä jo olemassa olevien luokitteluhypoteesien varmentamiseen. Tulokset mahdollistivat kaikkien viiden tutkitun viruksen kiistattoman luokittelun neljään aikaisemmin esitettyyn rakennepohjaiseen sukulinjaan: pikornavirusten kaltaisten virusten sukulinjaan, HK97-viruksen kaltaisten hännällisten faagien sukulinjaan, PRD1/adenovirus-sukulinjaan ja ikosahedraalisten kaksijuoste-RNA-virusten sukulinjaan. Lisäksi NORAV:n 2,7 Ångströmin tarkkuuden saavuttanut rakenne osoittaa että kyseinen virus edustaa aikaisemmin tuntematonta virusperhettä Picornavirales-lahkossa. Tutkimustulokseen ei olisi päästy pelkin geneettisin menetelmin ja se on erinomainen esimerkki rakennepohjaisesta virusluokittelusta

Structural insight into African horsesickness virus infection

African horsesickness (AHS) is a devastating disease of horses. The disease is caused by the double-stranded RNA-containing African horsesickness virus (AHSV). Using electron cryomicroscopy and three-dimensional image reconstruction, we determined the architecture of an AHSV serotype 4 (AHSV-4) reference strain. The structure revealed triple-layered AHS virions enclosing the segmented genome and transcriptase complex. The innermost protein layer contains 120 copies of VP3, with the viral polymerase, capping enzyme, and helicase attached to the inner surface of the VP3 layer on the 5-fold axis, surrounded by double-stranded RNA. VP7 trimers form a second, T 13 layer on top of VP3. Comparative analyses of the structures of bluetongue virus and AHSV-4 confirmed that VP5 trimers form globular domains and VP2 trimers form triskelions, on the virion surface. We also identified an AHSV-7 strain with a truncated VP2 protein (AHSV-7 tVP2) which outgrows AHSV-4 in culture. Comparison of AHSV-7 tVP2 to bluetongue virus and AHSV-4 allowed mapping of two domains in AHSV-4 VP2, and one in bluetongue virus VP2, that are important in infection. We also revealed a protein plugging the 5-fold vertices in AHSV-4. These results shed light on virus-host interactions in an economically important orbivirus to help the informed design of new vaccines

Diblock copolymers consisting of a polymerized ionic liquid and poly(N-isopropylacrylamide). Effects of PNIPAM block length and counter ion on self-assembling and thermal properties.

Amphiphilic diblock copolymers composed of a polymeric ionic liq., PIL, and poly(N-isopropylacrylamide), PNIPAM, have been synthesized using RAFT reactions. The length of the PIL block was kept const. while the mol. mass of the PNIPAM block was varied. The PIL was poly(2-(1-butylimidazolium-3-yl)ethyl methacrylate tetrafluoroborate) which is insol. in water owing to the bulky hydrophobic counterion. When the PNIPAM block was long enough, the polymers formed spherical micelles in water, which showed thermally responsive behavior. Colloidally stable particles could be prepd. also from the homopolymer PIL. PNIPAM affects noticeably the properties of the PIL, and also the polycation has a strong effect on the thermal properties of PNIPAM in aq. dispersions. As a ref., a polymer where bromide was the counterion instead of the tetrafluoroborate ion was synthesized, providing a water sol. PIL block. The core-shell micelles formed by amphiphilic block copolymers in pure water and those by double-hydrophilic polymers in aq. NaBF4 undergo partial structural inversion upon the thermal collapse of PNIPAM. [on SciFinder(R)

Electron Cryotomography of Tula Hantavirus Suggests a Unique Assembly Paradigm for Enveloped Viruses▿

Hantaviruses (family Bunyaviridae) are rodent-borne emerging viruses that cause a serious, worldwide threat to human health. Hantavirus diseases include hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Virions are enveloped and contain a tripartite single-stranded negative-sense RNA genome. Two types of glycoproteins, GN and GC, are embedded in the viral membrane and form protrusions, or “spikes.” The membrane encloses a ribonucleoprotein core, which consists of the RNA segments, the nucleocapsid protein, and the RNA-dependent RNA polymerase. Detailed information on hantavirus virion structure and glycoprotein spike composition is scarce. Here, we have studied the structures of Tula hantavirus virions using electron cryomicroscopy and tomography. Three-dimensional density maps show how the hantavirus surface glycoproteins, membrane, and ribonucleoprotein are organized. The structure of the GN-GC spike complex was solved to 3.6-nm resolution by averaging tomographic subvolumes. Each spike complex is a square-shaped assembly with 4-fold symmetry. Spike complexes formed ordered patches on the viral membrane by means of specific lateral interactions. These interactions may be sufficient for creating membrane curvature during virus budding. In conclusion, the structure and assembly principles of Tula hantavirus exemplify a unique assembly paradigm for enveloped viruses

Interaction of αVβ3 and αVβ6 Integrins with Human Parechovirus 1▿

Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to αV integrins. The interaction of HPEV1 with αV integrins is presumably mediated by a C-terminal RGD motif in the capsid protein VP1. We characterized the binding of integrins αVβ3 and αVβ6 to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, αVβ6 bound more efficiently than αVβ3 to immobilized HPEV1. Moreover, soluble αVβ6, but not αVβ3, blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD peptides. Using electron cryo-microscopy and image reconstruction, we showed that HPEV1 has the typical T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the 5-fold and 3-fold symmetry axes. This result does not match the RGD position predicted from the coxsackievirus A9 X-ray structure but is consistent with the predicted location of this motif in the shorter C terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than to significantly different viral footprints

Hexanol-Induced Order-Disorder Transitions in Lamellar Self-Assembling Aggregates of Bacteriochlorophyll c in Chlorobium tepidum Chlorosomes

Chlorosomes are light-harvesting complexes of green photosynthetic bacteria. Chlorosomes contain bacteriochlorophyll (BChl) c, d, or e aggregates that exhibit strong excitonic coupling. The short-range order, which is responsible for the coupling, has been proposed to be augmented by pigment arrangement into undulated lamellar structures with spacing between 2 and 3 nm. Treatment of chlorosomes with hexanol reversibly converts the aggregated chlorosome chlorophylls into a form with spectral properties very similar to that of the monomer. Although this transition has been extensively studied, the structural basis remains unclear due to variability in the obtained morphologies. Here we investigated hexanol-induced structural changes in the lamellar organization of BChl c in chlorosomes from Chlorobium tepidum by a combination of X-ray scattering, electron cryomicroscopy, and optical spectroscopy. At a low hexanol/pigment ratio, the lamellae persisted in the presence of hexanol while the short-range order and exciton interactions between chlorin rings were effectively eliminated, producing a monomer-like absorption. The result suggested that hexanol hydroxyls solvated the chlorin rings while the aliphatic tail partitioned into the hydrophobic part of the lamellar structure. This partitioning extended the chlorosome along its long axis. Further increase of the hexanol/pigment ratio produced round pigment-hexanol droplets, which lost all lamellar order. After hexanol removal the spectral properties were restored. In the samples treated under the high hexanol/pigment ratio, lamellae reassembled in small domains after hexanol removal while the shape and long-range order were irreversibly lost. Thus, all the interactions required for establishing the short-range order by self-assembly are provided by BChl c molecules alone. However, the long-range order and overall shape are imposed by an external structure, e.g., the proteinaceous chlorosome baseplate.This study was supported by the Spanish Ministry of Science and Education (Grant No. BFU2004-04914-C02-02 to J.B.A.), the Academy of Finland (Research Fellowship No. 1118462 to R.T.), the Czech Ministry of Education, Youth and Sports and Czech Science Foundation (Projects Nos. MSM0021620835 and 206/05/2739 to J.P.). Electron microscopy was carried out in the Electron Microscopy Unit of the Institute of Biotechnology, University of Helsinki.Peer reviewe