Hexasome-INO80 complex reveals structural basis of noncanonical nucleosome remodeling.

Loss of H2A-H2B histone dimers is a hallmark of actively transcribed genes, but how the cellular machinery functions in the context of noncanonical nucleosomal particles remains largely elusive. In this work, we report the structural mechanism for adenosine 5'-triphosphate-dependent chromatin remodeling of hexasomes by the INO80 complex. We show how INO80 recognizes noncanonical DNA and histone features of hexasomes that emerge from the loss of H2A-H2B. A large structural rearrangement switches the catalytic core of INO80 into a distinct, spin-rotated mode of remodeling while its nuclear actin module remains tethered to long stretches of unwrapped linker DNA. Direct sensing of an exposed H3-H4 histone interface activates INO80, independently of the H2A-H2B acidic patch. Our findings reveal how the loss of H2A-H2B grants remodelers access to a different, yet unexplored layer of energy-driven chromatin regulation.

Crystal structure and interactions of the Tof1-Csm3 (Timeless-Tipin) fork protection complex.

The Tof1-Csm3 fork protection complex has a central role in the replisome-it promotes the progression of DNA replication forks and protects them when they stall, while also enabling cohesion establishment and checkpoint responses. Here, I present the crystal structure of the Tof1-Csm3 complex from Chaetomium thermophilum at 3.1 Å resolution. The structure reveals that both proteins together form an extended alpha helical repeat structure, which suggests a mechanical or scaffolding role for the complex. Expanding on this idea, I characterize a DNA interacting region and a cancer-associated Mrc1 binding site. This study provides the molecular basis for understanding the functions of the Tof1-Csm3 complex, its human orthologue the Timeless-Tipin complex and additionally the Drosophila circadian rhythm protein Timeless.

Structure of the primed state of the ATPase domain of chromatin remodeling factor ISWI bound to the nucleosome.

ATP-dependent chromatin remodeling factors of SWI/SNF2 family including ISWI, SNF2, CHD1 and INO80 subfamilies share a conserved but functionally non-interchangeable ATPase domain. Here we report cryo-electron microscopy (cryo-EM) structures of the nucleosome bound to an ISWI fragment with deletion of the AutoN and HSS regions in nucleotide-free conditions and the free nucleosome at ∼ 4 Å resolution. In the bound conformation, the ATPase domain interacts with the super helical location 2 (SHL 2) of the nucleosomal DNA, with the N-terminal tail of H4 and with the α1 helix of H3. Density for other regions of ISWI is not observed, presumably due to disorder. Comparison with the structure of the free nucleosome reveals that although the histone core remains largely unchanged, remodeler binding causes perturbations in the nucleosomal DNA resulting in a bulge near the SHL2 site. Overall, the structure of the nucleotide-free ISWI-nucleosome complex is similar to the corresponding regions of the recently reported ADP bound ISWI-nucleosome structures, which are significantly different from that observed for the ADP-BeFx bound structure. Our findings are relevant to the initial step of ISWI binding to the nucleosome and provide additional insights into the nucleosome remodeling process driven by ISWI.

Solution structure and flexibility of the condensin HEAT-repeat subunit Ycg1.

High-resolution structural analysis of flexible proteins is frequently challenging and requires the synergistic application of different experimental techniques. For these proteins, small-angle X-ray scattering (SAXS) allows for a quantitative assessment and modeling of potentially flexible and heterogeneous structural states. Here, we report SAXS characterization of the condensin HEAT-repeat subunit Ycg1Cnd3 in solution, complementing currently available high-resolution crystallographic models. We show that the free Ycg1 subunit is flexible in solution but becomes considerably more rigid when bound to its kleisin-binding partner protein Brn1Cnd2 The analysis of SAXS and dynamic and static multiangle light scattering data furthermore reveals that Ycg1 tends to oligomerize with increasing concentrations in the absence of Brn1. Based on these data, we present a model of the free Ycg1 protein constructed by normal mode analysis, as well as tentative models of Ycg1 dimers and tetramers. These models enable visualization of the conformational transitions that Ycg1 has to undergo to adopt a closed rigid shape and thereby create a DNA-binding surface in the condensin complex.

Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.

Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1-3. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4-7. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.

3.2-Å-resolution structure of the 90S preribosome before A1 pre-rRNA cleavage.

The 40S small ribosomal subunit is cotranscriptionally assembled in the nucleolus as part of a large chaperone complex called the 90S preribosome or small-subunit processome. Here, we present the 3.2-Å-resolution structure of the Chaetomium thermophilum 90S preribosome, which allowed us to build atomic structures for 34 assembly factors, including the Mpp10 complex, Bms1, Utp14 and Utp18, and the complete U3 small nucleolar ribonucleoprotein. Moreover, we visualized the U3 RNA heteroduplexes with a 5' external transcribed spacer (5' ETS) and pre-18S RNA, and their stabilization by 90S factors. Overall, the structure explains how a highly intertwined network of assembly factors and pre-rRNA guide the sequential, independent folding of the individual pre-40S domains while the RNA regions forming the 40S active sites are kept immature. Finally, by identifying the unprocessed A1 cleavage site and the nearby Utp24 endonuclease, we suggest a proofreading model for regulated 5'-ETS separation and 90S-pre-40S transition.

Structural basis for 5'-ETS recognition by Utp4 at the early stages of ribosome biogenesis.

Eukaryotic ribosome biogenesis begins with the co-transcriptional assembly of the 90S pre-ribosome. The 'U three protein' (UTP) complexes and snoRNP particles arrange around the nascent pre-ribosomal RNA chaperoning its folding and further maturation. The earliest event in this hierarchical process is the binding of the UTP-A complex to the 5'-end of the pre-ribosomal RNA (5'-ETS). This oligomeric complex predominantly consists of ß-propeller and α-solenoidal proteins. Here we present the structure of the Utp4 subunit from the thermophilic fungus Chaetomium thermophilum at 2.15 Å resolution and analyze its function by UV RNA-crosslinking (CRAC) and in context of a recent cryo-EM structure of the 90S pre-ribosome. Utp4 consists of two orthogonal and highly basic ß-propellers that perfectly fit the EM-data. The Utp4 structure highlights an unusual Velcro-closure of its C-terminal ß-propeller as relevant for protein integrity and potentially Utp8 recognition in the context of the pre-ribosome. We provide a first model of the 5'-ETS RNA from the internally hidden 5'-end up to the region that hybridizes to the 3'-hinge sequence of U3 snoRNA and validate a specific Utp4/5'-ETS interaction by CRAC analysis.

Visualization of the structural changes in plywood and gypsum board during the growth of Chaetomium globosum and Stachybotrys chartarum.

Fungal growth in indoor environments is associated with many negative health effects. Many studies focus on brown- and white-rot fungi and their effect on wood, but there is none that reveals the influence of soft-rot fungi, such as Stachybotrys spp. and Chaetomium spp., on the structure of building materials such as plywood and gypsum wallboard. This study focuses on using micro-computed tomography (microCT) to investigate changes of the structure of plywood and gypsum wallboard during fungal degradation by S. chartarum and C. globosum. Changes in the materials as a result of dampness and fungal growth were determined by measuring porosity and pore shape via microCT. The results show that the composition of the building material influenced the level of penetration by fungi as shown by scanning electron microscopy (SEM). Plywood appeared to be the most affected, with the penetration of moisture and fungi throughout the whole thickness of the sample. Conversely, fungi grew only on the top cardboard in the gypsum wallboard and they did not have significant influence on the gypsum wallboard structure. The majority of the observed changes in gypsum wallboard occurred due to moisture. This paper suggests that the mycelium distribution within building materials and the structural changes, caused by dampness and fungal growth, depend on the type of the material.

Architecture of the fungal nuclear pore inner ring complex.

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation.

The genus Chaetomium in Iran, a phylogenetic study including six new species.

Twenty-one species of Chaetomium known from Iran were compared on the basis of morphological and molecular characters. Six new species are recognized, five isolated from cereals and one from nematode cysts. A combined sequence dataset of the ITS region, partial LSU rDNA, and ß-tubulin gene sufficiently resolved five species groups of Chaetomium that are largely concordant with combined features of peridium structure, ascospore shape and germ pore position. Among the new species C. undulatulum is a close relative of C. globosum, C. rectangulare is close to C. elatum, C. interruptum and C. grande are close to C. megalocarpum, altogether forming the C. globosum species group. Chaetomium iranianum and C. truncatulum are members of the C. carinthiacum species group, characterized by spirally coiled ascomatal hairs and fusiform ascospores. A chrysosporium-like anamorph is newly described for C. acropullum.

Fatal Chaetomium cerebritis in a bone marrow transplant patient.

The number of opportunistic infections in the central nervous system (CNS) has been steadily increasing because of a rising number of immunocompromised patients. A rare form of CNS infection can be caused by Chaetomium species, one of the largest genera of saprophytic ascomycetes. The CNS lesions in the present case were caused by Chaetomium atrobrunneum. The main characteristic of almost all Chaetomium species is presence of hairs or setae covering the ascomata. Microbiological studies are the only definitive way to correctly identify this fungal organism. The rapid evolvement of the cerebral infection suggests that the brain tissue provides a favorable environment for growth and proliferation of these fungi. This is the second documented case of a fatal brain abscess caused by Chaetomium atrobrunneum, and the first case report in a bone marrow transplant patient.

Ultrastructural alterations produced by sertaconazole on several opportunistic pathogenic fungi.

Sertaconazole is a new synthetic antifungal which has shown promising activity against numerous fungi. The morphological changes induced by the drug at two concentrations, the MIC and 10 times the MIC, during 24 and 48 h on the filamentous opportunistic fungi Aspergillus fumigatus, Chaetomium atrobrunneum and Scedosporium prolificans indicated that the intensity of the ultrastructural breakdown depended on the dose and not on the time of exposure. Common severe alterations of many fungal cells, which could be considered beyond repair, were seen at the cell wall, plasmalemma and cytoplasm levels. The results obtained underline the drug efficacy for those fungi and suggest its applicability as therapeutic agent in the human infections produced by them.