Core architecture of a bacterial type II secretion system

Bacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane. Here we use negative stain and cryo-electron microscopy to reveal the core architecture of an assembled T2SS from the pathogen Klebsiella pneumoniae. We show that 7 proteins form a ~2.4 MDa complex that spans the cell envelope. The outer membrane complex includes the secretin PulD, with all domains modelled, and the pilotin PulS. The inner membrane assembly platform components PulC, PulE, PulL, PulM and PulN have a relative stoichiometric ratio of 2:1:1:1:1. The PulE ATPase, PulL and PulM combine to form a flexible hexameric hub. Symmetry mismatch between the outer membrane complex and assembly platform is overcome by PulC linkers spanning the periplasm, with PulC HR domains binding independently at the secretin base. Our results show that the T2SS has a highly dynamic modular architecture, with implication for pseudo-pilus assembly and substrate loading

Structural Dynamics of the Vimentin Coiled-Coil Contact Regions involved in Filament Assembly as revealed by Hydrogen-Deuterium Exchange

Intermediate filaments (IF) are major constituents of the cytoskeleton of metazoan cells. They not only are responsible for the mechanical properties but also for various physiological activities in different cells and tissues. The building blocks of IFs are extended coiled-coil−forming proteins exhibiting a characteristic central α-helical domain (″rod″). The fundamental principles of the filament assembly mechanism and the network formation have been widely elucidated for the cytoplasmic IF protein vimentin. Also, a comprehensive structural model for the tetrameric complex of vimentin has been obtained by X-ray crystallography in combination with various biochemical and biophysical techniques. To extend these static data and investigate the dynamic properties of the full-length proteins in solution during the various assembly steps, we analyzed the patterns of hydrogen-deuterium exchange (HDex) in vimentin and in four variants carrying point mutations in the IF consensus motifs present at either end of theα-helical rod that cause an assembly arrest at the unit-length filament (ULF) stage. The results yielded unique insights into the structural properties of subdomains within full-length vimentin, in particular in regions of contact in α-helical and linker segments that stabilize different oligomeric forms such as tetramers, ULFs, and mature filaments. Moreover, HDex analysis of the point-mutated variants directly demonstrated the active role of the IF-consensus motifs in the oligomerization mechanism of tetramers during ULF formation. Ultimately, using molecular dynamics simulation procedures, we provide a structural model for the subdomain-mediated tetramer−tetramer interaction via ″cross-coiling″ as the first step of the assembly process

Electron microscopy study of the central retinal fovea in Pied flycatcher: evidence of a mechanism of light energy transmission through the retina

We present unique ultrastructural data on avian retinal cells. Presently and earlier (Zueva et al., 2016) we explored distribution of intermediate filaments (IFs) in retinal cells of the Pied flycatcher (Ficedula hypoleuca, Passeriformes, Aves) in the central foveolar zone. This retinal zone only contains single and double cone photoreceptors. Previously we found that continuous IFs span Müller cells (MC) lengthwise from the retinal inner limiting membrane (ILM) layer up to the outer limiting membrane (OLM) layer. Here we describe long cylindrical bundles of IFs (IFBs) inside the cone inner segments (CIS) adjoining the cone plasma membrane, with these IFBs following along the cone lengthwise, and surrounding the cone at equal spacing one from the other. Double cones form a combined unit, wherein they are separated by their respective plasma membranes. Double cones thus have a common external ring of IFBs, surrounding both cone components. In the layer of cilia, the IFBs that continue into the cone outer segment (COS) follow on to the cone apical tip along the direction of incident light, with single IFs separating from the IFB, touching, and sometimes passing in-between the light-sensitive lamellae of the COS. These new data support our previous hypothesis on the quantum mechanism of light energy propagation through the vertebrate retina (Zueva et al., 2016, 2019).info:eu-repo/semantics/publishedVersio

Self-consistent field theory for the interactions between keratin intermediate filaments

Background: Keratins are important structural proteins found in skin, hair and nails. Keratin Intermediate Filaments are major components of corneocytes, nonviable horny cells of the Stratum Corneum, the outermost layer of skin. It is considered that interactions between unstructured domains of Keratin Intermediate Filaments are the key factor in maintaining the elasticity of the skin. Results: We have developed a model for the interactions between keratin intermediate filaments based on self-consistent field theory. The intermediate filaments are represented by charged surfaces, and the disordered terminal domains of the keratins are represented by charged heteropolymers grafted to these surfaces. We estimate the system is close to a charge compensation point where the heteropolymer grafting density is matched to the surface charge density. Using a protein model with amino acid resolution for the terminal domains, we find that the terminal chains can mediate a weak attraction between the keratin surfaces. The origin of the attraction is a combination of bridging and electrostatics. The attraction disappears when the system moves away from the charge compensation point, or when excess small ions and/or NMF-representing free amino acids are added. Conclusions: These results are in concordance with experimental observations, and support the idea that the interaction between keratin filaments, and ultimately in part the elastic properties of the keratin-containing tissue, is controlled by a combination of the physico-chemical properties of the disordered terminal domains and the composition of the medium in the inter-filament region. Keywords: Stratum corneum, Skin keratins, Intermediate filaments, Unstructured terminal domains, Bridging attractio

Keratin: Structure, mechanical properties, occurrence in biological organisms, and efforts at bioinspiration

A ubiquitous biological material, keratin represents a group of insoluble, usually high-sulfur content and filament-forming proteins, constituting the bulk of epidermal appendages such as hair, nails, claws, turtle scutes, horns, whale baleen, beaks, and feathers. These keratinous materials are formed by cells filled with keratin and are considered 'dead tissues'. Nevertheless, they are among the toughest biological materials, serving as a wide variety of interesting functions, e.g. scales to armor body, horns to combat aggressors, hagfish slime as defense against predators, nails and claws to increase prehension, hair and fur to protect against the environment. The vivid inspiring examples can offer useful solutions to design new structural and functional materials. Keratins can be classified as α- and β-types. Both show a characteristic filament-matrix structure: 7 nm diameter intermediate filaments for α-keratin, and 3 nm diameter filaments for β-keratin. Both are embedded in an amorphous keratin matrix. The molecular unit of intermediate filaments is a coiled-coil heterodimer and that of β-keratin filament is a pleated sheet. The mechanical response of α-keratin has been extensively studied and shows linear Hookean, yield and post-yield regions, and in some cases, a high reversible elastic deformation. Thus, they can be also be considered 'biopolymers'. On the other hand, β-keratin has not been investigated as comprehensively. Keratinous materials are strain-rate sensitive, and the effect of hydration is significant. Keratinous materials exhibit a complex hierarchical structure: polypeptide chains and filament-matrix structures at the nanoscale, organization of keratinized cells into lamellar, tubular-intertubular, fiber or layered structures at the microscale, and solid, compact sheaths over porous core, sandwich or threads at the macroscale. These produce a wide range of mechanical properties: the Young's modulus ranges from 10 MPa in stratum corneum to about 2.5 GPa in feathers, and the tensile strength varies from 2 MPa in stratum corneum to 530 MPa in dry hagfish slime threads. Therefore, they are able to serve various functions including diffusion barrier, buffering external attack, energy-absorption, impact-resistance, piercing opponents, withstanding repeated stress and aerodynamic forces, and resisting buckling and penetration. A fascinating part of the new frontier of materials study is the development of bioinspired materials and designs. A comprehensive understanding of the biochemistry, structure and mechanical properties of keratins and keratinous materials is of great importance for keratin-based bioinspired materials and designs. Current bioinspired efforts including the manufacturing of quill-inspired aluminum composites, animal horn-inspired SiC composites, and feather-inspired interlayered composites are presented and novel avenues for research are discussed. The first inroads into molecular-based biomimicry are being currently made, and it is hoped that this approach will yield novel biopolymers through recombinant DNA and self-assembly. We also identify areas of research where knowledge development is still needed to elucidate structures and deformation/failure mechanisms

Stabilization of vimentin coil2 fragment via an engineered disulfide

Cytoskeletal intermediate filaments (IFs) assemble from the elementary dimers based on a segmented α-helical coiled-coil (CC) structure. Crystallographic studies of IF protein fragments remain the main route to access their atomic structure. To enable crystallization, such fragments must be sufficiently short. As a consequence, they often fail to assemble into the correct CC dimers. In particular, human vimentin fragment D3 corresponding to the first half of coil2 (residues 261-335) stays monomeric in solution. We have induced its dimerization via introducing a disulfide link between two cysteines engineered in the hydrophobic core of the CC close to its N-terminus. The 2.3Å crystal structure of the D3st (stabilized) fragment reveals a mostly parallel α-helical bundle structure in its N-terminal half which smoothly continues into a left-handed CC towards the C-terminus. This provides a direct evidence for a continuously α-helical structure of the coil2 segment and disproves the previously suggested existence of linker L2 separating it into two left-handed CCs. The general principles of CC dimer stabilization by disulfide introduction are also discussed.status: publishe

Crystallographic Studies of Intermediate Filament Proteins

Intermediate filaments (IFs), together with microtubules and actin microfilaments, are the three main cytoskeletal components in metazoan cells. IFs are formed by a distinct protein family, which is made up of 70 members in humans. Most IF proteins are tissue- or organelle specific, which includes lamins, the IF proteins of the nucleus. The building block of IFs is an elongated dimer, which consists of a central α-helical ‘rod’ domain flanked by flexible N- and C-terminal domains. The conserved rod domain is the ‘signature feature’ of the IF family. Bioinformatics analysis reveals that the rod domain of all IF proteins contains three α-helical segments of largely conserved length, interconnected by linkers. Moreover, there is a conserved pattern of hydrophobic repeats within each segment, which includes heptads and hendecads. This defines the presence of both left-handed and almost parallel coiled-coil regions along the rod length. Using X-ray crystallography on multiple overlapping fragments of IF proteins, the atomic structure of the nearly complete rod domain has been determined. Here, we discuss some specific challenges of this procedure, such as crystallization and diffraction data phasing by molecular replacement. Further insights into the structure of the coiled coil and the terminal domains have been obtained using electron paramagnetic resonance measurements on the full-length protein, with spin labels attached at specific positions. This atomic resolution information, as well as further interesting findings, such as the variation of the coiled-coil stability along the rod length, provide clues towards interpreting the data on IF assembly, collected by a range of methods. However, a full description of this process at the molecular level is not yet at hand.status: publishe

Intermediate filament structure: the bottom-up approach

Intermediate filaments (IFs) result from a key cytoskeletal protein class in metazoan cells, but currently there is no consensus as to their three-dimensional architecture. IF proteins form elongated dimers based on the coiled-coil structure within their central 'rod' domain. Here we focus on the atomic structure of this elementary dimer, elucidated using X-ray crystallography on multiple fragments and electron paramagnetic resonance experiments on spin-labelled vimentin samples. In line with conserved sequence features, the rod of all IF proteins is composed of three coiled-coil segments containing heptad and hendecad repeats and interconnected by linkers. In addition, the next assembly intermediate beyond the dimer, the tetramer, could be modelled. The impact of these structural results towards understanding the assembly mechanism is discussed.publisher: Elsevier articletitle: Intermediate filament structure: the bottom-up approach journaltitle: Current Opinion in Cell Biology articlelink: http://dx.doi.org/10.1016/j.ceb.2014.12.007 content_type: article copyright: Copyright © 2015 Elsevier Ltd. All rights reserved.status: publishe

How to Study Intermediate Filaments in Atomic Detail

Studies of the intermediate filament (IF) structure are a prerequisite of understanding their function. In addition, the structural information is indispensable if one wishes to gain a mechanistic view on the disease-related mutations in the IFs. Over the years, considerable progress has been made on the atomic structure of the elementary building block of all IFs, the coiled-coil dimer. Here, we discuss the approaches, methods and practices that have contributed to this advance. With abundant genetic information on hand, bioinformatics approaches give important insights into the dimer structure, including the head and tail regions poorly assessable experimentally. At the same time, the most important contribution has been provided by X-ray crystallography. Following the “divide-and-conquer” approach, many fragments from several IF proteins could be crystallized and resolved to atomic resolution. We will systematically cover the main procedures of these crystallographic studies, suggest ways to maximize their efficiency, and also discuss the possible pitfalls and limitations. In addition, electron paramagnetic resonance with site-directed spin labeling was another method providing a major impact toward the understanding of the IF structure. Upon placing the spin labels into specific positions within the full-length protein, one can evaluate the proximity of the labels and their mobility. This makes it possible to make conclusions about the dimer structure in the coiled-coil region and beyond, as well as to explore the dimer–dimer contacts.status: publishe