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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., Bacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane. Here, Chernyatina and Low use negative stain and cryo-electron microscopy to reveal the core architecture of an assembled T2SS from the pathogen Klebsiella pneumoniae.

The assembly of intermediate filaments (IFs) including nuclear lamins is driven by specific interactions of the elementary coiled-coil dimers in both lateral and longitudinal direction. The assembly mode A11 is dependent on lateral tetramerization of the second coiled-coil segment (coil1b) in antiparallel fashion. Recent cryo-electron microscopy studies pointed to 3.5 nm lamin filaments built from two antiparallel threads of longitudinally associated dimers but little molecular detail is available to date. Here we present the 2.6 Å resolution X-ray structure of a lamin A fragment including residues 65-222 which reveals the molecular basis of the A11 interaction. The crystal structure also indicates a continuous α-helical structure for the preceding linker L1 region. The middle part of the antiparallel tetramer reveals unique interactions due to the lamin-specific 42-residue insert in coil1b. At the same time, distinct characteristics of this insert provide for the preservation of common structural principles shared with lateral coil1b tetramers of vimentin and keratin K1/K10. In addition, structural analysis suggests that the A11 interaction in lamins is somewhat weaker than in cytoplasmic IFs, despite a 30% longer overlap. Establishing the structural detail of the A11 interaction across IF types is the first step towards a rational understanding of the IF assembly process which is indispensable for establishing the mechanism of disease-related mutations. ispartof: JOURNAL OF STRUCTURAL BIOLOGY vol:209 issue:1 ispartof: location:United States status: published

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.

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 2.3 A 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.

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.