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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

Together with actin filaments and microtubules, intermediate filaments (IFs) are the basic cytoskeletal components of metazoan cells. Over 80 human diseases have been linked to mutations in various IF proteins to date. However, the filament structure is far from being resolved at the atomic level, which hampers rational understanding of IF pathologies. The elementary building block of all IF proteins is a dimer consisting of an α-helical coiled-coil (CC) “rod” domain flanked by the flexible head and tail domains. Here we present three crystal structures of overlapping human vimentin fragments that comprise the first half of its rod domain. Given the previously solved fragments, a nearly complete atomic structure of the vimentin rod has become available. It consists of three α-helical segments (coils 1A, 1B, and 2) interconnected by linkers (L1 and L12). Most of the CC structure has a left-handed twist with heptad repeats, but both coil 1B and coil 2 also exhibit untwisted, parallel stretches with hendecad repeats. In the crystal structure, linker L1 was found to be α-helical without being involved in the CC formation. The available data allow us to construct an atomic model of the antiparallel tetramer representing the second level of vimentin assembly. Although the presence of the nonhelical head domains is essential for proper tetramer stabilization, the precise alignment of the dimers forming the tetramer appears to depend on the complementarity of their surface charge distribution patterns, while the structural plasticity of linker L1 and coil 1A plays a role in the subsequent IF assembly process.

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.