Bacterial type IV secretion systems (T4SSs) are a versatile group of nanomachines that can horizontally transfer DNA through conjugation and deliver effector proteins into a wide range of target ...cells. The components of T4SSs in gram-negative bacteria are organized into several large subassemblies: an inner membrane complex, an outer membrane core complex, and, in some species, an extracellular pilus. Cryo-electron tomography has been used to define the structures of T4SSs in intact bacteria, and high-resolution structural models are now available for isolated core complexes from conjugation systems, the Xanthomonas citri T4SS, the Helicobacter pylori Cag T4SS, and the Legionella pneumophila Dot/Icm T4SS. In this review, we compare the molecular architectures of these T4SSs, focusing especially on the structures of core complexes. We discuss structural features that are shared by multiple T4SSs as well as evolutionary strategies used for T4SS diversification. Finally, we discuss how structural variations among T4SSs may confer specialized functional properties.
The VacA toxin secreted by Helicobacter pylori enhances the ability of the bacteria to colonize the stomach and contributes to the pathogenesis of gastric adenocarcinoma and peptic ulcer disease. The ...amino acid sequence and structure of VacA are unrelated to corresponding features of other known bacterial toxins. VacA is classified as a pore-forming toxin, and many of its effects on host cells are attributed to formation of channels in intracellular sites. The most extensively studied VacA activity is its capacity to stimulate vacuole formation, but the toxin has many additional effects on host cells. Multiple cell types are susceptible to VacA, including gastric epithelial cells, parietal cells, T cells, and other types of immune cells. This review focuses on the wide range of VacA actions that are detectable in vitro, as well as actions of VacA in vivo that are relevant for H. pylori colonization of the stomach and development of gastric disease.
Colonization of the human stomach with Helicobacter pylori strains containing the cag pathogenicity island is a risk factor for development of gastric cancer. The cag pathogenicity island contains ...genes encoding a secreted effector protein (CagA) and components of a type IV secretion system (Cag T4SS). The molecular architecture of the H. pylori Cag T4SS is substantially more complex than that of prototype T4SSs in other bacterial species. In this review, we discuss recent discoveries pertaining to the structure and function of the Cag T4SS and its role in gastric cancer pathogenesis.
The Cag T4SS has a key role in the pathogenesis of H. pylori-associated gastric cancer.Recent advances in molecular imaging have facilitated investigation of the H. pylori Cag T4SS structure.The H. pylori Cag T4SS contains multiple components unrelated to components of T4SSs in other bacterial species, and the molecular architecture of the H. pylori Cag T4SS is substantially more complex than that of prototype T4SSs.T4SSs are a heterogeneous group of secretion systems with diverse functions. Despite a low level of sequence relatedness among corresponding components of T4SSs from different bacterial species, there are shared structural features among all T4SSs analyzed thus far.
The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric ...chromatin. However, little is known about how the CENP-A nucleosome affects the architecture of centromeric chromatin. In this study, we reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their 3D structures by cryoelectron microscopy. The H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture, with an outward-facing linker DNA path between nucleosomes. This is distinct from the H3-H3-H3 tri-nucleosome architecture, with an inward-facing DNA path. Intriguingly, the untwisted architecture may allow the CENP-A nucleosome to be exposed to the solvent in the condensed chromatin model. These results provide a structural basis for understanding the 3D configuration of CENP-A-containing chromatin, and may explain how centromeric proteins can specifically target the CENP-A nucleosomes buried in robust amounts of H3 nucleosomes in centromeres.
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•Cryo-EM structures of tri-nucleosomes with/without CENP-A have been determined•The CENP-A nucleosome adopts an untwisted architecture in the tri-nucleosome•Linker DNA length affects DNA path between central and peripheral nucleosomes
The centromere-specific histone H3 variant, CENP-A, is a crucial epigenetic marker for designating centromeres as sites for kinetochore assembly. Takizawa et al. reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their structures by single-particle cryo-EM.
Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two architectures: bundles and asters. Although both architectures are critical for cellular function, the molecular ...pathways that drive aster formation are poorly understood. Here, we study aster formation by human minus-end-directed kinesin-14 (HSET/KIFC1). We show that HSET is incapable of forming asters from preformed, nongrowing MTs, but rapidly forms MT asters in the presence of soluble (non-MT) tubulin. HSET binds soluble (non-MT) tubulin via its N-terminal tail domain to form heterogeneous HSET-tubulin clusters containing multiple motors. Cluster formation induces motor processivity and rescues the formation of asters from nongrowing MTs. We then show that excess soluble (non-MT) tubulin stimulates aster formation in HeLa cells overexpressing HSET during mitosis. We propose a model where HSET can toggle between MT bundle and aster formation in a manner governed by the availability of soluble (non-MT) tubulin.
The conserved multifunctional protein Gle1 regulates gene expression at multiple steps: nuclear mRNA export, translation initiation, and translation termination. A GLE1 mutation (FinMajor) is ...causally linked to human lethal congenital contracture syndrome-1 (LCCS1); however, the resulting perturbations on Gle1 molecular function were unknown. FinMajor results in a proline-phenylalanine-glutamine peptide insertion within the uncharacterized Gle1 coiled-coil domain. Here, we find that Gle1 self-associates both in vitro and in living cells via the coiled-coil domain. Electron microscopy reveals that high-molecular-mass Gle1 oligomers form ∼26 nm diameter disk-shaped particles. With the Gle1-FinMajor protein, these particles are malformed. Moreover, functional assays document a specific requirement for proper Gle1 oligomerization during mRNA export, but not for Gle1’s roles in translation. These results identify a mechanistic step in Gle1’s mRNA export function at nuclear pore complexes and directly implicate altered export in LCCS1 disease pathology.
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•Gle1 self-associates via its conserved, essential coiled-coil domain•Oligomerization of Gle1 is required for mRNA export and not translation•Gle1 oligomers form disk structures that are perturbed in the LCCS1 disease variant•mRNA export dysregulation at nuclear pore complexes is linked to LCCS1 pathology
The essential mRNA export factor Gle1 self-assembles to form large disk-like structures acting as an unexpected regulatory step at nuclear pore complexes. These structures are altered with the human lethal congenital contracture syndrome-1 disease protein.
Caveolins are an unusual family of membrane proteins whose primary biological function is to build small invaginated membrane structures at the surface of cells known as caveolae. Caveolins and ...caveolae regulate numerous signaling pathways, lipid homeostasis, intracellular transport, cell adhesion, and cell migration. They also serve as sensors and protect the plasma membrane from mechanical stress. Despite their many important functions, the molecular basis for how these 50–100 nm “little caves” are assembled and regulate cell physiology has perplexed researchers for 70 years. One major impediment to progress has been the lack of information about the structure of caveolin complexes that serve as building blocks for the assembly of caveolae. Excitingly, recent advances have finally begun to shed light on this long-standing question. In this review, we highlight new developments in our understanding of the structure of caveolin oligomers, including the landmark discovery of the molecular architecture of caveolin-1 complexes using cryo-electron microscopy.
Graphical Abstract
A unique aspect of arrestin-3 is its ability to support both receptor-dependent and receptor-independent signaling. Here, we show that inositol hexakisphosphate (IP
) is a non-receptor activator of ...arrestin-3 and report the structure of IP
-activated arrestin-3 at 2.4-Å resolution. IP
-activated arrestin-3 exhibits an inter-domain twist and a displaced C-tail, hallmarks of active arrestin. IP
binds to the arrestin phosphate sensor, and is stabilized by trimerization. Analysis of the trimerization surface, which is also the receptor-binding surface, suggests a feature called the finger loop as a key region of the activation sensor. We show that finger loop helicity and flexibility may underlie coupling to hundreds of diverse receptors and also promote arrestin-3 activation by IP
. Importantly, we show that effector-binding sites on arrestins have distinct conformations in the basal and activated states, acting as switch regions. These switch regions may work with the inter-domain twist to initiate and direct arrestin-mediated signaling.
Cryo-electron microscopy (cryo-EM), the structural analysis of samples embedded in vitreous ice, is a powerful approach for determining three-dimensional (3D) structures of biological specimens. Over ...the past two decades, this technique has been used to successfully calculate subnanometer (<10 Å) resolution and, in some cases, near-atomic resolution structures of highly symmetrical and stable complexes such as icosahedral viruses and ribosomes, as well as samples that form ordered two-dimensional or helical arrays. However, determining high-resolution 3D structures of smaller, less symmetrical, and dynamic samples remains a significant challenge. The recent development of electron microscopes with automated data collection capabilities and robust direct electron detection cameras, as well as new powerful image processing algorithms, has dramatically expanded the number of biological macromolecules amenable for study using cryo-EM. In addition, these new technological and computational developments have been used to successfully determine <5 Å resolution 3D structures of samples, such as membrane proteins and complexes with either low or no symmetry, that traditionally were not considered promising candidates for high-resolution cryo-EM. With these exciting new advances, cryo-EM is now on pace to determine atomic resolution 3D structures.
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