Structural biology is the study of the molecular architecture of proteins and nucleic acids, which are the basis for all life forms. Structural biology came into its own as a field during the 1950s ...when the atomic structures of DNA (1) and several globular proteins (2) were solved. Knowledge of these structures alone is not enough to understand their functions, but it has become clear that a detailed mechanistic picture of function is not possible without structural information. Studying structure can reveal how molecules have evolved, and this type of insight would otherwise be lost by looking at only the molecule's sequence.
The CARMA1/Bcl10/MALT1 (CBM) signalosome mediates antigen receptor-induced NF-κB signaling to regulate multiple lymphocyte functions. While CARMA1 and Bcl10 contain caspase recruitment domains ...(CARDs), MALT1 is a paracaspase with structural similarity to caspases. Here we show that the reconstituted CBM signalosome is a helical filamentous assembly in which substoichiometric CARMA1 nucleates Bcl10 filaments. Bcl10 filament formation is a highly cooperative process whose threshold is sensitized by oligomerized CARMA1 upon receptor activation. In cells, both cotransfected CARMA1/Bcl10 complex and the endogenous CBM signalosome are filamentous morphologically. Combining crystallography, nuclear magnetic resonance, and electron microscopy, we reveal the structure of the Bcl10 CARD filament and the mode of interaction between CARMA1 and Bcl10. Structure-guided mutagenesis confirmed the observed interfaces in Bcl10 filament assembly and MALT1 activation in vitro and NF-κB activation in cells. These data support a paradigm of nucleation-induced signal transduction with threshold response due to cooperativity and signal amplification by polymerization.
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•CARMA1 nucleates Bcl10 filaments in the CBM signalosome•The EM structure reveals mode of interactions in the CARMA1/Bcl10 complex•The interactions are validated by mutagenesis in vitro and in cells•The filamentous CBM complex implicates mechanism of signal amplification
Inflammasomes elicit host defense inside cells by activating caspase-1 for cytokine maturation and cell death. AIM2 and NLRP3 are representative sensor proteins in two major families of ...inflammasomes. The adaptor protein ASC bridges the sensor proteins and caspase-1 to form ternary inflammasome complexes, achieved through pyrin domain (PYD) interactions between sensors and ASC and through caspase activation and recruitment domain (CARD) interactions between ASC and caspase-1. We found that PYD and CARD both form filaments. Activated AIM2 and NLRP3 nucleate PYD filaments of ASC, which, in turn, cluster the CARD of ASC. ASC thus nucleates CARD filaments of caspase-1, leading to proximity-induced activation. Endogenous NLRP3 inflammasome is also filamentous. The cryoelectron microscopy structure of ASCPYD filament at near-atomic resolution provides a template for homo- and hetero-PYD/PYD associations, as confirmed by structure-guided mutagenesis. We propose that ASC-dependent inflammasomes in both families share a unified assembly mechanism that involves two successive steps of nucleation-induced polymerization.
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•AIM2 and NLRP3 inflammasomes are filamentous assemblies in vitro and in cells•3.8 Å cryo-EM structure of ASCPYD depicts the underlying oligomerization mechanism•Fluorescence polarization assays suggest nucleation-induced filament formation•ASC-dependent inflammasomes use a polymerization mechanism for caspase-1 activation
Extremely high-resolution cryoelectron microscopy analysis of inflammasomes establishes that assembly of these complexes, which are integral to the innate immune response to pathogens, requires two successive steps of nucleation-induced polymerization to bring inflammasome sensor proteins together and to activate downstream signaling events.
Helical reconstruction, again Egelman, Edward H.
Current opinion in structural biology,
April 2024, 2024-04-00, 20240401, Letnik:
85
Journal Article
Recenzirano
Many protein and nucleoprotein complexes exist as helical polymers. As a result, much effort has been invested in developing methods for using electron microscopy to determine the structure of these ...assemblies. With the revolution in cryo-electron microscopy (cryo-EM), it has now become routine to reach a near-atomic level of resolution for these structures, and it is the exception when this is not possible. However, the greatest challenge is frequently determining the correct symmetry. This review focuses on why this can be so difficult and the current absence of a better approach than trial-and-error.
Bacteria use rapid contraction of a long sheath of the type VI secretion system (T6SS) to deliver effectors into a target cell. Here, we present an atomic-resolution structure of a native contracted ...Vibrio cholerae sheath determined by cryo-electron microscopy. The sheath subunits, composed of tightly interacting proteins VipA and VipB, assemble into a six-start helix. The helix is stabilized by a core domain assembled from four β strands donated by one VipA and two VipB molecules. The fold of inner and middle layers is conserved between T6SS and phage sheaths. However, the structure of the outer layer is distinct and suggests a mechanism of interaction of the bacterial sheath with an accessory ATPase, ClpV, that facilitates multiple rounds of effector delivery. Our results provide a mechanistic insight into assembly of contractile nanomachines that bacteria and phages use to translocate macromolecules across membranes.
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•Atomic structure of the bacterial T6SS sheath was solved by cryo-EM•β-strand-mediated intermolecular interactions stabilize six-start helical assembly•Structural alignments with phage sheaths indicate conserved mechanism of assembly•Arrangement of the outer domain of the sheath facilitates recycling by ClpV ATPase
The atomic structure of a bacterial type VI secretion system shows similarities to phage homologs in the sheath core architecture while a distinct outer layer facilitates interactions with the ClpV ATPase that enables multiple rounds of sheath use.
RIG-I activates interferon signaling pathways by promoting filament formation of the adaptor molecule, MAVS. Assembly of the MAVS filament is mediated by its CARD domain (CARDMAVS), and requires its ...interaction with the tandem CARDs of RIG-I (2CARDRIG-I). However, the precise nature of the interaction between 2CARDRIG-I and CARDMAVS, and how this interaction leads to CARDMAVS filament assembly, has been unclear. Here we report a 3.6 Å electron microscopy structure of the CARDMAVS filament and a 3.4 Å crystal structure of the 2CARDRIG-I:CARDMAVS complex, representing 2CARDRIG-I “caught in the act” of nucleating the CARDMAVS filament. These structures, together with functional analyses, show that 2CARDRIG-I acts as a template for the CARDMAVS filament assembly, by forming a helical tetrameric structure and recruiting CARDMAVS along its helical trajectory. Our work thus reveals that signal activation by RIG-I occurs by imprinting its helical assembly architecture on MAVS, a previously uncharacterized mechanism of signal transmission.
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•The CARDMAVS filament is a left-handed, single-stranded helix•The CARDMAVS filament shares the same helical symmetry as the 2CARDRIG-I tetramer•The 2CARDRIG-I tetramer serves as a template to nucleate the CARDMAVS filament•CARDMAVS utilizes the same surface to interact with both 2CARDRIG-I and CARDMAVS
A viral RNA sensor, RIG-I, activates interferon signaling pathways by promoting filament formation of the adaptor molecule, MAVS. Wu et al. show that RIG-I acts as a template for the CARDMAVS filament assembly by forming a helical tetrameric structure and recruiting individual CARDMAVS along its extended helical trajectory.
Actin Filaments as Tension Sensors Galkin, Vitold E.; Orlova, Albina; Egelman, Edward H.
Current biology,
02/2012, Letnik:
22, Številka:
3
Journal Article
Recenzirano
Odprti dostop
The field of mechanobiology has witnessed an explosive growth over the past several years as interest has greatly increased in understanding how mechanical forces are transduced by cells and how ...cells migrate, adhere and generate traction. Actin, a highly abundant and anomalously conserved protein, plays a large role in forming the dynamic cytoskeleton that is so essential for cell form, motility and mechanosensitivity. While the actin filament (F-actin) has been viewed as dynamic in terms of polymerization and depolymerization, new results suggest that F-actin itself may function as a highly dynamic tension sensor. This property may help explain the unusual conservation of actin's sequence, as well as shed further light on actin's essential role in structures from sarcomeres to stress fibers.
•Direct electron detectors allow for near-atomic resolution for many polymers.•Three types of bacterial pili discussed: chaperone-usher, mating, and Type IV.•Homology of an archaeal ‘adhesion ...filament’ to a flagellar filament has been shown.•Insights into homology of N-terminal domains in Type IV pili and archaeal flagella.
Recent advances in cryo-electron microscopy (cryo-EM) have opened up the possibility that a large class of biological structures, helical polymers, may now be readily reconstructed at near-atomic resolution. This will have a huge impact, since most of these structures are unlikely to be crystallized. This review focuses on new cryo-EM studies involving three classes of bacterial pili (chaperone-usher, mating, and Type IV) as well as on archaeal flagellar filaments. While it has long been known that one domain within archaeal flagellar filaments is homologous to a domain within bacterial Type IV pilins, the new studies shed light on how homologous and even highly conserved subunits can pack together in different ways with only small changes in sequence.
Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the ...DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.
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•Caspase-8 tDED assembles into filaments through quasi-equivalent contacts•The assembly of caspase-8 filaments is nucleated by the upstream Fas/FADD complex•cFLIP tDED also forms filaments, which interact with caspase-8 by comingling•MC159 inhibits caspase-8 filament assembly by a unique capping mechanism
How caspase-8 is activated has been a long-standing question. Fu et al. show that its tDED forms filaments using quasi-equivalent interactions. Cryo-EM structure of the filament reveals mechanisms of caspase-8 activation and its regulation by cFLIP and MC159.
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