Sphingolipid and cholesterol-rich Triton X-100-insoluble membrane fragments (detergent-resistant membranes, DRMs) containing lipids in a state similar to the liquid-ordered phase can be isolated from mammalian cells, and probably exist as discrete domains or rafts in intact membranes. We postulated that proteins with a high affinity for such an ordered lipid environment might be targeted to rafts. Saturated acyl chains should prefer an extended conformation that would fit well in rafts. In contrast, prenyl groups, which are as hydrophobic as acyl chains but have a branched and bulky structure, should be excluded from rafts. Here, we showed that at least half of the proteins in Increasing evidence suggests that cholesterol and sphingolipid-rich lipid microdomains or rafts exist in eukaryotic cell membranes and have important functions there (1-3). These rafts are likely to be important in the structure and function of caveolae, plasma membrane invaginations that are implicated in signal transduction (4, 5), endocytosis (6), transcytosis across endothelial cells (7,8), and cholesterol trafficking (9 -11). However, rafts are not restricted to caveolae (2, 3, 12) and recent evidence suggests that they act in signal transduction in cells that lack distinct caveolae, such as T lymphocytes (13-16) and basophils (17)(18)(19). Rafts have also been implicated in protein and lipid sorting in the secretory and endocytic pathways (1, 20 -22).Cholesterol and sphingolipid-rich detergent-resistant membranes (DRMs) 1 can be isolated from mammalian cells (23). DRM lipids are in a state similar to the liquid-ordered (l o ) phase (3, 24 -26). The l o phase, which requires cholesterol to form, is favored by lipids like sphingolipids, whose long saturated acyl chains give them a high degree of order and a high acyl-chain melting temperature (3). Acyl chain order explains the detergent-insolubility of DRMs (3). We hypothesize that DRMs are an in vitro correlate of rafts in intact membranes. It is important to note that detergent insolubility can underestimate the association of proteins and lipids with the l o phase; some proteins and lipids that are in rafts can be solubilized (25). Nevertheless, DRM association provides a powerful tool for identifying molecules that are likely to have a high affinity for rafts. DRMs isolated from cells contain a number of proteins (27-29) which are undoubtedly crucial for the function of the domains in vivo. For this reason, it is important to determine how proteins associate with DRMs. Three DRM targeting signals have been defined. First, glycosylphosphatidylinositol (GPI)-anchored proteins are targeted to DRMs through acyl chain interactions (23)(24)(25)30). An N-terminal Met-Gly-Cys motif that is present in some Src family kinases and heterotrimeric G protein ␣ subunits, in which Gly is myristoylated and Cys is palmitoylated, can also serve as a DRM targeting signal (31,32). Third, dual palmitoylated Cys residues are required for raft association of the T cell adaptor protein LAT (15) and the neu...
The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3 – 0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.
Non-enveloped viruses of different types have evolved distinct mechanisms for penetrating a cellular membrane during infection. Rotavirus penetration appears to occur by a process resembling enveloped-virus fusion: membrane distortion linked to conformational changes in a viral protein. Evidence for such a mechanism comes from crystallographic analyses of fragments of VP4, the rotavirus-penetration protein, and infectivity analyses of structure-based VP4 mutants. We describe here the structure of an infectious rotavirus particle determined by electron cryomicroscopy (cryoEM) and single-particle analysis at about 4.3 Å resolution. The cryoEM image reconstruction permits a nearly complete trace of the VP4 polypeptide chain, including the positions of most side chains. It shows how the two subfragments of VP4 (VP8* and VP5*) retain their association after proteolytic cleavage, reveals multiple structural roles for the b-barrel domain of VP5*, and specifies interactions of VP4 with other capsid proteins. The virion model allows us to integrate structural and functional information into a coherent mechanism for rotavirus entry.
The treatment of helical objects as a string of single particles has become an established technique to resolve their three-dimensional (3D) structure using electron cryo-microscopy. It can be applied to a wide range of helical particles such as viruses, microtubules and helical filaments. We have made improvements to this approach using Tobacco Mosaic Virus (TMV) as a test specimen and obtained a map from 210,000 asymmetric units at a resolution better than 5 A. This was made possible by performing a full correction of the contrast transfer function of the microscope. Alignment of helical segments was helped by constraints derived from the helical symmetry of the virus. Furthermore, symmetrization was implemented by multiple inclusions of symmetry-related views in the 3D reconstruction. We used the density map to build an atomic model of TMV. The model was refined using a real-space refinement strategy that accommodates multiple conformers. The atomic model shows significant deviations from the deposited model for the helical form of TMV at the lower-radius region (residues 88 to 109). This region appears more ordered with well-defined secondary structure, compared with the earlier helical structure. The RNA phosphate backbone is sandwiched between two arginine side-chains, stabilizing the interaction between RNA and coat protein. A cluster of two or three carboxylates is buried in a hydrophobic environment isolating it from neighboring subunits. These carboxylates may represent the so-called Caspar carboxylates that form a metastable switch for viral disassembly. Overall, the observed differences suggest that the new model represents a different, more stable state of the virus, compared with the earlier published model.
The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FlgE, and its function as a nano-sized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FlgE31, a major proteolytic fragment of FlgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.
Orderly termination of sister-chromatid cohesion during mitosis is critical for accurate chromosome segregation. During prophase, mitotic kinases phosphorylate cohesin and its protector sororin, triggering Wapl-dependent cohesin release from chromosome arms. The shugoshin (Sgo1)–PP2A complex protects centromeric cohesin until its cleavage by separase at anaphase onset. Here, we report the crystal structure of a human cohesin subcomplex comprising SA2 and Scc1. Multiple HEAT repeats of SA2 form a dragon-shaped structure. Scc1 makes extensive contacts with SA2, with one binding hotspot. Sgo1 and Wapl compete for binding to a conserved site on SA2–Scc1. Mutations of SA2 residues at this site that disrupt Wapl binding bypass Sgo1 requirement in cohesion protection. Thus, besides recruiting PP2A to dephosphorylate cohesin and sororin, Sgo1 physically shields cohesin from Wapl. This unexpected, direct antagonism between Sgo1 and Wapl augments centromeric cohesion protection.
Significance SML-8-73-1 (SML) is the first example, to our knowledge, of a GTP-competitive inhibitor of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras). A high-resolution structure of K-Ras G12C bound to SML shows K-Ras in an inactive conformation. In situ proteomic-based chemical profiling of SML demonstrates that SML is highly selective for K-Ras G12C over other small GTPases. A novel chemosensor-based assay allows measurement of covalent reaction rates between K-Ras G12C and SML and enables characterization of this reaction in the context of millimolar concentrations of GTP and GDP, well in exccss of what is found in living cells. These results demonstrate that even in the presence of high concentrations of GTP and GDP, SML is able to exchange into the GN site.
The ubiquitous C2 domain is a conserved Ca2+ triggered membrane-docking module that targets numerous signaling proteins to membrane surfaces where they regulate diverse processes critical for cell signaling. In this study, we quantitatively compared the equilibrium and kinetic parameters of C2 domains isolated from three functionally distinct signaling proteins: cytosolic phospholipase A2-alpha (cPLA2-alpha), protein kinase C-beta (PKC-beta), and synaptotagmin-IA (Syt-IA). The results show that equilibrium C2 domain docking to mixed phosphatidylcholine and phosphatidylserine membranes occurs at micromolar Ca2+ concentrations for the cPLA2-alpha C2 domain, but requires 3- and 10-fold higher Ca2+ concentrations for the PKC-beta and Syt-IA C2 domains ([Ca2+](1/2) = 4.7, 16, 48 microM, respectively). The Ca2+ triggered membrane docking reaction proceeds in at least two steps: rapid Ca2+ binding followed by slow membrane association. The greater Ca2+ sensitivity of the cPLA2-alpha domain results from its higher intrinsic Ca2+ affinity in the first step compared to the other domains. Assembly and disassembly of the ternary complex in response to rapid Ca2+ addition and removal, respectively, require greater than 400 ms for the cPLA2-alpha domain, compared to 13 ms for the PKC-beta domain and only 6 ms for the Syt-IA domain. Docking of the cPLA2-alpha domain to zwitterionic lipids is triggered by the binding of two Ca2+ ions and is stabilized via hydrophobic interactions, whereas docking of either the PKC-beta or the Syt-IA domain to anionic lipids is triggered by at least three Ca2+ ions and is maintained by electrostatic interactions. Thus, despite their sequence and architectural similarity, C2 domains are functionally specialized modules exhibiting equilibrium and kinetic parameters optimized for distinct Ca2+ signaling applications. This specialization is provided by the carefully tuned structural and electrostatic parameters of their Ca2+ and membrane-binding loops, which yield distinct patterns of Ca2+ coordination and contrasting mechanisms of membrane docking.
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