DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas ...and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.
The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth in response to the nutrient and energy status of the cell, and its deregulation is common in human cancers. Little is known ...about the overall architecture and subunit organization of this essential signaling complex. We have determined the three-dimensional (3D) structure of the fully assembled human mTORC1 by cryo-electron microscopy (cryo-EM). Our analyses reveal that mTORC1 is an obligate dimer with an overall rhomboid shape and a central cavity. The dimeric interfaces are formed by interlocking interactions between the mTOR and raptor subunits. Extended incubation with FKBP12-rapamycin compromises the structural integrity of mTORC1 in a stepwise manner, leading us to propose a model in which rapamycin inhibits mTORC1-mediated phosphorylation of 4E-BP1 and S6K1 through different mechanisms.
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► The cryo-EM structure of human mTORC1 was determined at a resolution of 26 Å ► Interlocking mTOR-raptor interactions maintain the dimeric architecture of mTORC1 ► FKBP12-rapamycin profoundly disrupts the structural integrity of mTORC1
Conserved from yeast to humans, Elongator is a protein complex implicated in multiple processes including transcription regulation, α-tubulin acetylation, and tRNA modification, and its defects have ...been shown to cause human diseases such as familial dysautonomia. Elongator consists of two copies of six core subunits (Elp1, Elp2, Elp3, Elp4, Elp5, and Elp6) that are organized into two subcomplexes: Elp1/2/3 and Elp4/5/6 and form a stable assembly of ~ 850 kDa in size. Although the catalytic subunit of Elongator is Elp3, which contains a radical
S
-adenosyl-
l
-methionine (SAM) domain and a putative histone acetyltransferase domain, the Elp4/5/6 subcomplex also possesses ATP-modulated tRNA binding activity. How at the molecular level, Elongator performs its multiple functions and how the different subunits regulate Elongator’s activities remains poorly understood. Here, we provide an overview of the proposed functions of Elongator and describe how recent structural studies provide new insights into the mechanism of action of this multifunctional complex.
Self-templated protein aggregation and intracerebral deposition of aggregates, sometimes in the form of amyloid fibrils, is a hallmark of mammalian prion diseases. What distinguishes amyloid fibrils ...formed by prions from those formed by other proteins is not clear. On the basis of previous studies on yeast prions that correlated high intrinsic fragmentation rates of fibrils with prion propagation efficiency, it has been hypothesized that the nanomechanical properties of prion amyloid such as strength and elastic modulus may be the distinguishing feature. Here, we reveal that fibrils formed by mammalian prions are relatively soft and clearly in a different class of rigidities when compared to nanofibrils formed by nonprions. We found that amyloid fibrils made of both wild-type and mutant mouse recombinant PrP(23-231) have remarkably low axial elastic moduli of 0.1–1.4 GPa. We demonstrate that even the proteinase K resistant core of these fibrils has similarly low intrinsic rigidities. Using a new mode of atomic force microscopy called AM-FM mode, we estimated the radial modulus of PrP fibrils at ∼0.6 GPa, consistent with the axial moduli derived by using an ensemble method. Our results have far-reaching implications for the understanding of protein-based infectivity and the design of amyloid biomaterials.
Spt-Ada-Gcn5-Acetyltransferase (SAGA) is a conserved multi-subunit complex that activates RNA polymerase II-mediated transcription by acetylating and deubiquitinating nucleosomal histones and by ...recruiting TATA box binding protein (TBP) to DNA. The prototypical yeast Saccharomyces cerevisiae SAGA contains 19 subunits that are organized into Tra1, core, histone acetyltransferase, and deubiquitination modules. Recent cryo-electron microscopy studies have generated high-resolution structural information on the Tra1 and core modules of yeast SAGA. However, the two catalytical modules were poorly resolved due to conformational flexibility of the full assembly. Furthermore, the high sample requirement created a formidable barrier to further structural investigations of SAGA. Here, we report a workflow for isolating/stabilizing yeast SAGA and preparing cryo-EM specimens at low protein concentration using a graphene oxide support layer. With this procedure, we were able to determine a cryo-EM reconstruction of yeast SAGA at 3.1 Å resolution and examine its conformational landscape with the neural network-based algorithm cryoDRGN. Our analysis revealed that SAGA adopts a range of conformations with its HAT module and central core in different orientations relative to Tra1.
There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) ...pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.
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•HDX-MS identifies epitopes of single-chain nanobodies toward class IB PI3K complexes•A nanobody targeting p101 facilitates high-resolution cryo-EM of the p110γ/p101 complex•Nanobodies can block p110γ/p101 and p110γ/p84 activation by Ras and Gβγ
Rathinaswamy and Fleming et al. describe a mass spectrometry-based approach for characterizing nanobodies targeting the lipid kinase phosphoinositide-3-kinase (PIK3CG, PI3Kγ) that modulated enzyme regulation and enabled high-resolution cryoelectron microscopy.
Mycobacterium tuberculosis (Mtb) uses the ESX-1 type VII secretion system to export virulence proteins across its lipid-rich cell wall, which helps permeabilize the host’s macrophage phagosomal ...membrane, facilitating the escape and cell-to-cell spread of Mtb. ESX-1 membranolytic activity depends on a set of specialized secreted Esp proteins, the structure and specific roles of which are not currently understood. Here, we report the X-ray and electron microscopic structures of the ESX-1-secreted EspB. We demonstrate that EspB adopts a PE/PPE-like fold that mediates oligomerization with apparent heptameric symmetry, generating a barrel-shaped structure with a central pore that we propose contributes to the macrophage killing functions of EspB. Our structural data also reveal unexpected direct interactions between the EspB bipartite secretion signal sequence elements that form a unified aromatic surface. These findings provide insight into how specialized proteins encoded within the ESX-1 locus are targeted for secretion, and for the first time indicate an oligomerization-dependent role for Esp virulence factors.
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•The crystal structure of EspB reveals a fused PE/PPE homology domain•EspB has a stabilized bipartite secretion signal that targets the EccCb1 ATPase•EspB oligomerizes to form a ring-shaped heptamer•A model of the heptamer was fit to EM density and crosslinking data
Mycobacterium tuberculosis exports virulence factors to its surface using the ESX-1 secretion system, progressing the infection of human macrophages. Solomonson et al. show that one of these factors, EspB, adopts a PE/PPE-like fold and oligomerizes to form a barrel-shaped structure with heptameric symmetry.
Macroautophagy (also known as autophagy) is a major pathway for selective degradation of misfolded/aggregated proteins and damaged organelles and non-selective degradation of cytoplasmic constituents ...for the generation of power during nutrient deprivation. The multi-step degradation process, from sequestering cytoplasmic cargo into the double-membrane vesicle termed autophagosome to the delivery of the autophagosome to the lysosome or lytic vacuole for breakdown, is mediated by the core autophagy machinery composed of multiple Atg proteins, as well as the divergent sequence family of selective autophagy receptors. Single-particle electron microscopy (EM) is a molecular imaging approach that has become an increasingly important tool in the structural characterization of proteins and macromolecular complexes. This article summarizes the contributions single-particle EM have made in advancing our understanding of the core autophagy machinery and selective autophagy receptors. We also discuss current technical challenges and roadblocks, as well as look into the future of single-particle EM in autophagy research.
Vesicle trafficking requires membrane fusion, mediated by SNARE proteins, and upstream events that probably include “tethering,” an initial long-range attachment between a vesicle and its target ...organelle. Among the factors proposed to mediate tethering are a set of multisubunit tethering complexes (MTCs). The Dsl1 complex, with only three subunits, is the simplest known MTC and is essential for the retrograde traffic of COPI-coated vesicles from the Golgi to the ER. To elucidate structural principles underlying MTC function, we have determined the structure of the Dsl1 complex, revealing a tower containing at its base the binding sites for two ER SNAREs and at its tip a flexible lasso for capturing vesicles. The Dsl1 complex binds to individual SNAREs via their N-terminal regulatory domains and also to assembled SNARE complexes; moreover, it is capable of accelerating SNARE complex assembly. Our results suggest that even the simplest MTC may be capable of orchestrating vesicle capture, uncoating, and fusion.
Several members of the
(
) produce a conserved horizontal gene transfer vector, called the gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study ...GTA biology is the
GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the
and is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production in
We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in
and identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the
Members of the
are abundant in ocean and freshwater environments. The conserved GTA produced by many
may have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA in
extends the model of CtrA regulation from
to a member of the
We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.
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