The coronavirus (CoV) spike (S) protein, involved in viral-host cell fusion, is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The ...highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, we implemented a structure-based vector analysis of available β-CoV S-protein structures. Despite an overall similarity in domain organization, we found that S-proteins from different β-CoVs display distinct configurations. Based on this analysis, we developed two soluble ectodomain constructs for the SARS-CoV-2 S-protein, in which the highly immunogenic and mobile receptor binding domain (RBD) is either locked in the all-RBDs 'down' position or adopts 'up' state conformations more readily than the wild-type S-protein. These results demonstrate that the conformation of the S-protein can be controlled via rational design and can provide a framework for the development of engineered CoV S-proteins for vaccine applications.
Transient receptor potential melastatin member 8 (TRPM8) is a calcium ion (Ca
)-permeable cation channel that serves as the primary cold and menthol sensor in humans. Activation of TRPM8 by cooling ...compounds relies on allosteric actions of agonist and membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP
), but lack of structural information has thus far precluded a mechanistic understanding of ligand and lipid sensing by TRPM8. Using cryo-electron microscopy, we determined the structures of TRPM8 in complex with the synthetic cooling compound icilin, PIP
, and Ca
, as well as in complex with the menthol analog WS-12 and PIP
Our structures reveal the binding sites for cooling agonists and PIP
in TRPM8. Notably, PIP
binds to TRPM8 in two different modes, which illustrate the mechanism of allosteric coupling between PIP
and agonists. This study provides a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents.
NPR1 is a master regulator of the defence transcriptome induced by the plant immune signal salicylic acid
. Despite the important role of NPR1 in plant immunity
, understanding of its regulatory ...mechanisms has been hindered by a lack of structural information. Here we report cryo-electron microscopy and crystal structures of Arabidopsis NPR1 and its complex with the transcription factor TGA3. Cryo-electron microscopy analysis reveals that NPR1 is a bird-shaped homodimer comprising a central Broad-complex, Tramtrack and Bric-à-brac (BTB) domain, a BTB and carboxyterminal Kelch helix bundle, four ankyrin repeats and a disordered salicylic-acid-binding domain. Crystal structure analysis reveals a unique zinc-finger motif in BTB for interacting with ankyrin repeats and mediating NPR1 oligomerization. We found that, after stimulation, salicylic-acid-induced folding and docking of the salicylic-acid-binding domain onto ankyrin repeats is required for the transcriptional cofactor activity of NPR1, providing a structural explanation for a direct role of salicylic acid in regulating NPR1-dependent gene expression. Moreover, our structure of the TGA3
-NPR1
-TGA3
complex, DNA-binding assay and genetic data show that dimeric NPR1 activates transcription by bridging two fatty-acid-bound TGA3 dimers to form an enhanceosome. The stepwise assembly of the NPR1-TGA complex suggests possible hetero-oligomeric complex formation with other transcription factors, revealing how NPR1 reprograms the defence transcriptome.
HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of ...fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we describe the contrasting structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic. Binding of trimeric HIV-1 BaL Env to either soluble CD4 or 17b alone, is sufficient to trigger formation of the open quaternary conformation of Env. In contrast, VRC01 locks Env in the closed state, while b12 binding requires a partial opening in the quaternary structure of trimeric Env. Our results show that, despite general similarities in regions of the HIV-1 gp120 polypeptide that contact CD4, VRC01, VRC03 and b12, there are important differences in quaternary structures of the complexes these ligands form on native trimeric Env, and potentially explain differences in the neutralizing breadth and potency of antibodies with similar specificities. From cryo-electron microscopic analysis at ∼9 Å resolution of a cleaved, soluble version of trimeric Env, we show that a structural signature of the open Env conformation is a three-helix motif composed of α-helical segments derived from highly conserved, non-glycosylated N-terminal regions of the gp41 trimer. The three N-terminal gp41 helices in this novel, activated Env conformation are held apart by their interactions with the rest of Env, and are less compactly packed than in the post-fusion, six-helix bundle state. These findings suggest a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage.
CorA, the major Mg2+ uptake system in prokaryotes, is gated by intracellular Mg2+ (KD ∼1–2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg2+-bound and Mg2+-free ...conditions, but EPR spectroscopic studies reveal large Mg2+-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg2+-bound closed conformation and in two open Mg2+-free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg2+, four of the five subunits are displaced to variable extents (∼10–25 Å) by hinge-like motions as large as ∼35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg2+, open, asymmetric conformational states is, thus, the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.
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•Find 3.8 Å resolution cryo-EM structure of the ∼200 kDa magnesium channel CorA•Mg2+-free CorA exhibits dramatic loss of symmetry in the cytoplasmic domain•Identify two discrete, asymmetric conformations of Mg2+-free CorA•Inter-subunit Mg2+ is important for stabilizing the closed state of CorA
Cryo-EM structure of the magnesium channel CorA in its closed and open states reveals an unexpected loss of symmetry within the cytoplasmic domain during the transition between these conformations, providing insights into the gating mechanism of this family of ion channels.
Abstract
Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae ...revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3′ of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here, we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3′ of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA, and will aid in the development of new anti-viral therapeutics.
Transient receptor potential vanilloid member 1 (TRPV1) is a Ca
-permeable cation channel that serves as the primary heat and capsaicin sensor in humans. Using cryo-EM, we have determined the ...structures of apo and capsaicin-bound full-length rat TRPV1 reconstituted into lipid nanodiscs over a range of temperatures. This has allowed us to visualize the noxious heat-induced opening of TRPV1 in the presence of capsaicin. Notably, noxious heat-dependent TRPV1 opening comprises stepwise conformational transitions. Global conformational changes across multiple subdomains of TRPV1 are followed by the rearrangement of the outer pore, leading to gate opening. Solvent-accessible surface area analyses and functional studies suggest that a subset of residues form an interaction network that is directly involved in heat sensing. Our study provides a glimpse of the molecular principles underlying noxious physical and chemical stimuli sensing by TRPV1, which can be extended to other thermal sensing ion channels.
The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection. ...Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with three-dimensional image classification and averaging, we report the three-dimensional structures of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells.
Histone H3 lysine 79 (H3K79) methylation is enriched on actively transcribed genes, and its misregulation is a hallmark of leukemia. Methylation of H3K79, which resides on the structured disk face of ...the nucleosome, is mediated by the Dot1L methyltransferase. Dot1L activity is part of a trans-histone crosstalk pathway, requiring prior histone H2B ubiquitylation of lysine 120 (H2BK120ub) for optimal activity. However, the molecular details describing both how Dot1L binds to the nucleosome and why Dot1L is activated by H2BK120 ubiquitylation are unknown. Here, we present the cryoelectron microscopy (cryo-EM) structure of Dot1L bound to a nucleosome reconstituted with site-specifically ubiquitylated H2BK120. The structure reveals that Dot1L engages the nucleosome acidic patch using a variant arginine anchor and occupies a conformation poised for methylation. In this conformation, Dot1L and ubiquitin interact directly through complementary hydrophobic surfaces. This study establishes a path to better understand Dot1L function in normal and leukemia cells.
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•Cryo-EM structure of Dot1L bound in a poised state to a ubiquitylated nucleosome•Dot1L engages the nucleosome acidic patch using a variant arginine anchor•Dot1L-ubiquitin hydrophobic interactions enhance histone H3K79 methylation
Dot1L is a histone H3K79-specific methyltransferase that is critical to the pathogenesis of leukemia. Here, Anderson et al. report the cryo-EM structure of Dot1L in complex with a ubiquitylated nucleosome, providing molecular details of how Dot1L binds its nucleosome substrate and is activated by ubiquitin.
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