Structural basis for Ca2+-independence and activation by homodimerization of tomato subtilase 3.
Ontology highlight
ABSTRACT:
SUBMITTER: Ottmann C
PROVIDER: S-EPMC2749846 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Pathogenic strains of Escherichia coli produce a number of toxins that belong to the AB5 toxin family, which comprise a catalytic A-subunit that induces cellular dysfunction and a B-pentamer that recognizes host glycans. Although the molecular actions of many of the individual subunits of AB5 toxins are well understood, how they self-associate and the effect of this association on cytotoxicity are poorly understood. Here we have solved the structure of the holo-SubAB toxin that, in contrast to other AB5 toxins whose molecular targets are located in the cytosol, cleaves the endoplasmic reticulum chaperone BiP. SubA interacts with SubB in a similar manner to other AB5 toxins via the A2 helix and a conserved disulfide bond that joins the A1 domain with the A2 helix. The structure revealed that the active site of SubA is not occluded by the B-pentamer, and the B-pentamer does not enhance or inhibit the activity of SubA. Structure-based sequence comparisons with other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, show how the hydrophobic patch on top of the B-pentamer plays a dominant role in binding the A-subunit. The structure of SubAB and the accompanying functional characterization of various mutants of SubAB provide a framework for understanding the important role of the B-pentamer in the assembly and the intracellular trafficking of this AB5 toxin.
| S-EPMC3779744 | biostudies-literature
Project description:Plant metacaspases mediate programmed cell death in development, biotic and abiotic stresses, damage-induced immune response, and resistance to pathogen attack. Most metacaspases require Ca2+ for their activation and substrate processing. However, the Ca2+-dependent activation mechanism remains elusive. Here we report the crystal structures of Metacaspase 4 from Arabidopsis thaliana (AtMC4) that modulates Ca2+-dependent, damage-induced plant immune defense. The AtMC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of AtMC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca2+. Our analysis provides insight into the Ca2+-dependent activation of AtMC4 and lays the basis for tuning its activity in response to stresses for engineering of more sustainable crops for food and biofuels.
| S-EPMC7206013 | biostudies-literature
Project description:Chemically-induced dimerization (CID) systems are essential tools to interrogate and control biological systems. AcVHH is a single domain antibody homo-dimerizing upon caffeine binding. AcVHH has a strong potential for clinical applications through caffeine-mediated in vivo control of therapeutic gene networks. Here we provide the structural basis for caffeine-induced homo-dimerization of acVHH.
| S-EPMC6372657 | biostudies-literature
Project description:The estrogen receptor ? (ER-?) regulates expression of target genes implicated in development, metabolism, and breast cancer. Calcium-dependent regulation of ER-? is critical for activating gene expression and is controlled by calmodulin (CaM). Here, we present the NMR structures for the two lobes of CaM each bound to a localized region of ER-? (residues 287-305). A model of the complete CaM·ER-? complex was constructed by combining these two structures with additional data. The two lobes of CaM both compete for binding at the same site on ER-? (residues 292, 296, 299, 302, and 303), which explains why full-length CaM binds two molecules of ER-? in a 1:2 complex and stabilizes ER-? dimerization. Exposed glutamate residues in CaM (Glu(11), Glu(14), Glu(84), and Glu(87)) form salt bridges with key lysine residues in ER-? (Lys(299), Lys(302), and Lys(303)), which are likely to prevent ubiquitination at these sites and inhibit degradation of ER-?. Mutants of ER-? at the CaM-binding site (W292A and K299A) weaken binding to CaM, and I298E/K299D disrupts estrogen-induced transcription. CaM facilitates dimerization of ER-? in the absence of estrogen, and stimulation of ER-? by either Ca(2+) and/or estrogen may serve to regulate transcription in a combinatorial fashion.
| S-EPMC3308758 | biostudies-literature
Project description:The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca2+-activated scramblases, but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here, we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca2+-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.
| S-EPMC6355197 | biostudies-literature
Project description:The Drosophila neural receptor Dscam1 (Down syndrome cell adhesion molecule 1) plays an essential role in neuronal wiring and self-avoidance. Dscam1 potentially encodes 19,008 ectodomains through alternative RNA splicing and exhibits exquisite isoform-specific homophilic binding, which makes it an exceptional example for studying protein binding specificity. However, structural information on Dscam1 is limited, which hinders illumination of the mechanism of Dscam1 isoform-specific recognition. Whether different Dscam1 isoforms adopt the same dimerization mode remains a subject of debate. We present 12 Dscam1 crystal structures, provide direct evidence indicating that all isoforms adopt a conserved homodimer geometry in a modular fashion, identify two mechanisms for the Ig2 binding domain to dispel electrostatic repulsion during dimerization, decode Ig2 binding specificity by a central motif at its symmetry center, uncover the role of glycosylation in Dscam1 homodimerization, and find electrostatic potential complementarity to help define the binding region and the antiparallel binding mode. We then propose a concept that the context of a protein may set restrictions to regulate its binding specificity, which provides a better understanding of protein recognition.
| S-EPMC4928987 | biostudies-literature
Project description:In arthropods, zinc finger-associated domains (ZADs) are found at the N-termini of many DNA-binding proteins with tandem arrays of Cys2-His2 zinc fingers (ZAD-C2H2 proteins). ZAD-C2H2 proteins undergo fast evolutionary lineage-specific expansion and functional diversification. Here, we show that all ZADs from Drosophila melanogaster form homodimers, but only certain ZADs with high homology can also heterodimerize. CG2712, for example, is unable to heterodimerize with its paralog, the previously characterized insulator protein Zw5, with which it shares 46% homology. We obtained a crystal structure of CG2712 protein's ZAD domain that, in spite of a low sequence homology, has similar spatial organization with the only known ZAD structure (from Grauzone protein). Steric clashes prevented the formation of heterodimers between Grauzone and CG2712 ZADs. Using detailed structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that rapid evolutionary acquisition of interaction specificity was mediated by the more energy-favorable formation of homodimers in comparison to heterodimers, and that this specificity was achieved by multiple amino acid substitutions resulting in the formation or breaking of stabilizing interactions. We speculate that specific homodimerization of ZAD-C2H2 proteins is important for their architectural role in genome organization.
| S-EPMC7913770 | biostudies-literature
Project description:Sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) Ca(2+) transporters pump cytosolic Ca(2+) into the endoplasmic reticulum, maintaining a Ca(2+) gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for Ca(2+) ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca(2+) affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure-function analysis of SERCA2b mutants and SERCA1a2b chimera revealed how the 2b-tail controls Ca(2+) affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the Ca(2+)-bound E1 conformation and alters Ca(2+)-transport kinetics, which provides the rationale for the higher apparent Ca(2+) affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the alpha- and beta-subunits of Na(+),K(+)-ATPase. The 2b-tail interaction site may represent a novel target to increase the Ca(2+) affinity of malfunctioning SERCA2a in the failing heart to improve contractility.
| S-EPMC2774012 | biostudies-literature
Project description:Astrocytic Ca2+ signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the spongiform domain of astrocytes where most tripartite synapses are located. Using 3D-STED microscopy in living organotypic brain slices, we imaged the spongiform domain of astrocytes and observed a reticular meshwork of nodes and shafts that often formed loop-like structures. These anatomical features were also observed in acute hippocampal slices and in barrel cortex in vivo. The majority of dendritic spines were contacted by nodes and their sizes were correlated. FRAP experiments and Ca2+ imaging showed that nodes were biochemical compartments and Ca2+ microdomains. Mapping astrocytic Ca2+ signals onto STED images of nodes and dendritic spines showed they were associated with individual synapses. Here, we report on the nanoscale organization of astrocytes, identifying nodes as a functional astrocytic component of tripartite synapses that may enable synapse-specific communication between neurons and astrocytes.
| S-EPMC7170846 | biostudies-literature
Project description:The bacterium, Francisella tularensis (Ft), is one of the most infectious agents known and classified as a category A bioweapon. Ft virulence is controlled by a unique set of transcription regulators, the MglA-SspA heterodimer, PigR, and the stress signal, ppGpp. These factors activate Francisella pathogenicity island (FPI) gene expression, which is required for virulence. MglA-SspA is expressed during infection and constitutively associates with the σ70 associated RNAP holoenzyme (RNAPσ70), indicating that RNAPσ70-(MglA-SspA) is a virulence specific polymerase. How virulence activation is mediated by these components, however, is unknown. Here we report cryo-EM structures of FtRNAPσ70, FtRNAPσ70-(MglA-SspA) and RNAPσ70-(MglA-SspA)-ppGpp-PigR complexes with promoter DNA. FtRNAPσ70-DNA and FtRNAPσ70-(MglA-SspA)-DNA structures and RT-PCR analyses show MglA-SspA stabilizes σ70 binding to DNA to regulate FPI-independent, virulence-enhancing genes. Strikingly, an Escherichia coli RNAPσ70 complex with EcSspA suggests this is a general mechanism for SspA-like regulation of bacterial RNAPσ70. Finally, our FtRNAP-σ70-(MglA-SspA)-ppGpp-PigR-DNA structure reveals that ppGpp binds to MglA-SspA to tether the DNA-binding activator, PigR, to FPI promoters. PigR in turn recruits FtRNAP CTDs to two DNA upstream (UP) elements, generating stable FPI transcription complexes. Thus, these studies unveil a novel paradigm for pathogenesis in Ft involving a virulence-specific RNAP that employs two (MglA-SspA)-based strategies to activate virulence genes.
2020-11-05 | GSE150932 | GEO