Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. Here, we report successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion.

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The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

Microbiology ◽

10.1099/mic.0.26997-0 ◽

2004 ◽

Vol 150 (7) ◽

pp. 2055-2068 ◽

Cited By ~ 10

Author(s):

Daniel V. Zurawski ◽

Murry A. Stein

Keyword(s):

Type Iii Secretion ◽

Coiled Coil ◽

Amphipathic Helix ◽

Yeast Two Hybrid ◽

C Terminus ◽

N Terminus ◽

Domain Mapping ◽

Self Association ◽

Discrete Domains ◽

Two Hybrid

SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization.

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Hydrogen-bond networks between the C-terminus and Arg from the first α-helix stabilize photoprotein molecules

Photochemical & Photobiological Sciences ◽

10.1039/c3pp50369k ◽

2014 ◽

Vol 13 (3) ◽

pp. 541 ◽

Cited By ~ 9

Author(s):

Elena V. Eremeeva ◽

Ludmila P. Burakova ◽

Vasilisa V. Krasitskaya ◽

Alexander N. Kudryavtsev ◽

Osamu Shimomura ◽

...

Keyword(s):

Hydrogen Bond ◽

C Terminus ◽

Hydrogen Bond Networks ◽

Α Helix

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The Unique Structure of Haemophilus influenzae Protein E Reveals Multiple Binding Sites for Host Factors

Infection and Immunity ◽

10.1128/iai.01111-12 ◽

2012 ◽

Vol 81 (3) ◽

pp. 801-814 ◽

Cited By ~ 35

Author(s):

Birendra Singh ◽

Tamim Al-Jubair ◽

Matthias Mörgelin ◽

Marjolein M. Thunnissen ◽

Kristian Riesbeck

Keyword(s):

Haemophilus Influenzae ◽

Disulfide Bridge ◽

Lung Epithelial Cells ◽

Binding Region ◽

Content Type ◽

Bacterial Surface ◽

Hydrogen Bonding Pattern ◽

Functional Regions ◽

C Terminus ◽

Α Helix

ABSTRACTHaemophilus influenzaeprotein E (PE) is a multifunctional adhesin involved in direct interactions with lung epithelial cells and host proteins, including plasminogen and the extracellular matrix proteins vitronectin and laminin. We recently crystallized PE and successfully collected X-ray diffraction data at 1.8 Å. Here, we solved the structure of a recombinant version of PE and analyzed different functional regions. It is a dimer in solution and in the asymmetric unit of the crystals. The dimer has a structure that resembles a flattened β-barrel. It is, however, not a true β-barrel, as there are differences in both the hydrogen-bonding pattern and the shape. Each monomer consisted of a 6-stranded antiparallel β-sheet with a rigid α-helix at the C terminus tethered to the concave side of the sheet by a disulfide bridge. The laminin/plasminogen binding region (residues 41 to 68) is exposed, while the vitronectin binding region (residues 84 to 108) is partially accessible in the dimer. The dimerized PE explains the simultaneous interaction with laminin and vitronectin. In addition, we found this unique adhesin to be present in many bacterial genera of the familyPasteurellaceaeand also orthologues in other, unrelated species (Enterobacter cloacaeandListeria monocytogenes). Peptides corresponding to the surface-exposed regions PE 24 to 37, PE 74 to 89, and PE 134 to 156 were immunogenic in the mouse. Importantly, these peptide-based antibodies also recognized PE at the bacterial surface. Taken together, our detailed structure of PE explains how this important virulence factor ofH. influenzaesimultaneously interacts with host vitronectin, laminin, or plasminogen, promoting bacterial pathogenesis.

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An excitatory scorpion toxin with a distinctive feature: an additional α helix at the C terminus and its implications for interaction with insect sodium channels

Structure ◽

10.1016/s0969-2126(98)00111-7 ◽

1998 ◽

Vol 6 (9) ◽

pp. 1095-1103 ◽

Cited By ~ 53

Author(s):

Deena A Oren ◽

Oren Froy ◽

Efrat Amit ◽

Nurit Kleinberger-Doron ◽

Michael Gurevitz ◽

...

Keyword(s):

Sodium Channels ◽

Distinctive Feature ◽

Scorpion Toxin ◽

C Terminus ◽

Α Helix

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NCK-associated protein 1 like (nckap1l) minor splice variant regulates intrahepatic biliary network morphogenesis

PLoS Genetics ◽

10.1371/journal.pgen.1009402 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009402

Author(s):

Kimia Ghaffari ◽

Lain X. Pierce ◽

Maria Roufaeil ◽

Isabel Gibson ◽

Kevin Tae ◽

...

Keyword(s):

Epithelial Cells ◽

Network Formation ◽

Stop Codon ◽

Cell Lineage ◽

Adaptor Protein ◽

Specific Protein ◽

Chemical Mutagenesis ◽

Biliary Epithelial Cells ◽

Regulatory Complex ◽

Splice Isoform

Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in thenckap1lgene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediatednckap1ldeletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver ofnckap1lmutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show thatnckap1lgenetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that althoughnckap1lwas previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.

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An amphipathic helix enables septins to sense micrometer-scale membrane curvature

The Journal of Cell Biology ◽

10.1083/jcb.201807211 ◽

2019 ◽

Vol 218 (4) ◽

pp. 1128-1137 ◽

Cited By ~ 26

Author(s):

Kevin S. Cannon ◽

Benjamin L. Woods ◽

John M. Crutchley ◽

Amy S. Gladfelter

Keyword(s):

Membrane Curvature ◽

Amphipathic Helix ◽

Curvature Sensing ◽

C Terminus ◽

Number Of Binding Sites ◽

Curved Membranes ◽

Necessary And Sufficient ◽

Micrometer Scale

Cell shape is well described by membrane curvature. Septins are filament-forming, GTP-binding proteins that assemble on positive, micrometer-scale curvatures. Here, we examine the molecular basis of curvature sensing by septins. We show that differences in affinity and the number of binding sites drive curvature-specific adsorption of septins. Moreover, we find septin assembly onto curved membranes is cooperative and show that geometry influences higher-order arrangement of septin filaments. Although septins must form polymers to stay associated with membranes, septin filaments do not have to span micrometers in length to sense curvature, as we find that single-septin complexes have curvature-dependent association rates. We trace this ability to an amphipathic helix (AH) located on the C-terminus of Cdc12. The AH domain is necessary and sufficient for curvature sensing both in vitro and in vivo. These data show that curvature sensing by septins operates at much smaller length scales than the micrometer curvatures being detected.

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The Histone Variant MacroH2A1 Regulates Key Genes for Myogenic Cell Fusion in a Splice-Isoform Dependent Manner

Cells ◽

10.3390/cells9051109 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1109

Author(s):

Sarah Hurtado-Bagès ◽

Melanija Posavec Marjanovic ◽

Vanesa Valero ◽

Roberto Malinverni ◽

David Corujo ◽

...

Keyword(s):

Gene Regulation ◽

Ex Vivo ◽

Histone Variants ◽

Histone Variant ◽

C2c12 Cells ◽

Dependent Manner ◽

Chronic Injury ◽

Splice Isoforms ◽

Splice Isoform

MacroH2A histone variants have functions in differentiation, somatic cell reprogramming and cancer. However, at present, it is not clear how macroH2As affect gene regulation to exert these functions. We have parted from the initial observation that loss of total macroH2A1 led to a change in the morphology of murine myotubes differentiated ex vivo. The fusion of myoblasts to myotubes is a key process in embryonic myogenesis and highly relevant for muscle regeneration after acute or chronic injury. We have focused on this physiological process, to investigate the functions of the two splice isoforms of macroH2A1. Individual perturbation of the two isoforms in myotubes forming in vitro from myogenic C2C12 cells showed an opposing phenotype, with macroH2A1.1 enhancing, and macroH2A1.2 reducing, fusion. Differential regulation of a subset of fusion-related genes encoding components of the extracellular matrix and cell surface receptors for adhesion correlated with these phenotypes. We describe, for the first time, splice isoform-specific phenotypes for the histone variant macroH2A1 in a physiologic process and provide evidence for a novel underlying molecular mechanism of gene regulation.

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Solution Structure of the Carbon Storage Regulator Protein CsrA from Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.10.3496-3501.2005 ◽

2005 ◽

Vol 187 (10) ◽

pp. 3496-3501 ◽

Cited By ~ 62

Author(s):

Pablo Gutiérrez ◽

Yan Li ◽

Michael J. Osborne ◽

Ekaterina Pomerantseva ◽

Qian Liu ◽

...

Keyword(s):

Carbon Storage ◽

Solution Structure ◽

Rna Binding ◽

Binding Motif ◽

Binding Properties ◽

Nmr Titration ◽

C Terminus ◽

Regulator Protein ◽

Carbon Storage Regulator ◽

Α Helix

ABSTRACT The carbon storage regulator A (CsrA) is a protein responsible for the repression of a variety of stationary-phase genes in bacteria. In this work, we describe the nuclear magnetic resonance (NMR)-based structure of the CsrA dimer and its RNA-binding properties. CsrA is a dimer of two identical subunits, each composed of five strands, a small α-helix and a flexible C terminus. NMR titration experiments suggest that the β1-β2 and β3-β4 loops and the C-terminal helix are important elements in RNA binding. Even though the β3-β4 loop contains a highly conserved RNA-binding motif, GxxG, typical of KH domains, our structure excludes CsrA from being a member of this protein family, as previously suggested. A mechanism for the recognition of mRNAs downregulated by CsrA is proposed.

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