scholarly journals Structure of the ALS Mutation Target Annexin A11 Reveals a Stabilising N-Terminal Segment

2020 ◽  
Author(s):  
Peder August Gudmundsen Lillebostad ◽  
Arne Raasakka ◽  
Silje Johannessen Hjellbrekke ◽  
Sudarshan Patil ◽  
Trude Røstbø ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Main Chain ◽  
Structural Data ◽  
Terminal Segment ◽  
Hydrogen Bonding Interactions ◽  
C Terminus ◽  
Folded Core ◽  
Annexin A11 ◽  
Neuronal Transport ◽  
Lateral Sclerosis

AbstractThe functions of the annexin family of proteins involve binding to Ca2+, lipid membranes, other proteins, and RNA, and the annexins share a common folded core structure at the C terminus. Annexin A11 (AnxA11) has a long N-terminal region, which is predicted to be disordered, binds RNA, and forms membraneless organelles involved in neuronal transport. Mutations in AnxA11 have been linked to amyotrophic lateral sclerosis (ALS). We studied the structure and stability of AnxA11 and identified a short stabilising segment in the N-terminal end of the folded core, which links domains I and IV. Crystal structure of the AnxA11 core highlights main-chain hydrogen bonding interactions formed through this bridging segment, which are likely conserved in most annexins. The structure was also used to study the currently known ALS mutations in AnxA11. Three of these mutations correspond to buried Arg residues highly conserved in the annexin family, indicating central roles in annexin folding. The structural data provide starting points for detailed structure-function studies of both full-length AnxA11 and the disease variants being identified in ALS.

scholarly journals Structure of the ALS Mutation Target Annexin A11 Reveals a Stabilising N-Terminal Segment

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 660
Author(s):  
Peder A. G. Lillebostad ◽  
Arne Raasakka ◽  
Silje J. Hjellbrekke ◽  
Sudarshan Patil ◽  
Trude Røstbø ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Main Chain ◽  
Structural Data ◽  
Terminal Segment ◽  
Hydrogen Bonding Interactions ◽  
C Terminus ◽  
Folded Core ◽  
Annexin A11 ◽  
Neuronal Transport ◽  
Lateral Sclerosis

The functions of the annexin family of proteins involve binding to Ca2+, lipid membranes, other proteins, and RNA, and the annexins share a common folded core structure at the C terminus. Annexin A11 (AnxA11) has a long N-terminal region, which is predicted to be disordered, binds RNA, and forms membraneless organelles involved in neuronal transport. Mutations in AnxA11 have been linked to amyotrophic lateral sclerosis (ALS). We studied the structure and stability of AnxA11 and identified a short stabilising segment in the N-terminal end of the folded core, which links domains I and IV. The crystal structure of the AnxA11 core highlights main-chain hydrogen bonding interactions formed through this bridging segment, which are likely conserved in most annexins. The structure was also used to study the currently known ALS mutations in AnxA11. Three of these mutations correspond to buried Arg residues highly conserved in the annexin family, indicating central roles in annexin folding. The structural data provide starting points for detailed structure–function studies of both full-length AnxA11 and the disease variants being identified in ALS.

Limitations of Allotopic Expression of Mitochondrial Genes in Mammalian Cells

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 707-720
Author(s):  
Jose Oca-Cossio ◽  
Lesley Kenyon ◽  
Huiling Hao ◽  
Carlos T Moraes
Keyword(s):  
Mitochondrial Dna ◽  
Mammalian Cells ◽  
Mitochondrial Genes ◽  
Terminal Segment ◽  
Universal Genetic Code ◽  
Apocytochrome B ◽  
Dna Mutations ◽  
C Terminus ◽  
Cytoplasmic Compartment ◽  
Allotopic Expression

Abstract The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria.

Crystal structure of hexanoyl-CoA bound to β-ketoacyl reductase FabG4 of Mycobacterium tuberculosis

2013 ◽  
Vol 450 (1) ◽  
pp. 127-139 ◽  
Author(s):  
Debajyoti Dutta ◽  
Sudipta Bhattacharyya ◽  
Amlan Roychowdhury ◽  
Rupam Biswas ◽  
Amit Kumar Das
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Ternary Complex ◽  
Catalytic Mechanism ◽  
Acyl Carrier Protein ◽  
Structural Data ◽  
Acid Synthesis ◽  
Binary Complex ◽  
C Terminus ◽  
Covalently Linked

FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β-oxoacyl-ACP (acyl-carrier protein) into β-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal ‘flavodoxintype’ domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-oxoacyl-CoA to β-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4–NADH binary complex and the FabG4–NAD+–hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg146 and Arg445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

scholarly journals NRas slows the rate at which a model lipid bilayer phase separates

2014 ◽  
Vol 169 ◽  
pp. 209-223 ◽  
Author(s):  
Elizabeth Jefferys ◽  
Mark S. P. Sansom ◽  
Philip W. Fowler
Keyword(s):  
Lipid Bilayer ◽  
Cell Signalling ◽  
Line Tension ◽  
Structural Data ◽  
Coarse Grained ◽  
Ras Proteins ◽  
C Terminus ◽  
Ras Family ◽  
Multiple Copies ◽  
Dynamics Simulations

The Ras family of small membrane-associated GTP-ases are important components in many different cell signalling cascades. They are thought to cluster on the cell membrane through association with cholesterol-rich nanodomains. This process remains poorly understood. Here we test the effect of adding multiple copies of NRas, one of the canonical Ras proteins, to a three-component lipid bilayer that rapidly undergoes spinodal decomposition (i.e.unmixing), thereby creating ordered and disordered phases. Coarse-grained molecular dynamics simulations of a large bilayer containing 6000 lipids, with and without protein, are compared. NRas preferentially localises to the interface between the domains and slows the rate at which the domains grow. We infer that this doubly-lipidated cell signalling protein is reducing the line tension between the ordered and disordered regions. This analysis is facilitated by our use of techniques borrowed from image-processing. The conclusions above are contingent upon several assumptions, including the use of a model lipid with doubly unsaturated tails and the limited structural data available for the C-terminus of NRas, which is where the lipid anchors are found.

scholarly journals Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis

2017 ◽  
Vol 9 (388) ◽  
pp. eaad9157 ◽  
Author(s):  
Bradley N. Smith ◽  
Simon D. Topp ◽  
Claudia Fallini ◽  
Hideki Shibata ◽  
Han-Jou Chen ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Vesicular Trafficking ◽  
Annexin A11 ◽  
Lateral Sclerosis

Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase

2011 ◽  
Vol 435 (2) ◽  
pp. 441-450 ◽  
Author(s):  
Evgeni Yu. Zernii ◽  
Konstantin E. Komolov ◽  
Sergei E. Permyakov ◽  
Tatiana Kolpakova ◽  
Daniele Dell'orco ◽  
...  
Keyword(s):  
Structural Control ◽  
Target Recognition ◽  
Computational Modelling ◽  
Structural Similarity ◽  
Terminal Segment ◽  
Site Directed Mutagenesis ◽  
Resonance Spectroscopy ◽  
Dependent Manner ◽  
C Terminus ◽  
Rhodopsin Kinase

NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin–kinase complex provided insight into the protein–protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation–π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.

scholarly journals The contribution of the C-terminal sequence to the catalytic activity of GST2, a human Alpha-class glutathione transferase

1991 ◽  
Vol 275 (1) ◽  
pp. 171-174 ◽  
Author(s):  
P G Board ◽  
B Mannervik
Keyword(s):  
Catalytic Activity ◽  
Glutathione Transferase ◽  
Specific Activity ◽  
Affinity Purification ◽  
Cumene Hydroperoxide ◽  
Plasmid Vector ◽  
Competitive Inhibitor ◽  
Terminal Segment ◽  
Truncated Form ◽  
C Terminus

A plasmid vector was constructed that encodes the expression in Escherichia coli of a truncated form of GST2, a human Alpha-class glutathione transferase. The truncated enzyme, GST2del210, has 12 residues deleted from the C-terminus and has the last two residues of the new C-terminal mutated from aspartic acid and glutamic acid to histidine and glycine respectively. GST2del210 has substantially diminished specific activity with either 1-chloro-2,4-dinitrobenzene or cumene hydroperoxide as substrate. The affinity of the truncated enzyme for a GSH-agarose matrix was also diminished, but sufficient interaction remained to enable affinity purification. Inhibition of GST2del210 by bromosulphophthalein was not altered. In contrast, this truncated form was not inhibited by S-pentylglutathione, a competitive inhibitor of the wild-type GST2 isoenzyme. The results show that the C-terminal segment of the Alpha-class glutathione transferases may form a component of the hydrophobic substrate-binding site. In contrast, this region appears not to be directly involved in GSH binding and is not absolutely essential for catalytic activity.

scholarly journals Redetermination of ammonium metavanadate

IUCrData ◽  
2018 ◽  
Vol 3 (8) ◽  
Author(s):  
Aarón Pérez-Benítez ◽  
Sylvain Bernès
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonding ◽  
Solid State ◽  
Single Crystal ◽  
Structural Data ◽  
Ammonium Cation ◽  
Ammonium Metavanadate ◽  
Starting Material ◽  
X Ray ◽  
Hydrogen Bonding Interactions

The crystal structure of ammonium metavanadate, NH4VO3, a compound widely used as a starting material for the synthesis of vanadium and polyoxidovanadate compounds, had been determined twice using single-crystal X-ray data [Syneček & Hanic (1954). Czech. J. Phys. 4, 120–129 (Weissenberg data); Hawthorne & Calvo (1977). J. Solid State Chem. 22, 157–170 (four-circle diffractometer data)]. Its structure is now redetermined at higher resolution using Ag Kα radiation, and the result is compared with the former refinements. Structural data for the polymeric [VO3]∞ chain remain unchanged, while more accurate parameters are obtained for the ammonium cation, improving the description of hydrogen-bonding interactions in the crystal structure.

scholarly journals 3D architecture and structural flexibility revealed in the subfamily of large glutamate dehydrogenases by a mycobacterial enzyme

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Melisa Lázaro ◽  
Roberto Melero ◽  
Charlotte Huet ◽  
Jorge P. López-Alonso ◽  
Sandra Delgado ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Structural Data ◽  
Terminal Segment ◽  
Low Molecular Weight ◽  
Structural Flexibility ◽  
Catalytic Core ◽  
Bacterial Physiology ◽  
Functional Studies ◽  
3D Architecture ◽  
Function Relationship

AbstractGlutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs180) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH180 (mL-GDH180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs.

scholarly journals Novel HIV-1 Protease Inhibitors (PIs) Containing a Bicyclic P2 Functional Moiety, Tetrahydropyrano-Tetrahydrofuran, That Are Potent against Multi-PI-Resistant HIV-1 Variants

2011 ◽  
Vol 55 (4) ◽  
pp. 1717-1727 ◽  
Author(s):  
Kazuhiko Ide ◽  
Manabu Aoki ◽  
Masayuki Amano ◽  
Yasuhiro Koh ◽  
Ravikiran S. Yedidi ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Protease Inhibitors ◽  
Main Chain ◽  
Hydrophobic Interactions ◽  
Drug Resistant ◽  
Hydrogen Bonding Interactions ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACTWe identified GRL-1388 and -1398, potent nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing a bicyclic P2 functional moiety, tetrahydropyrano-tetrahydrofuran (Tp-THF). GRL-1388 was as potent as darunavir (DRV) against various drug-resistant HIV-1 laboratory strains with 50% effective concentration (EC50s) of 2.6 to 32.6 nM. GRL-1398 was significantly more potent against such variants than DRV with EC50s of 0.1 to 5.7 nM. GRL-1388 and -1398 were also potent against multiple-PI-resistant clinical HIV-1 variants (CLHIV-1MDR) with EC50s ranging from 2.7 to 21.3 nM and from 0.3 to 4.8 nM, respectively. A highly DRV-resistant HIV-1 variant selectedin vitroremained susceptible to GRL-1398 with the EC50of 21.9 nM, while the EC50of DRV was 214.1 nM. When HIV-1NL4-3was selected with GRL-1398, four amino acid substitutions—leucine to phenylalanine at a position 10 (L10F), A28S, L33F, and M46I—emerged, ultimately enabling the virus to replicate in the presence of >1.0 μM the compound beyond 57 weeks of selection. When a mixture of 10 differentCLHIV-1MDRstrains was selected, the emergence of resistant variants was more substantially delayed with GRL-1398 than with GRL-1388 and DRV. Modeling analyses revealed that GRL-1398 had greater overall hydrogen bonding and hydrophobic interactions than GRL-1388 and DRV and that GRL-1388 and -1398 had hydrogen bonding interactions with the main chain of the active-site amino acids (Asp29 and Asp30) of protease. The present findings warrant that GRL-1398 be further developed as a potential drug for treating individuals with HIV-1 infection.

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