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1.
J Mol Biol ; 419(1-2): 4-21, 2012 May 25.
Article in English | MEDLINE | ID: mdl-22366302

ABSTRACT

The formation of the CBM (CARD11-BCL10-MALT1) complex is pivotal for antigen-receptor-mediated activation of the transcription factor NF-κB. Signaling is dependent on MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), which not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. It remained unclear how the CBM activates MALT1. Here, we provide biochemical and structural evidence that MALT1 activation is dependent on its dimerization and show that mutations at the dimer interface abrogate activity in cells. The unliganded protease presents itself in a dimeric yet inactive state and undergoes substantial conformational changes upon substrate binding. These structural changes also affect the conformation of the C-terminal Ig-like domain, a domain that is required for MALT1 activity. Binding to the active site is coupled to a relative movement of caspase and Ig-like domains. MALT1 binding partners thus may have the potential of tuning MALT1 protease activity without binding directly to the caspase domain.


Subject(s)
Caspases/chemistry , Caspases/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , B-Cell CLL-Lymphoma 10 Protein , Catalytic Domain , Cells, Cultured , Dimerization , Enzyme Activation , HEK293 Cells , Humans , Ligands , Mice , Models, Molecular , Molecular Sequence Data , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Mutation , NF-kappa B/genetics , NF-kappa B/metabolism , Protein Binding , Protein Interaction Domains and Motifs/genetics , Protein Structure, Tertiary , Receptors, Antigen/chemistry , Receptors, Antigen/genetics , Receptors, Antigen/metabolism , Signal Transduction , Structure-Activity Relationship
2.
Biochem J ; 423(3): 429-39, 2009 Oct 12.
Article in English | MEDLINE | ID: mdl-19694615

ABSTRACT

Neurodegenerative diseases pose one of the most pressing unmet medical needs today. It has long been recognized that caspase-6 may play a role in several neurodegenerative diseases for which there are currently no disease-modifying therapies. Thus it is a potential target for neurodegenerative drug development. In the present study we report on the biochemistry and structure of caspase-6. As an effector caspase, caspase-6 is a constitutive dimer independent of the maturation state of the enzyme. The ligand-free structure shows caspase-6 in a partially mature but latent conformation. The cleaved inter-domain linker remains partially inserted in the central groove of the dimer, as observed in other caspases. However, in contrast with the structures of other caspases, not only is the catalytic machinery misaligned, but several structural elements required for substrate recognition are missing. Most importantly, residues forming a short anti-parallel beta-sheet abutting the substrate in other caspase structures are part of an elongation of the central alpha-helix. Despite the dramatic structural changes that are required to adopt a canonical catalytically competent conformation, the pre-steady-state kinetics exhibit no lag phase in substrate turnover. This suggests that the observed conformation does not play a regulatory role in caspase-6 activity. However, targeting the latent conformation in search for specific and bio-available caspase-6 inhibitors might offer an alternative to active-site-directed approaches.


Subject(s)
Axons/enzymology , Caspase 6/chemistry , Neurodegenerative Diseases/enzymology , Protein Multimerization , Humans , Protein Structure, Quaternary , Protein Structure, Secondary , Structure-Activity Relationship
3.
Pest Manag Sci ; 60(10): 945-58, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15481821

ABSTRACT

Neonicotinoids bind selectively to insect nicotinic acetylcholine receptors with nanomolar affinity to act as potent insecticides. While the members of the neonicotinoid class have many structural features in common, it is not known whether they also share the same mode of binding to the target receptor. Previous competition studies with [3H]imidacloprid, the first commercialised neonicotinoid, indicated that thiamethoxam, representing a novel structural sub-class, may bind in a different way from that of other neonicotinoids. In the present work we analysed the mode of [3H]imidacloprid displacement by established neonicotinoids and newly synthesized analogues in the aphids Myzus persicae Sulzer and Aphis craccivora Koch. We found two classes of neonicotinoids with distinct modes of interference with [3H]imidacloprid, described as direct competitive inhibition and non-competitive inhibition, respectively. Competitive neonicotinoids were acetamiprid, nitenpyram, thiacloprid, clothianidin and nithiazine, whereas thiamethoxam and the N-methyl analogues of imidacloprid and clothianidin showed non-competitive inhibition. The chloropyridine or chlorothiazole heterocycles, the polar pharmacophore parts, such as nitroimino, cyanoimino and nitromethylene, and the cyclic or acyclic structure of the pharmacophore were not relevant for the mode of inhibition. Consensus structural features of the neonicotinoids were defined for the two mechanisms of interaction with [3H]imidacloprid binding. Furthermore, two sub-classes of non-competitive inhibitors can be discriminated on the basis of their Hill coefficients for imidacloprid displacement. We conclude from the present data that the direct competitors share the binding site with imidacloprid, whereas non-competitive compounds, like thiamethoxam, bind to a different site or in a different mode.


Subject(s)
Anabasine/chemistry , Aphids , Insecticides/chemistry , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/metabolism , Anabasine/metabolism , Anabasine/pharmacology , Animals , Binding Sites , Cell Membrane/metabolism , Imidazoles/chemistry , Imidazoles/pharmacology , Insecticides/metabolism , Insecticides/pharmacology , Neonicotinoids , Nitro Compounds , Receptors, Nicotinic/drug effects , Species Specificity , Structure-Activity Relationship , Tritium
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