Structural determinants of calmodulin binding to the intracellular C-terminal domain of the metabotropic glutamate receptor 7A.
ABSTRACT Calmodulin (CaM) binds in a Ca2+-dependent manner to the intracellular C-terminal domains of most group III metabotropic glutamate receptors (mGluRs). Here we combined mutational and biophysical approaches to define the structural basis of CaM binding to mGluR 7A. Ca2+/CaM was found to interact with mGluR 7A primarily via its C-lobe at a 1:1 CaM:C-tail stoichiometry. Pulldown experiments with mutant CaM and mGluR 7A C-tail constructs and high resolution NMR with peptides corresponding to the CaM binding region of mGluR 7A allowed us to define hydrophobic and ionic interactions required for Ca2+/CaM binding and identified a 1-8-14 CaM-binding motif. The Ca2+/CaM.mGluR 7A peptide complex displays a classical wraparound structure that closely resembles that formed by Ca2+/CaM upon binding to smooth muscle myosin light chain kinase. Our data provide insight into how Ca2+/CaM regulates group III mGluR signaling via competition with intracellular proteins for receptor-binding sites.
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ABSTRACT: Here we present a novel NMR method for the structure determination of calcium-calmodulin (Ca(2+)-CaM)-peptide complexes from a limited set of experimental restraints. A comparison of solved CaM-peptide structures reveals invariability in CaM's backbone conformation and a structural plasticity in CaM's domain orientation enabled by a flexible linker. Knowing this, the collection and analysis of an extensive set of NOESY spectra is redundant. Although RDCs can define CaM domain orientation in the complex, they lack the translational information required to position the domains on the bound peptide and highlight the necessity of intermolecular NOEs. Here we employ a specific isotope labeling strategy in which the role of methionine in CaM-peptide interactions is exploited to collect these critical NOEs. By (1)H, (13)C-labeling the methyl groups of deuterated methionine against a (2)H, (12)C background, we can acquire a (13)C-edited NOESY characterized by simplified, easily analyzable spectra. Together with measured CaM backbone H(N)-N RDCs and intrapeptide NOE-based distances, these intermolecular NOEs provide restraints for a low temperature torsion-angle dynamics and simulated annealing protocol used to calculate the complex structure. We have applied our method to a CaM complex previously solved through X-ray crystallography: Ca(2+)-CaM bound to the CaM kinase I peptide (PDB code: 1MXE). The resulting structure has a backbone RMSD of 1.6 Å to that previously published. We have also used this test complex to investigate the importance of homologous model selection on the calculated outcome. In addition to having application for fast complex structure determination, this method can be used to determine the structures of difficult complexes characterized by chemical shift overlap and broad signals for which the traditional method based on the use of fully (13)C, (15)N-labeled CaM fails.Journal of Biomolecular NMR 03/2011; 50(1):71-81. · 2.85 Impact Factor
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ABSTRACT: Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.Frontiers in Molecular Neuroscience 01/2012; 5:52.
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ABSTRACT: Background and purpose: The mGlu(7) receptors are strategically located at the site of vesicle fusion where they modulate the release of the main excitatory and inhibitory neurotransmitters. Consequently, they are implicated in the underlying pathophysiology of CNS diseases such as epilepsy and stress-related psychiatric disorders. Here, we characterized a selective, potent and functional anti- mGlu(7) monoclonal antibody, MAB1/28, that triggers receptor internalization. Experimental approach: MAB1/28's activity was investigated using Western Blot and direct immunofluorescence on live cells, in-vitro pharmacology by functional cAMP and GTPγ[(35) S]-binding assays, the kinetics of IgG-induced internalization by image-analysis and the activation of the extracellular-related kinase 1/2 (ERK1/2) by ELISA. Key results: mGlu(7) /mGlu(6) chimeric studies located the MAB1/28 binding site at the extracellular amino-terminus of mGlu(7) . MAB1/28 potently antagonizes both orthosteric and allosteric agonist-induced inhibition of cAMP accumulation (pIC(50) of 8.62). The potency of the antagonistic actions is similar to the potency in triggering receptor internalizations (pEC(50) of 8.95). The internalization mechanism occurs via a pertussis toxin-insensitive pathway and does not require Gα(i) protein activation. MAB1/28 activates ERK1/2 with an activity pEC(50) of 8.12, which is similar in potency to that of EC(50) receptor internalization. The requirement of a bivalent receptor binding mode for receptor internalizations suggests that MAB1/28 modulates mGlu(7) dimers. Conclusions and implications: We show evidence for an allosteric biased agonist activity triggered by MAB1/28, which activates a novel IgG-mediated GPCR internalization pathway that is not utilized by small molecule, orthosteric or allosteric agonists. Thus, MAB1/28 provides an invaluable biological tool for probing mGlu(7) function and selective activation of its intracellular trafficking. © 2012 F. Hoffmann-La Roche AG, Basel, Switzerland. British Journal of Pharmacology © 2012 The British Pharmacological Society.British Journal of Pharmacology 07/2012; · 5.07 Impact Factor