Dataset Information

G Protein binding sites on Calnuc (nucleobindin 1) and NUCB2 (nucleobindin 2) define a new class of G(alpha)i-regulatory motifs.

ABSTRACT: Heterotrimeric G proteins are molecular switches modulated by families of structurally and functionally related regulators. GIV (G?-interacting vesicle-associated protein) is the first non-receptor guanine nucleotide exchange factor (GEF) that activates G?(i) subunits via a defined, evolutionarily conserved motif. Here we found that Calnuc and NUCB2, two highly homologous calcium-binding proteins, share a common motif with GIV for G?(i) binding and activation. Bioinformatics searches and structural analysis revealed that Calnuc and NUCB2 possess an evolutionarily conserved motif with sequence and structural similarity to the GEF sequence of GIV. Using in vitro pulldown and competition assays, we demonstrate that this motif binds preferentially to the inactive conformation of G?(i1) and G?(i3) over other G? subunits and, like GIV, docks onto the ?3/switch II cleft. Calnuc binding was impaired when Lys-248 in the ?3 helix of G?(i3) was replaced with M, the corresponding residue in G?(o), which does not bind to Calnuc. Moreover, mutation of hydrophobic residues in the conserved motif predicted to dock on the ?3/switch II cleft of G?(i3) impaired the ability of Calnuc and NUCB2 to bind and activate G?(i3) in vitro. We also provide evidence that calcium binding to Calnuc and NUCB2 abolishes their interaction with G?(i3) in vitro and in cells, probably by inducing a conformational change that renders the G?(i)-binding residues inaccessible. Taken together, our results identify a new type of G?(i)-regulatory motif named the GBA motif (for G?-binding and -activating motif), which is conserved across different proteins throughout evolution. These findings provide the structural basis for the properties of Calnuc and NUCB2 binding to G? subunits and its regulation by calcium ions.

SUBMITTER: Garcia-Marcos M

PROVIDER: S-EPMC3151059 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

G Protein binding sites on Calnuc (nucleobindin 1) and NUCB2 (nucleobindin 2) define a new class of G(alpha)i-regulatory motifs.

Garcia-Marcos Mikel M Kietrsunthorn Patrick S PS Wang Honghui H Ghosh Pradipta P Farquhar Marilyn G MG

The Journal of biological chemistry 20110608 32

Heterotrimeric G proteins are molecular switches modulated by families of structurally and functionally related regulators. GIV (Gα-interacting vesicle-associated protein) is the first non-receptor guanine nucleotide exchange factor (GEF) that activates Gα(i) subunits via a defined, evolutionarily conserved motif. Here we found that Calnuc and NUCB2, two highly homologous calcium-binding proteins, share a common motif with GIV for Gα(i) binding and activation. Bioinformatics searches and structu ...[more]

PMID: 21653697

Similar Datasets

Project description:BackgroundClassically, models of DNA-transcription factor binding sites (TFBSs) have been based on relatively few known instances and have treated them as sites of fixed length using position weight matrices (PWMs). Various extensions to this model have been proposed, most of which take account of dependencies between the bases in the binding sites. However, some transcription factors are known to exhibit some flexibility and bind to DNA in more than one possible physical configuration. In some cases this variation is known to affect the function of binding sites. With the increasing volume of ChIP-seq data available it is now possible to investigate models that incorporate this flexibility. Previous work on variable length models has been constrained by: a focus on specific zinc finger proteins in yeast using restrictive models; a reliance on hand-crafted models for just one transcription factor at a time; and a lack of evaluation on realistically sized data sets.ResultsWe re-analysed binding sites from the TRANSFAC database and found motivating examples where our new variable length model provides a better fit. We analysed several ChIP-seq data sets with a novel motif search algorithm and compared the results to one of the best standard PWM finders and a recently developed alternative method for finding motifs of variable structure. All the methods performed comparably in held-out cross validation tests. Known motifs of variable structure were recovered for p53, Stat5a and Stat5b. In addition our method recovered a novel generalised version of an existing PWM for Sp1 that allows for variable length binding. This motif improved classification performance.ConclusionsWe have presented a new gapped PWM model for variable length DNA binding sites that is not too restrictive nor over-parameterised. Our comparison with existing tools shows that on average it does not have better predictive accuracy than existing methods. However, it does provide more interpretable models of motifs of variable structure that are suitable for follow-up structural studies. To our knowledge, we are the first to apply variable length motif models to eukaryotic ChIP-seq data sets and consequently the first to show their value in this domain. The results include a novel motif for the ubiquitous transcription factor Sp1.

Dataset Information

G Protein binding sites on Calnuc (nucleobindin 1) and NUCB2 (nucleobindin 2) define a new class of G(alpha)i-regulatory motifs.

Publications

G Protein binding sites on Calnuc (nucleobindin 1) and NUCB2 (nucleobindin 2) define a new class of G(alpha)i-regulatory motifs.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets