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Structural Analysis of PTM Hotspots (SAPH-ire)--A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families.

Dewhurst HM, Choudhury S, Torres MP - Mol. Cell Proteomics (2015)

Bottom Line: Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins).To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit-N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo.These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Georgia Institute of Technology; School of Biology; 310 Ferst Drive; Atlanta, Georgia 30332.

No MeSH data available.


Related in: MedlinePlus

Intrinsic structural disorder and phosphorylation are conserved in Gγ N-terminal tails.A, Frequency distribution of PTM load for six different protein families essential for G protein signaling – Gα, Gβ, Gγ, RGS protein, and GPCR. PTM load calculated by dividing total number of PTMs observed for a uniprot entry by the number of residues in the full-length protein. B, Table of protein sequence identity, PTM load and phosphorylation load within the Gγ family. For comparison, sequence identity and PTM statistics for Gγ-Nt have been compared with the remainder of the Gγ protein (Gγ-Nt/Gγ). C, Disorder tendency for all reviewed Uniprot entries for Gγ subunits (blue lines). Yeast Gγ/Ste18 (inlay). Structural disorder is predicted for tendency values greater than 0.5 using IUPred (29). D, 3D structure of mammalian heterotrimeric G protein (1GP2) with a threaded model of Gγ-Nt and PTM heatmap. Sidechains with projected hotspots are shown with van der Waals radii (vdw = 1.0).
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Figure 5: Intrinsic structural disorder and phosphorylation are conserved in Gγ N-terminal tails.A, Frequency distribution of PTM load for six different protein families essential for G protein signaling – Gα, Gβ, Gγ, RGS protein, and GPCR. PTM load calculated by dividing total number of PTMs observed for a uniprot entry by the number of residues in the full-length protein. B, Table of protein sequence identity, PTM load and phosphorylation load within the Gγ family. For comparison, sequence identity and PTM statistics for Gγ-Nt have been compared with the remainder of the Gγ protein (Gγ-Nt/Gγ). C, Disorder tendency for all reviewed Uniprot entries for Gγ subunits (blue lines). Yeast Gγ/Ste18 (inlay). Structural disorder is predicted for tendency values greater than 0.5 using IUPred (29). D, 3D structure of mammalian heterotrimeric G protein (1GP2) with a threaded model of Gγ-Nt and PTM heatmap. Sidechains with projected hotspots are shown with van der Waals radii (vdw = 1.0).

Mentions: To our surprise, we found that the Gγ family exhibits twice the PTM load (#PTMs/protein length) of any other heterotrimeric G protein family in addition to GPCRs and RGS protein families (Fig. 5A). Unexpectedly, 41% of all Gγ PTMs are located within Gγ-Nt – 18 of which correspond to 56% of all observed phosphorylations in the Gγ family (Fig. 5B). In addition, we found that 100% of the N-terminal tails from Gγ family members contain multiple serine and/or threonine residues, which are enriched by 1.4 times the next most abundant residue. The N-terminal tails of Gγ subunits have not been resolved by x-ray crystallography, presumably because of a high degree of intrinsic disorder, which we confirmed using the IUPred prediction algorithm (29). We found that all known reviewed Gγ subunits, including the yeast Gγ subunit Ste18, exhibit intrinsic disorder throughout the first 8 to 15 residues of their N termini (Fig. 5C), which we visualized using a threaded structural model of the gamma subunit in the 1GP2 crystal structure (Fig. 5D). Thus, much like the disordered C-terminal tails of GPCRs (30), the intrinsically disordered N-terminal tails of Gγ subunits are phosphorylation hotspots in the canonical heterotrimeric G protein complex.


Structural Analysis of PTM Hotspots (SAPH-ire)--A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families.

Dewhurst HM, Choudhury S, Torres MP - Mol. Cell Proteomics (2015)

Intrinsic structural disorder and phosphorylation are conserved in Gγ N-terminal tails.A, Frequency distribution of PTM load for six different protein families essential for G protein signaling – Gα, Gβ, Gγ, RGS protein, and GPCR. PTM load calculated by dividing total number of PTMs observed for a uniprot entry by the number of residues in the full-length protein. B, Table of protein sequence identity, PTM load and phosphorylation load within the Gγ family. For comparison, sequence identity and PTM statistics for Gγ-Nt have been compared with the remainder of the Gγ protein (Gγ-Nt/Gγ). C, Disorder tendency for all reviewed Uniprot entries for Gγ subunits (blue lines). Yeast Gγ/Ste18 (inlay). Structural disorder is predicted for tendency values greater than 0.5 using IUPred (29). D, 3D structure of mammalian heterotrimeric G protein (1GP2) with a threaded model of Gγ-Nt and PTM heatmap. Sidechains with projected hotspots are shown with van der Waals radii (vdw = 1.0).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4528253&req=5

Figure 5: Intrinsic structural disorder and phosphorylation are conserved in Gγ N-terminal tails.A, Frequency distribution of PTM load for six different protein families essential for G protein signaling – Gα, Gβ, Gγ, RGS protein, and GPCR. PTM load calculated by dividing total number of PTMs observed for a uniprot entry by the number of residues in the full-length protein. B, Table of protein sequence identity, PTM load and phosphorylation load within the Gγ family. For comparison, sequence identity and PTM statistics for Gγ-Nt have been compared with the remainder of the Gγ protein (Gγ-Nt/Gγ). C, Disorder tendency for all reviewed Uniprot entries for Gγ subunits (blue lines). Yeast Gγ/Ste18 (inlay). Structural disorder is predicted for tendency values greater than 0.5 using IUPred (29). D, 3D structure of mammalian heterotrimeric G protein (1GP2) with a threaded model of Gγ-Nt and PTM heatmap. Sidechains with projected hotspots are shown with van der Waals radii (vdw = 1.0).
Mentions: To our surprise, we found that the Gγ family exhibits twice the PTM load (#PTMs/protein length) of any other heterotrimeric G protein family in addition to GPCRs and RGS protein families (Fig. 5A). Unexpectedly, 41% of all Gγ PTMs are located within Gγ-Nt – 18 of which correspond to 56% of all observed phosphorylations in the Gγ family (Fig. 5B). In addition, we found that 100% of the N-terminal tails from Gγ family members contain multiple serine and/or threonine residues, which are enriched by 1.4 times the next most abundant residue. The N-terminal tails of Gγ subunits have not been resolved by x-ray crystallography, presumably because of a high degree of intrinsic disorder, which we confirmed using the IUPred prediction algorithm (29). We found that all known reviewed Gγ subunits, including the yeast Gγ subunit Ste18, exhibit intrinsic disorder throughout the first 8 to 15 residues of their N termini (Fig. 5C), which we visualized using a threaded structural model of the gamma subunit in the 1GP2 crystal structure (Fig. 5D). Thus, much like the disordered C-terminal tails of GPCRs (30), the intrinsically disordered N-terminal tails of Gγ subunits are phosphorylation hotspots in the canonical heterotrimeric G protein complex.

Bottom Line: Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins).To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit-N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo.These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Georgia Institute of Technology; School of Biology; 310 Ferst Drive; Atlanta, Georgia 30332.

No MeSH data available.


Related in: MedlinePlus