A P-loop mutation in Gα subunits prevents transition to the active state : implications for G-protein signaling in fungal pathogenesis
Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, where...
| Main Authors: | , , , , , , |
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| Format: | Journal Article |
| Language: | English |
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2013
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| Online Access: | https://hdl.handle.net/10356/100154 http://hdl.handle.net/10220/10955 |
| _version_ | 1811679739730984960 |
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| author | Siderovski, David P. Bosch, Dustin E. Willard, Francis S. Kimple, Adam J. Willard, Melinda D. Naqvi, Naweed Issak. Ramanujam, Ravikrishna. |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Siderovski, David P. Bosch, Dustin E. Willard, Francis S. Kimple, Adam J. Willard, Melinda D. Naqvi, Naweed Issak. Ramanujam, Ravikrishna. |
| author_sort | Siderovski, David P. |
| collection | NTU |
| description | Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gαi1(G42R) binding to GDP·AlF4− or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gβγ and GoLoco motifs in any nucleotide state. The corresponding Gαq(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants. |
| first_indexed | 2024-10-01T03:13:57Z |
| format | Journal Article |
| id | ntu-10356/100154 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T03:13:57Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | ntu-10356/1001542023-02-28T17:02:56Z A P-loop mutation in Gα subunits prevents transition to the active state : implications for G-protein signaling in fungal pathogenesis Siderovski, David P. Bosch, Dustin E. Willard, Francis S. Kimple, Adam J. Willard, Melinda D. Naqvi, Naweed Issak. Ramanujam, Ravikrishna. School of Biological Sciences DRNTU::Science::Biological sciences Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gαi1(G42R) binding to GDP·AlF4− or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gβγ and GoLoco motifs in any nucleotide state. The corresponding Gαq(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants. Published version 2013-07-05T01:33:07Z 2019-12-06T20:17:33Z 2013-07-05T01:33:07Z 2019-12-06T20:17:33Z 2012 2012 Journal Article Bosch, D. E., Willard, F. S., Ramanujam, R., Kimple, A. J., Willard, M. D., Naqvi, N. I., et al. (2012). A P-loop mutation in Gα subunits prevents transition to the active state: Implications for G-protein signaling in fungal pathogenesis. PLoS Pathogens, 8(2), e1002553. 1553-7366 https://hdl.handle.net/10356/100154 http://hdl.handle.net/10220/10955 10.1371/journal.ppat.1002553 22383884 en PLoS pathogens © 2012 The Authors. This paper was published in PLoS Pathogens and is made available as an electronic reprint (preprint) with permission of The Authors. The paper can be found at the following official DOI: [http://dx.doi.org/10.1371/journal.ppat.1002553]. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law. application/pdf |
| spellingShingle | DRNTU::Science::Biological sciences Siderovski, David P. Bosch, Dustin E. Willard, Francis S. Kimple, Adam J. Willard, Melinda D. Naqvi, Naweed Issak. Ramanujam, Ravikrishna. A P-loop mutation in Gα subunits prevents transition to the active state : implications for G-protein signaling in fungal pathogenesis |
| title | A P-loop mutation in Gα subunits prevents transition to the active state : implications for G-protein signaling in fungal pathogenesis |
| title_full | A P-loop mutation in Gα subunits prevents transition to the active state : implications for G-protein signaling in fungal pathogenesis |
| title_fullStr | A P-loop mutation in Gα subunits prevents transition to the active state : implications for G-protein signaling in fungal pathogenesis |
| title_full_unstemmed | A P-loop mutation in Gα subunits prevents transition to the active state : implications for G-protein signaling in fungal pathogenesis |
| title_short | A P-loop mutation in Gα subunits prevents transition to the active state : implications for G-protein signaling in fungal pathogenesis |
| title_sort | p loop mutation in gα subunits prevents transition to the active state implications for g protein signaling in fungal pathogenesis |
| topic | DRNTU::Science::Biological sciences |
| url | https://hdl.handle.net/10356/100154 http://hdl.handle.net/10220/10955 |
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