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The robustness of a signaling complex to domain rearrangements facilitates network evolution.

Sato PM, Yoganathan K, Jung JH, Peisajovich SG - PLoS Biol. (2014)

Bottom Line: Our results indicate that, while domain rearrangement events that result in the loss of catalytic activities within the signaling complex are not tolerated, domain rearrangements can drastically alter protein interactions without impairing function.Furthermore, we also found that the ability of the complex to tolerate changes in interaction partners does not depend on long disordered linkers that often connect domains.Taken together, our results suggest that some signaling complexes are dynamic ensembles with loose spatial constraints that could be easily re-shaped by evolution and, therefore, are ideal targets for cellular engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, and Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
The rearrangement of protein domains is known to have key roles in the evolution of signaling networks and, consequently, is a major tool used to synthetically rewire networks. However, natural mutational events leading to the creation of proteins with novel domain combinations, such as in frame fusions followed by domain loss, retrotranspositions, or translocations, to name a few, often simultaneously replace pre-existing genes. Thus, while proteins with new domain combinations may establish novel network connections, it is not clear how the concomitant deletions are tolerated. We investigated the mechanisms that enable signaling networks to tolerate domain rearrangement-mediated gene replacements. Using as a model system the yeast mitogen activated protein kinase (MAPK)-mediated mating pathway, we analyzed 92 domain-rearrangement events affecting 11 genes. Our results indicate that, while domain rearrangement events that result in the loss of catalytic activities within the signaling complex are not tolerated, domain rearrangements can drastically alter protein interactions without impairing function. This suggests that signaling complexes can maintain function even when some components are recruited to alternative sites within the complex. Furthermore, we also found that the ability of the complex to tolerate changes in interaction partners does not depend on long disordered linkers that often connect domains. Taken together, our results suggest that some signaling complexes are dynamic ensembles with loose spatial constraints that could be easily re-shaped by evolution and, therefore, are ideal targets for cellular engineering.

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Exploring the mechanisms that enable signaling complexes to tolerate rearrangement-mediated gene replacements.(A) Differentiating between two alternative hypotheses: removal of IDRs should negatively impact signaling function if the signaling complex possesses well-defined spatial constraints and therefore a rather rigid structure (left). In contrast, removal of IDRs could be tolerated if the complex is flexible and can adopt a wide ensemble of conformations (right). (B) Schematic representation of the IDR deletion variants. (C) Mating pathway function in yeast strains with IDR-deleted Ste11, Ste20, or Ste50 variants (“Short”), relative to their corresponding full-length variants. (D) Mating pathway function in yeast strains with pairs of simultaneously IDR-deleted variants (either Ste11 and Ste20, or Ste11 and Ste50) in the respective double KO, relative to their corresponding full-length variants. (E) Mating pathway function in yeast strains with IDR-deleted domain rearrangement variants. Statistically significant differences are marked with asterisks. Data shown in Data S1.
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pbio-1002012-g004: Exploring the mechanisms that enable signaling complexes to tolerate rearrangement-mediated gene replacements.(A) Differentiating between two alternative hypotheses: removal of IDRs should negatively impact signaling function if the signaling complex possesses well-defined spatial constraints and therefore a rather rigid structure (left). In contrast, removal of IDRs could be tolerated if the complex is flexible and can adopt a wide ensemble of conformations (right). (B) Schematic representation of the IDR deletion variants. (C) Mating pathway function in yeast strains with IDR-deleted Ste11, Ste20, or Ste50 variants (“Short”), relative to their corresponding full-length variants. (D) Mating pathway function in yeast strains with pairs of simultaneously IDR-deleted variants (either Ste11 and Ste20, or Ste11 and Ste50) in the respective double KO, relative to their corresponding full-length variants. (E) Mating pathway function in yeast strains with IDR-deleted domain rearrangement variants. Statistically significant differences are marked with asterisks. Data shown in Data S1.

Mentions: We then investigated the mechanisms that enable the signaling complex to tolerate changes in recruitment interactions. Domain-domain interactions depend on specific binding interfaces, thus they are unlikely to tolerate drastic changes in interaction partners. Thus, if alternative recruitment has to maintain specific domain-domain interactions, two hypotheses are possible (Figure 4A): (i) rearrangements are tolerated because, even though signaling complexes possess precisely defined spatial constraints that result in fairly rigid 3D structures, domains are connected by long and flexible linkers (e.g., Ste5, Ste7, Ste11, Ste20, and Ste50 are predicted to contain intrinsically disordered regions [IDRs] [30] ranging from ∼74 to ∼207 amino acids long, within their inter-domain linkers, see Figure S14); or (ii) rearrangements are tolerated because signaling complexes do not possess rigid spatial constraints, but are rather diffuse ensembles of dynamically interacting proteins [31]–[33]. We reasoned that, if signaling complexes had defined spatial constraints and thus IDRs were required for networks to tolerate domain-rearrangement-mediated replacements, deletion of IDRs located within inter-domain linkers should substantially reduce pathway function. In contrast, if signaling complexes do not possess tight spatial constraints, but are rather loosely defined regions in which multiple weak interactions create higher local concentrations of signaling proteins, then deletion of inter-domain IDRs should not be detrimental to pathway function. To differentiate between these two hypotheses, we deleted segments of 171 amino acids from Ste20's IDR, 97 amino acids from Ste11's IDR, and 74 amino acids from Ste50's IDR and determined the ability of the shortened proteins to mediate pathway activation, as compared to their respective full-length variants. Note that we did not analyze IDRs present in Ste7's N-t and Ste5's N-t or C-terminus (C-t) because they do not connect pairs of domains, and thus are not likely to facilitate inter-domain flexibility. As shown in Figure 4C, all three shortened variants are still capable of mediating pathway activation (the decrease observed with Ste20's short variant is expected, as Ste20's IDR contains a proline-rich motif needed for proper binding of Bem1, a Cdc42 interaction partner, that when mutated has been shown to reduce pathway activation by ∼50% [34]). To further explore the role of IDRs in pathway function, we simultaneously replaced two WT proteins for their corresponding shortened variants. As shown in Figure 4D, co-expression of IDR-deleted Ste11 and IDR-deleted Ste20 variants effectively mediates pathway activation in the double deletion strain Ste20Δ Ste11Δ; similarly, co-expression of IDR-deleted Ste11 and IDR-deleted Ste50 variants effectively mediates pathway activation in the double deletion strain Ste50Δ Ste11Δ. These results indicate that the mating signaling complex can tolerate simultaneous deletions of IDRs in at least two proteins. Finally, we asked whether IDRs were necessary to tolerate domain rearrangement-mediated replacements, by measuring pathway activation for IDR-deleted domain-rearranged variants, as compared to their respective full-length variants. As shown in Figure 4E, deletion of the IDRs does not reduce pathway activity for most of the domain rearrangement variants tested, suggesting that IDRs are not needed to tolerate domain rearrangement-mediated replacements. Taken together, our results suggest that the yeast mating signaling complex does not possess a rigid, precisely defined spatial geometry, or that at least multiple alternative conformations are functional. Though not the focus of this study, we also noticed that, in some instances, deletion of the IDRs increased basal levels of pathway activation (Figure S15). This observation suggests that, while IDRs are not required for pathway function, they might have regulatory roles.


The robustness of a signaling complex to domain rearrangements facilitates network evolution.

Sato PM, Yoganathan K, Jung JH, Peisajovich SG - PLoS Biol. (2014)

Exploring the mechanisms that enable signaling complexes to tolerate rearrangement-mediated gene replacements.(A) Differentiating between two alternative hypotheses: removal of IDRs should negatively impact signaling function if the signaling complex possesses well-defined spatial constraints and therefore a rather rigid structure (left). In contrast, removal of IDRs could be tolerated if the complex is flexible and can adopt a wide ensemble of conformations (right). (B) Schematic representation of the IDR deletion variants. (C) Mating pathway function in yeast strains with IDR-deleted Ste11, Ste20, or Ste50 variants (“Short”), relative to their corresponding full-length variants. (D) Mating pathway function in yeast strains with pairs of simultaneously IDR-deleted variants (either Ste11 and Ste20, or Ste11 and Ste50) in the respective double KO, relative to their corresponding full-length variants. (E) Mating pathway function in yeast strains with IDR-deleted domain rearrangement variants. Statistically significant differences are marked with asterisks. Data shown in Data S1.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4260825&req=5

pbio-1002012-g004: Exploring the mechanisms that enable signaling complexes to tolerate rearrangement-mediated gene replacements.(A) Differentiating between two alternative hypotheses: removal of IDRs should negatively impact signaling function if the signaling complex possesses well-defined spatial constraints and therefore a rather rigid structure (left). In contrast, removal of IDRs could be tolerated if the complex is flexible and can adopt a wide ensemble of conformations (right). (B) Schematic representation of the IDR deletion variants. (C) Mating pathway function in yeast strains with IDR-deleted Ste11, Ste20, or Ste50 variants (“Short”), relative to their corresponding full-length variants. (D) Mating pathway function in yeast strains with pairs of simultaneously IDR-deleted variants (either Ste11 and Ste20, or Ste11 and Ste50) in the respective double KO, relative to their corresponding full-length variants. (E) Mating pathway function in yeast strains with IDR-deleted domain rearrangement variants. Statistically significant differences are marked with asterisks. Data shown in Data S1.
Mentions: We then investigated the mechanisms that enable the signaling complex to tolerate changes in recruitment interactions. Domain-domain interactions depend on specific binding interfaces, thus they are unlikely to tolerate drastic changes in interaction partners. Thus, if alternative recruitment has to maintain specific domain-domain interactions, two hypotheses are possible (Figure 4A): (i) rearrangements are tolerated because, even though signaling complexes possess precisely defined spatial constraints that result in fairly rigid 3D structures, domains are connected by long and flexible linkers (e.g., Ste5, Ste7, Ste11, Ste20, and Ste50 are predicted to contain intrinsically disordered regions [IDRs] [30] ranging from ∼74 to ∼207 amino acids long, within their inter-domain linkers, see Figure S14); or (ii) rearrangements are tolerated because signaling complexes do not possess rigid spatial constraints, but are rather diffuse ensembles of dynamically interacting proteins [31]–[33]. We reasoned that, if signaling complexes had defined spatial constraints and thus IDRs were required for networks to tolerate domain-rearrangement-mediated replacements, deletion of IDRs located within inter-domain linkers should substantially reduce pathway function. In contrast, if signaling complexes do not possess tight spatial constraints, but are rather loosely defined regions in which multiple weak interactions create higher local concentrations of signaling proteins, then deletion of inter-domain IDRs should not be detrimental to pathway function. To differentiate between these two hypotheses, we deleted segments of 171 amino acids from Ste20's IDR, 97 amino acids from Ste11's IDR, and 74 amino acids from Ste50's IDR and determined the ability of the shortened proteins to mediate pathway activation, as compared to their respective full-length variants. Note that we did not analyze IDRs present in Ste7's N-t and Ste5's N-t or C-terminus (C-t) because they do not connect pairs of domains, and thus are not likely to facilitate inter-domain flexibility. As shown in Figure 4C, all three shortened variants are still capable of mediating pathway activation (the decrease observed with Ste20's short variant is expected, as Ste20's IDR contains a proline-rich motif needed for proper binding of Bem1, a Cdc42 interaction partner, that when mutated has been shown to reduce pathway activation by ∼50% [34]). To further explore the role of IDRs in pathway function, we simultaneously replaced two WT proteins for their corresponding shortened variants. As shown in Figure 4D, co-expression of IDR-deleted Ste11 and IDR-deleted Ste20 variants effectively mediates pathway activation in the double deletion strain Ste20Δ Ste11Δ; similarly, co-expression of IDR-deleted Ste11 and IDR-deleted Ste50 variants effectively mediates pathway activation in the double deletion strain Ste50Δ Ste11Δ. These results indicate that the mating signaling complex can tolerate simultaneous deletions of IDRs in at least two proteins. Finally, we asked whether IDRs were necessary to tolerate domain rearrangement-mediated replacements, by measuring pathway activation for IDR-deleted domain-rearranged variants, as compared to their respective full-length variants. As shown in Figure 4E, deletion of the IDRs does not reduce pathway activity for most of the domain rearrangement variants tested, suggesting that IDRs are not needed to tolerate domain rearrangement-mediated replacements. Taken together, our results suggest that the yeast mating signaling complex does not possess a rigid, precisely defined spatial geometry, or that at least multiple alternative conformations are functional. Though not the focus of this study, we also noticed that, in some instances, deletion of the IDRs increased basal levels of pathway activation (Figure S15). This observation suggests that, while IDRs are not required for pathway function, they might have regulatory roles.

Bottom Line: Our results indicate that, while domain rearrangement events that result in the loss of catalytic activities within the signaling complex are not tolerated, domain rearrangements can drastically alter protein interactions without impairing function.Furthermore, we also found that the ability of the complex to tolerate changes in interaction partners does not depend on long disordered linkers that often connect domains.Taken together, our results suggest that some signaling complexes are dynamic ensembles with loose spatial constraints that could be easily re-shaped by evolution and, therefore, are ideal targets for cellular engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, and Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
The rearrangement of protein domains is known to have key roles in the evolution of signaling networks and, consequently, is a major tool used to synthetically rewire networks. However, natural mutational events leading to the creation of proteins with novel domain combinations, such as in frame fusions followed by domain loss, retrotranspositions, or translocations, to name a few, often simultaneously replace pre-existing genes. Thus, while proteins with new domain combinations may establish novel network connections, it is not clear how the concomitant deletions are tolerated. We investigated the mechanisms that enable signaling networks to tolerate domain rearrangement-mediated gene replacements. Using as a model system the yeast mitogen activated protein kinase (MAPK)-mediated mating pathway, we analyzed 92 domain-rearrangement events affecting 11 genes. Our results indicate that, while domain rearrangement events that result in the loss of catalytic activities within the signaling complex are not tolerated, domain rearrangements can drastically alter protein interactions without impairing function. This suggests that signaling complexes can maintain function even when some components are recruited to alternative sites within the complex. Furthermore, we also found that the ability of the complex to tolerate changes in interaction partners does not depend on long disordered linkers that often connect domains. Taken together, our results suggest that some signaling complexes are dynamic ensembles with loose spatial constraints that could be easily re-shaped by evolution and, therefore, are ideal targets for cellular engineering.

Show MeSH
Related in: MedlinePlus