Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While some mutants show enhanced binding affinity to the foreign pheromone, others only display weakened interactions with the network's negative regulators. Importantly, the latter changes have a limited impact on overall pathway regulation, despite their considerable effect on sensitivity. Our results demonstrate that a new receptor–ligand pair can evolve through network-altering mutations independently of receptor–ligand binding, and suggest a potential role for such mutations in disease

BBF RFC 28: A method for combinatorial multi-part assembly based on the Type IIs restriction enzyme AarI

This BioBricks Foundation Request for Comments (BBF RFC) describes an alternative assembly standard based on the Type IIS restriction enzyme AarI

Diseño y caracterización de una forma parcialmente plegada de la iFABP

Las conformaciones no nativas de las proteínas son de gran interés ya que su estudio puede ayudar a comprender el proceso de plegamiento proteico. En este Seminario de Licenciatura se eligió como modelo para estudios de plegamiento a la proteína transportadora de ácidos grasos de intestino de rata (iFABP), de 131 aminoácidos. Con el propósito de estabilizar una forma no nativa de esta proteína, se decidió remover los tres últimos residuos del extremo C-terminal. La hipótesis planteada fue que Ia truncación, al eliminar cuatro puentes hidrógeno entre cordonesB adyacentes y dos puentes salinos entre cadenas laterales, permitiría el acceso del solvente al interior de la molécula, desestabilizando su estructura nativa. Para facilitarel análisis espectroscópico de la proteína truncada, se construyó también una variante con uno de los dos triptofanos de la molécula, el ubicado en posición seis, reemplazado por fenilalanina. Asimismo, los efectos del reemplazo del triptofano per se, se analizaron construyendo una variante de longitud completa con el reemplazo triptofano 6-fenilalanina. Luego de ser expresadas y purificadas a partir de cultivos bacterianos, las proteínas fueron analizadas utilizando distintas técnicas para establecer sus conformaciones. Los resultados obtenidos muestran que se pudieron estabilizar formas compactas no nativas de la iFABP, que pueden ser relevantes en el proceso de plegamiento. Las variantes truncadas de la iFABP constituyen un modelo adecuado para el estudio de la estabilidad y conformación tridimensional de proteínas parcialmente plegadas.Fil: Peisajovich, Sergio G.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Evolution of Synthetic Signaling Scaffolds by Recombination of Modular Protein Domains

Signaling scaffolds are proteins that interact via modular domains with multiple partners, regulating signaling networks in space and time and providing an ideal platform from which to alter signaling functions. However, to better exploit scaffolds for signaling engineering, it is necessary to understand the full extent of their modularity. We used a directed evolution approach to identify, from a large library of randomly shuffled protein interaction domains, variants capable of rescuing the signaling defect of a yeast strain in which Ste5, the scaffold in the mating pathway, had been deleted. After a single round of selection, we identified multiple synthetic scaffold variants with diverse domain architectures, able to mediate mating pathway activation in a pheromone-dependent manner. The facility with which this signaling network accommodates changes in scaffold architecture suggests that the mating signaling complex does not possess a single, precisely defined geometry into which the scaffold has to fit. These relaxed geometric constraints may facilitate the evolution of signaling networks, as well as their engineering for applications in synthetic biology

Introduction of Premature Stop Codons as an Evolutionary Strategy To Rescue Signaling Network Function

The cellular concentrations of key components of signaling networks are tightly regulated, as deviations from their optimal ranges can have negative effects on signaling function. For example, overexpression of the yeast mating pathway mitogen-activated protein kinase (MAPK) Fus3 decreases pathway output, in part by sequestering individual components away from functional multiprotein complexes. Using a synthetic biology approach, we investigated potential mechanisms by which selection could compensate for a decrease in signaling activity caused by overexpression of Fus3. We overexpressed a library of random mutants of Fus3 and used cell sorting to select variants that rescued mating pathway activity. Our results uncovered that one remarkable way in which selection can compensate for protein overexpression is by introducing premature stop codons at permitted positions. Because of the low efficiency with which premature stop codons are read through, the resulting cellular concentration of active Fus3 returns to values within the range required for proper signaling. Our results underscore the importance of interpreting genotypic variation at the systems rather than at the individual gene level, as mutations can have opposite effects on protein and network function

The Robustness of a Signaling Complex to Domain Rearrangements Facilitates Network Evolution

<div><p>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 <i>in frame</i> 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.</p></div

The yeast mating pathway is robust to domain rearrangement-mediated gene replacements.

<p>(A) Mating pathway activation was determined by flow cytometry, measuring the fluorescence intensity of a GFP reporter controlled by a mating-responsive <i>FUS1</i> promoter, 2 hours after addition of 1 µM α-factor. (B) As expected, individual deletions of the pathway components Ste50, Ste20, Ste7, Ste4, Ste11, Ste5, or Ste18 eliminate pathway activation <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002012#pbio.1002012-Hartwell1" target="_blank">[51]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002012#pbio.1002012-Jenness1" target="_blank">[52]</a>. (C) Mating pathway activation for the library of domain rearrangement variants expressed in individual deletion strains. Rearrangement events that recreate WT genes are marked as “WT.” Repeated attempts to transform variant Ste50[N]-Ste18[C] failed, suggesting that it may result in cell toxicity. For a statistical analysis of the results see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002012#pbio.1002012.s004" target="_blank">Figure S4</a>. Data shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002012#pbio.1002012.s017" target="_blank">Data S1</a>.</p

Signaling complexes can tolerate multiple rearrangement-mediated gene replacements.

<p>(A) Schematic representation of the reciprocally rearranged variants. (B) Co-expression of Ste20[N]-Ste11[C]+Ste11[N]-Ste20[C] restores pathway activation in the Ste20Δ Ste11Δ strain, co-expression of Ste7[N]-Ste11[C]+Ste11[N]-Ste7[C] restores pathway activation in the Ste7Δ Ste11Δ strain, and co-expression of Ste50[N]-Ste11[C]+Ste11[N]-Ste50[C] restores pathway activation in the Ste50Δ Ste11Δ strain. (C) Changes in network topology resulting from domain rearrangement events in our experiments, mimic changes in network topology that have occurred during evolution. (D) Expression of the domain rearranged variant Ste20[N]-Ste11[C] in the double deletion strain Ste50Δ Ste11Δ rescues pathway activation. Statistically significant differences are marked with asterisks. Data shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002012#pbio.1002012.s017" target="_blank">Data S1</a>.</p

Exploring the mechanisms that enable signaling complexes to tolerate rearrangement-mediated gene replacements.

<p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002012#pbio.1002012.s017" target="_blank">Data S1</a>.</p