Eukaryotic signaling proteins display highly diverse and divergent allosteric regulation. Although any one genome might contain many evolutionarily related signaling molecules, such as protein kinases, individual family members usually display divergent substrate specificity and unique allosteric regulation by various partner proteins. By controlling when and where signaling proteins are activated, these allosteric regulatory interactions play a central role in determining the specific “wiring” of the molecular networks that control cellular behavior ().
Fungal Erk kinase signaling repertoires provide a model system for biochemically interrogating the evolution of novel and divergent allosteric activation mechanisms
Despite their importance, little is known about how these complex allosteric regulatory partnerships in signaling networks evolve. The molecular complexity of these systems represents a challenge for evolution: allosteric activators and the target proteins that they act on must seemingly acquire their complementary regulatory properties simultaneously for these systems to be functional and provide a selective advantage. These allosteric activators must also be specific enough to ensure that they do not inadvertently target homologous signaling components in the cell. The viable paths by which such multicomponent regulatory systems can evolve are therefore unclear.
In other complex systems, many new features appear to evolve by taking advantage of pre-existing or latent behavior: an active site that catalyzes a particular reaction can, with increased promiscuity, perform similar reactions on other substrates; a binding pocket that favors binding of one nuclear hormone can be adapted accommodate a yet-to-be-evolved hormone with somewhat similar structural features (Aharoni et al., 2005
; Baker et al., 2012
; Bridgham et al., 2006
; O’Brien and Herschlag, 1999
; Khersonsky and Tawfik, 2010
; Wise et al., 2005
). While such latent capacities provide clear toeholds for new enzymatic activities or ligand binding capacities, these changes represent a shift in an already well-established and constitutive molecular activity. It is thus unclear the extent to which these evolutionary models apply to allosteric systems in which new protein partnerships must develop that are unrelated to any existing form of regulation and that must produce complex structural reorganization. Computational and protein engineering studies suggest that certain features of protein structure and dynamics may endow proteins with some latent capacity for allosteric regulation (Lee et al., 2008; Reynolds et al., 2011
). Whether natural systems have harnessed such latent features to produce new allosteric regulation during evolution, however, has not been established.
Comparative studies that track the appearance of specific molecular properties across related species were instrumental in uncovering the role of latent protein features in the evolution of other systems and have provided great insights into how new enzymatic activities, receptor/ligand pairs, and transcriptional circuits evolve (Afriat et al., 2006
; Booth et al., 2010
; Gerlt and Babbitt, 2001
; O’Brien and Herschlag, 2001; Roodveldt and Tawfik, 2005
; Taylor Ringia et al., 2004
; Thornton, 2001). However, applying these approaches to multi-component allosteric regulation of signaling proteins has been hindered by a lack of model systems that can be biochemically interrogated over species spanning a considerable window of evolutionary time.
The budding yeast MAP kinase network presents a unique model system with which to take a comparative approach to understand how complex multi-component allosteric regulation might have evolved. Prior biochemical studies have shown that, in S. cerevisiae
, the function of the mating-pathway specific MAP kinase Fus3 requires its allosteric activation by the scaffold protein Ste5 (). This scaffold-mediated allosteric activation ensures that Fus3 is only activated in signaling complexes that are organized in response to pheromone stimulation, thus preventing inappropriate cross-talk in which distinct MAP kinase mediated pathways trigger mating (Zalatan et al., 2012
). Interestingly, the closely related starvation-responsive MAP kinase, Kss1, functions independent of Ste5 regulation, despite the fact that Fus3 and Kss1 are 55 % identical, are both targets of the MAPKK Ste7, and likely arose from duplication of the same Erk-like MAP kinase ancestor () (Madhani and Fink, 1998
Given their common MAPK ancestor, how did Fus3 become dependent on allosteric regulation while Kss1 did not? The availability of a large number of sequenced fungal genomes provides an opportunity to gain insights into this evolutionary question by exploring the regulatory properties of orthologs from scaffold and MAP kinase species throughout the fungal tree. Comparison of Erk-like MAP kinase sequences from across the Ascomycota fungi (to which S. cer.
belongs) indicates that these kinases are highly divergent and fall into distinct classes that are associated with specific fungal lineages (Fig. S1A–C
). Interestingly, only those species that have both a Fus3 and Kss1 ortholog also have a Ste5 scaffold ortholog (; detailed in supplement
). It is unclear how both a potent allosteric activator (Ste5) and its regulated target (Fus3) could simultaneously evolve as a two-part complementary system. However, because we have access to signaling repertoires from species that clearly diverged from the S. cer.
lineage prior to the appearance of Fus3, Kss1 and Ste5, we have the potential to uncover the mechanism by which this allosteric partnership evolved.
Here we expressed and purified Erk-like MAP kinases and Ste5 orthologs (if present) from 13 diverse fungal species that span from S. pombe to S. cer. (~1 billion years of divergence—comparable to the divergence between sea squirt and human). Using an in vitro reconstituted system, we determined the ability of these orthologs to cross-activate one another, even for species that do not contain a Ste5 protein. These quantitative data allowed us to determine when specific kinase and scaffold biochemical features arose during evolution and to formulate a model for the evolution of the allosteric regulatory schemes observed in S. cer.
First, we find that the Ste5 allosteric interaction required for Fus3 activation by the MAPKK Ste7 (Ste5-VWA) is a conserved scaffold feature of all Fus3/Kss1 containing species, while a second allosteric region in Ste5 (Ste5-FBD) that tunes the ultrasensitiviy of the mating response is, in general, not conserved outside of S. cer. This is consistent with a model in which a core function of the Ste5 scaffold protein has been to functionally insulate Fus3 and Kss1 since their divergence, but also suggests that Ste5/Fus3 interactions might continue to evolve to meet specific organismal needs. Second, and surprisingly, we find that the Ste5 scaffold can allosterically activate orthologous MAP kinases from species that diverged prior to the evolution of Ste5, i.e. kinases that are likely to never have co-existed with the Ste5 scaffold. This result suggests that the Ste5 allosteric interactions evolved by tapping into latent, pre-existing dynamic properties of the MAP kinase. The magnitude of this latent allostery appears to drift significantly within the pre-Ste5 MAP kinases—some orthologs are primed for Fus3-like regulation (strong allosteric response) while others are primed for Kss1-like regulation (inability to respond). We propose that hidden diversity in these latent allosteric properties provides a toehold that new partner molecules can exploit to develop novel, component-specific allosteric regulatory relationships, simplifying the evolutionary paths to allosteric controls that shape pathway behavior and distinguish functional identity.