A functionally divergent intrinsically disordered region underlying the conservation of stochastic signaling

Stochastic signaling dynamics expand living cells' information processing capabilities. An increasing number of studies report that regulators encode information in their pulsatile dynamics. The evolutionary mechanisms that lead to complex signaling dynamics remain uncharacterized, perhaps because key interactions of signaling proteins are encoded in intrinsically disordered regions (IDRs), whose evolution is difficult to analyze. Here we focused on the IDR that controls the stochastic pulsing dynamics of Crz1, a transcription factor in fungi downstream of the widely conserved calcium signaling pathway. We find that Crz1 IDRs from anciently diverged fungi can all respond transiently to calcium stress; however, only Crz1 IDRs from the Saccharomyces clade support pulsatility, encode extra information, and rescue fitness in competition assays, while the Crz1 IDRs from distantly related fungi do none of the three. On the other hand, we find that Crz1 pulsing is conserved in the distantly related fungi, consistent with the evolutionary model of stabilizing selection on the signaling phenotype. Further, we show that a calcineurin docking site in a specific part of the IDRs appears to be sufficient for pulsing and show evidence for a beneficial increase in the relative calcineurin affinity of this docking site. We propose that evolutionary flexibility of functionally divergent IDRs underlies the conservation of stochastic signaling by stabilizing selection. Author summary During the last decade or so, it has been appreciated that transcription factors sometimes control gene expression by entering the nucleus in short, stochastic "pulses", as opposed to (seemingly simpler) stable nuclear localization. The amino acid sequences that encode this type of dynamic regulation are found in so-called intrinsically disordered regions that do not fold into stable 3D conformations. Thusfar, little is known about the evolution of transcription factor pulsing. Here we studied the rapidly evolving intrinsically disordered reigons underlying pulsing of the yeast transcription factor, Crz1. We find that the intrinsically disordered regions are highly diverged in their amino acid sequences, but for some species they can still perform the pulsing function when expressed in yeast, while for more distantly related species they can not. Surprisingly, for these distantly related species, we found that Crz1 does pulse in the cells of the distantly related species. We propose that there has been compensatory evolution to preserve pulsing, even though the intrinsically disordered regions have diverged. This type of evolutionary preservation is called "stabilizing selection," and we believe that it is working to preserve the functional output of signaling pathways (in this case pulsing) even when the protein components (or at least their intrinsically disordered regions) are changing.