Rugged fitness landscapes minimize promiscuity in the evolution of transcriptional repressors

Our understanding of how proteins evolve over time and across fitness landscapes of variable ruggedness remains incomplete. Our ability to generate and characterize expansive and sufficiently diverse datasets to examine this question has been limited, which has hampered efforts to understand why some fitness landscapes are more rugged than others and how they are a product of protein structure and function. Here we describe the use of ancestral sequence reconstruction to generate a 1158 sequence phylogeny of the DNA-binding domain (DBD) of the LacI/GalR transcriptional repressor family. This revealed a rugged fitness landscape, which we rationalize through biophysical analysis to be intrinsic to the structure and function of DBDs: the asymmetry of the operator-DBD complex means that the fitness landscape is in fact a composite of two separate fitness landscapes. This ruggedness underlies the rapid evolution of orthogonal specificity within DBDs, which is an essential part of their function.