Multiple Co-Evolutionary Networks Are Supported by the Common Tertiary Scaffold of the LacI/GalR Proteins

Abstract

Protein families might evolve paralogous functions on their common tertiary scaffold in two ways. First, the locations of functionally-important sites might be “hard-wired” into the structure, with novel functions evolved by altering the amino acid (e.g. Ala vs Ser) at these positions. Alternatively, the tertiary scaffold might be adaptable, accommodating a unique set of functionally important sites for each paralogous function. To discriminate between these possibilities, we compared the set of functionally important sites in the six largest paralogous subfamilies of the LacI/GalR transcription repressor family. LacI/GalR paralogs share a common tertiary structure, but have low sequence identity (≤30%), and regulate a variety of metabolic processes. Functionally important positions were identified by conservation and co-evolutionary sequence analyses. Results showed that conserved positions use a mixture of the “hard-wired” and “accommodating” scaffold frameworks, but that the co-evolution networks were highly dissimilar between any pair of subfamilies. Therefore, the tertiary structure can accommodate multiple networks of functionally important positions. This possibility should be included when designing and interpreting sequence analyses of other protein families. Software implementing conservation and co-evolution analyses is available at https://sourceforge.net/projects/coevolutils/.

Engineering allostery

“…Indeed, most co-evolving residues involved in allostery within a subfamily were found in spatially disconnected regions, showing that complex epistatic networks participate in allostery [22, 23]. In LacI, these positions are scattered across the N- and C-terminal dimerization interfaces, core-pivot and hinge regions, and DNA-binding domain, consistent with genetic and biochemical studies of the mutants [21]. …”

“…In order to preserve protein function, co-evolving pairs undergo mutually compensatory changes during evolution, which provides insight into residue connectivity [20]. The LacI family of bacterial transcription factors share the same protein architecture yet have a sequence similarity less than 30% [21]. Although residues in the ligand-binding pocket and at the DNA-binding interface vary depending on inducer and operator sequence, residues involved in allostery have lower sequence entropy.…”

“…Comparison across more distant families shows that approximately 19% of the residues are conserved, suggesting that these positions may be indispensable or “hardwired” for structural integrity and function for that protein fold family [21]. As the scaffold evolved to acquire specific functions, it may have adapted to a particular niche through minor alterations in mechanism.…”

AlloRep: A Repository of Sequence, Structural and Mutagenesis Data for the LacI/GalR Transcription Regulators

“…Separate tables contain whole-family and subfamily alignments, so that the user can search for conservation patterns at these distinct phylogenetic levels [23]. …”

“…For 34 subfamilies, the sequence alignment construction was described in [23,25]; where possible, these sequence alignments were benchmarked against structure-based sequence alignments. The AlloRep database also includes additional sequences for (a) the AscG, CytR, FruR, and LacI subfamilies and (b) 11 new LacI/GalR subfamilies that were nucleated from 11 unpublished structures deposited in the Protein Data Bank by the Protein Structure Initiative (PDB: 3bil, 3cs3, 3d8u, 3e3m, 3gv0, 3h5t, 3hs3, 3jvd, 3jy6, 3k4h and 3kjx) [17,18].…”