Sequence-encoded dynamics diversify allostery within a conserved protein scaffold.

Proteins evolve allosteric mechanisms through sequence variation while preserving a conserved structural fold. How these substitutions reshape the protein's conformational landscape to drive functional diversification remains poorly understood. Members of the cAMP receptor protein family (CRP), a ubiquitous bacterial transcription factor, share a common architecture yet differentially couple cAMP binding to DNA interactions. Here, our results support a model in which allosteric diversification in CRP arises from the tuning of sequence-encoded protein's conformational dynamics. Comparative analysis of sequence conservation and helical propensity across proteobacterial and actinobacterial CRPs reveals lineage-specific distributions of conformational heterogeneity within the dimerization C-helix. These evolutionary patterns map onto the proteobacterial Escherichia coli (E. coli) CRP and the actinobacterial Mycobacterium tuberculosis (MTB) CRP, two protein homologs that respond to cAMP through distinct allosteric mechanisms. Guided by functional and evolutionary differences, we introduced CRPE. coli-inspired C-helix substitutions into CRPMTB to identify sequence, structural and conformational features that contribute to the divergent allosteric behavior in CRPMTB. The resulting C-helix variants preserved the global fold, but displayed altered thermodynamic stability, cAMP binding affinity and cooperativity, DNA recognition properties, and promoter-associated oligomerization. Among these, the C-helix hinge residue, N135, emerges as an allosteric node that redistributes cAMP-induced conformational responses, modulating DNA binding. Together, our results identify the C-helix as an evolvable hotspot where sequence changes reweight conformational ensembles and generate distinct allosteric outputs within a conserved protein scaffold.