Conformational Tuning Shapes the Balance between Functional Promiscuity and Specialization in Paralogous Plasmodium Acyl-CoA Binding Proteins

Paralogous proteins confer enhanced fitness to organisms via complex sequence-conformation codes that shape functional divergence, specialization, or promiscuity. Here, we resolve the underlying mechanism of promiscuous binding versus partial sub-functionalization in paralogs by studying structurally-identical Acyl-CoA Binding Proteins (ACBPs) from Plasmodium falciparum that serve as promising drug targets due to their high expression during the protozoan proliferative phase. Combining spectroscopic measurements, solution NMR, SPR and simulations on two of the paralogs, A16 and A749, we show that minor sequence differences shape nearly every local and global conformational feature. A749 displays a broader and heterogeneous native ensemble, weaker thermodynamic coupling and cooperativity, enhanced fluctuations, and a larger binding-pocket volume, compared to A16. Site-specific tryptophan probes signal a graded reduction in the sampling of substates in the holo form, which is particularly more apparent in A749, hinting at conformational-selection-like mechanism of binding. The paralogs exhibit a spectrum of binding affinities to different acyl-CoAs with A749, the more promiscuous and hence the likely ancestor, binding 1000-fold stronger to Lauroyl-CoA under physiological conditions. We thus demonstrate how minor sequence changes modulate the extent of long-range interactions and dynamics, effectively contributing to the molecular evolution of contrasting functional repertoires in paralogs.