Conflicting Interfacial Electrostatic Interactions as a Design Principle to Modulate Long-Range Interdomain Communication.

The extent and molecular basis of interdomain communication in multidomain proteins, central to understanding allostery and function, is an open question. One simple evolutionary strategy could involve the selection of either conflicting or favorable electrostatic interactions across the interface of two closely spaced domains to tune the magnitude of interdomain connectivity. Here, we study a bilobed domain FF34 from the eukaryotic p190A RhoGAP protein to explore one such design principle that mediates interdomain communication. We find that while the individual structural units in wild-type FF34 are marginally coupled, they exhibit distinct intrinsic stabilities and low cooperativity, manifesting as slow folding. The FF3-FF4 interface harbors a frustrated network of highly conserved electrostatic interactions-a charge troika-that promotes the population of multiple, decoupled, and non-native structural modes on a rugged native landscape. Perturbing this network via a charge-reversal mutation not only enhances stability and cooperativity but also dampens the fluctuations globally and speeds up the folding rate by at least an order of magnitude. Our work highlights how a conserved but nonoptimal network of interfacial electrostatic interactions shapes the native ensemble of a bilobed protein, a feature that could be exploited in designing molecular systems with long-range connectivity and enhanced cooperativity.

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 dissect the underlying mechanism of promiscuous binding versus partial subfunctionalization in paralogues 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 paralogues, 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 apparent in A749. The paralogues 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 paralogues.