Identification of core allosteric sites through temperature- and nucleus-invariant chemical shift covariance.

Allosteric regulation is essential to control biological function. In addition, allosteric sites offer a promising venue for selective drug targeting. However, accurate mapping of allosteric sites remains challenging since allostery relies on often subtle, yet functionally relevant, structural and dynamical changes. A viable approach proposed to overcome such challenge is chemical shift covariance analysis (CHESCA). Although CHESCA offers an exhaustive map of allosteric networks, it is critical to define the core allosteric sites to be prioritized in subsequent functional studies or in the design of allosteric drugs. Here, we propose two new CHESCA-based methodologies, called temperature CHESCA (T-CHESCA) and CLASS-CHESCA, aimed at narrowing down allosteric maps to the core allosteric residues. Both T- and CLASS-CHESCAs rely on the invariance of core inter-residue correlations to changes in the chemical shifts of the active and inactive conformations interconverting in fast exchange. In T-CHESCA the chemical shifts of such states are modulated through temperature changes, while in CLASS-CHESCA through variations in the spin-active nuclei involved in pairwise correlations. T- and CLASS-CHESCAs, as well as complete-linkage CHESCA, were applied to the cAMP-binding domain of the exchange protein directly activated by cAMP (EPAC), which serves as a prototypical allosteric switch. Residues consistently identified by the three CHESCA methods were found in previously identified EPAC allosteric core sites. Hence, T-, CLASS-, and CL-CHESCA provide a toolset to establish allosteric site hierarchy and triage allosteric sites to be further analyzed by mutations and functional assays. Furthermore, the core allosteric networks selectively revealed through T- and CLASS-CHESCA are expected to facilitate the mechanistic understanding of disease-related mutations and the design of selective allosteric modulators.

Divergent allostery reveals critical differences between structurally homologous regulatory domains of Plasmodium falciparum and human protein kinase G.

Malaria is a life-threatening infectious disease primarily caused by the Plasmodium falciparum parasite. The increasing resistance to current antimalarial drugs and their side effects has led to an urgent need for novel malaria drug targets, such as the P. falciparum cGMP-dependent protein kinase (pfPKG). However, PKG plays an essential regulatory role also in the human host. Human cGMP-dependent protein kinase (hPKG) and pfPKG are controlled by structurally homologous cGMP-binding domains (CBDs). Here, we show that despite the structural similarities between the essential CBDs in pfPKG and hPKG, their respective allosteric networks differ significantly. Through comparative analyses of chemical shift covariance analyses, molecular dynamics simulations, and backbone internal dynamics measurements, we found that conserved allosteric elements within the essential CBDs are wired differently in pfPKG and hPKG to implement cGMP-dependent kinase activation. Such pfPKG versus hPKG rewiring of allosteric networks was unexpected because of the structural similarity between the two essential CBDs. Yet, such finding provides crucial information on which elements to target for selective inhibition of pfPKG versus hPKG, which may potentially reduce undesired side effects in malaria treatments.