In this work, we have examined the molecular mechanisms of allosteric regulation of the ABL tyrosine kinase at the atomic level. Atomistic modeling of the ABL complexes with a panel of allosteric modulators has been performed using a combination of molecular dynamics simulations, structural residue perturbation scanning, and a novel community analysis of the residue interaction networks. Our results have indicated that allosteric inhibitors and activators may exert a differential control on allosteric signaling between the kinase binding sites and functional regions. While the inhibitor binding can strengthen the closed ABL state and induce allosteric communications directed from the allosteric pocket to the ATP binding site, the DPH activator may induce a more dynamic open form and activate allosteric couplings between the ATP and substrate binding sites. By leveraging a network-centric theoretical framework, we have introduced a novel community analysis method and global topological parameters that have unveiled the hierarchical modularity and the intercommunity bridging sites in the residue interaction network. We have found that allosteric functional hotspots responsible for the kinase regulation may serve the intermodular bridges in the global interaction network. The central conclusion from this analysis is that the regulatory switch centers play a fundamental role in the modular network organization of ABL as the unique intercommunity bridges that connect the SH2 and SH3 domains with the catalytic core into a functional kinase assembly. The hierarchy of network organization in the ABL regulatory complexes may allow for the synergistic action of dense intercommunity links required for the robust signal transfer in the catalytic core and sparse network bridges acting as the regulatory control points that orchestrate allosteric transitions between the inhibited and active kinase forms.

Structural and biochemical studies of Hsp70 chaperones have provided a molecular view of the chaperone biochemical cycle by revealing a complex interplay between allosteric conformational states that controls the feedback loop between stimulation of the adenosinetriphosphatase (ATPase) activity and the substrate release. Allosteric regulation in the Hsp70 chaperones and efficient substrate targeting are mediated by J-domain cochaperones through a dynamic interaction network controlled by the regulatory hotspots. In the current work, we have simulated conformational landscapes and residue interaction networks in the open, closed, and cochaperone-bound DnaK structures. The results of this work have shown that J-domain can selectively enhance direction-specific signal propagation from the substrate-binding domain to the catalytic center and promote the structural environment required for ATP hydrolysis. By employing different network-based approaches, we examined the role and contribution of post-translational modification sites in allosteric regulation of human Hsp70. The central finding of this analysis indicated that conserved phosphorylation sites localized preferentially in the nucleotide-binding domain regions are often aligned with the allosteric control points and serve as effector centers in Hsp70. We have found that cooperation of post-translational modifications sites is based on the governing role of phosphorylation sites in dictating regulatory switching functions, whereas the bulk of acetylation sites can be involved in sensing the long-range signals and executing allosteric changes during the ATPase cycle. The results of this study highlight the important role of phosphorylation sites in exerting control over allosteric changes in Hsp70. The network-centric framework for the analysis of conformational dynamics and chaperone landscapes can explain a range of structural and functional experiments, providing a robust dynamic model of Hsp70 regulation by cochaperones and sites of post-translational modifications.

Combating malaria is almost a never-ending battle, as Plasmodium parasites develop resistance to the drugs used against them, as observed recently in artemisinin-based combination therapies. The main concern now is if the resistant parasite strains spread from Southeast Asia to Africa, the continent hosting most malaria cases. To prevent catastrophic results, we need to find non-conventional approaches. Allosteric drug targeting sites and modulators might be a new hope for malarial treatments. Heat shock proteins (HSPs) are potential malarial drug targets and have complex allosteric control mechanisms. Yet, studies on designing allosteric modulators against them are limited. Here, we identified allosteric modulators (SANC190 and SANC651) against P. falciparum Hsp70-1 and Hsp70-x, affecting the conformational dynamics of the proteins, delicately balanced by the endogenous ligands. Previously, we established a pipeline to identify allosteric sites and modulators. This study also further investigated alternative approaches to speed up the process by comparing all atom molecular dynamics simulations and dynamic residue network analysis with the coarse-grained (CG) versions of the calculations. Betweenness centrality (BC) profiles for PfHsp70-1 and PfHsp70-x derived from CG simulations not only revealed similar trends but also pointed to the same functional regions and specific residues corresponding to BC profile peaks.

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