Yu Zhu

Ca2+-release activated Ca2+ (CRAC) channels, formed by stromal interaction molecule (STIM) and Orai proteins, mediate store-operated Ca2+ entry and are implicated in autoimmune diseases, acute pancreatitis and cancer. Some small molecule inhibitors of these channels such as BTP2, Pyr6, Synta66 and GSK-7975A, are widely used as experimental tools, yet their precise mechanisms of action remain incompletely understood. Using whole-cell patch-clamp electrophysiology in rat basophilic leukaemia (RBL-1) cells, we determined whether each of these inhibitors acts from the extracellular or intracellular side of the plasma membrane. We found that BTP2 and GSK-7975A could suppress CRAC currents only when applied extracellularly, whereas Pyr6 and Synta66 were equally effective from either side. Molecular docking and molecular dynamics simulations using a rat Orai1 channel model identified plausible extracellular binding sites for all four inhibitors and additional putative intracellular binding sites for Pyr6 and Synta66. These predicted sites are located near regions that are likely to influence pore geometry and channel gating. Our findings suggest that, despite considerable structural similarity, these popular CRAC channel inhibitors exhibit mechanistically distinct interactions with Orai1, with important implications for their use as research tools and for future CRAC channel drug discovery.

Missense mutations in multidomain signalling proteins often produce divergent functional phenotypes despite preserved structural integrity, posing a fundamental challenge for interpreting human genetic variation. This problem is exemplified by the type 1 inositol 1,4,5-trisphosphate receptor, IP ₃ R1, the principal neuronal IP ₃ -gated Ca ²⁺ release channel and a recurrent locus of pathogenic variation in ITPR1 -associated ataxias and neurodevelopmental disorders. Although disease mutations cluster within the N-terminal suppression domain (SD) and IP ₃ -binding core (IBC), how neighbouring substitutions can drive either gain- or loss-of-function remains unresolved. Here, we establish a unified ensemble-allosteric framework for IP ₃ R1 channelopathy by integrating mutational constraint analysis, ensemble structural modelling, adaptive molecular dynamics, Markov state modelling and dynamical network analysis. We show that pathogenic variants preferentially occupy a stability-preserving regime, perturbing local sequence-structure compatibility without inducing global structural collapse. Thus, disease arises not from loss of fold, but from corruption of the conformational and allosteric logic that links ligand recognition to channel gating. The IP ₃ -binding pocket forms a discrete multistate ensemble whose populations, physicochemical properties and kinetic connectivity are selectively remodelled by mutation. The loss-of-function variant R269W disrupts a cationic IP ₃ -coordinating site, enriches non-permissive binding-pocket states and diverts conformational exchange through indirect kinetic routes. By contrast, the gain-of-function variant R36C preserves local pocket competence but weakens suppressor-domain restraint by rerouting long-range communication through extended, less efficient allosteric pathways. These findings reconcile opposing disease phenotypes within a single mechanistic model, showing that pathogenic variation can be encoded in altered ensemble probabilities and information flow rather than in static structural lesions.

Inositol 1,4,5-trisphosphate receptor (IP3R) represents a major family of intracellular Ca2+-release channels. Distal to its pore-forming region lies its cytoplasmic N-terminus (NT) that harbours the agonist-binding pocket. Although inositol 1,4,5-trisphosphate (IP3) is the endogenous agonist, the fungal metabolite adenophostin A (AdA) is known to function as a ‘super-agonist’, displaying roughly 10-fold higher affinity on binding. Using all-atom molecular dynamics simulations of rat IP3R1 NT in apo, IP3-bound and AdA-bound states, we here show that both agonists alter NT’s flexibility, yet principal component analysis reveals that AdA drives the domain into broader and distinct conformational substates that are visited by neither the apo nor the IP3-bound form. AdA occupies a more spacious, hydrophobic pocket, engaging a wider spectrum of transient polar and non-polar contacts and favouring an entropy-dominated binding mode despite fewer enduring hydrogen bonds. Dynamic cross-correlation analysis further demonstrates that AdA enhances long-range correlated motions across the NT domain. Taken together, the data indicate that AdA’s superior potency arises from its unique capacity to remodel NT dynamics more profoundly than IP3, thereby pre-configuring or priming IP3R for activation gating. These findings refine current models of ligand efficacy and offer a framework for the rational design of next-generation IP3R modulators.

DNA methyltransferases (DNMTs) including DNMT1 are a conserved family of cytosine methylases that play crucial roles in epigenetic regulation. The versatile functions of DNMT1 rely on allosteric networks between its different interacting partners, emerging as novel therapeutic targets. In this work, based on the modeling structures of DNMT1-ubiquitylated H3 (H3Ub)/ubiquitin specific peptidase 7 (USP7) complexes, we have used a combination of elastic network models, molecular dynamics simulations, structural residue perturbation, network modeling, and pocket pathway analysis to examine their molecular mechanisms of allosteric regulation. The comparative intrinsic and conformational dynamics analysis of three DNMT1 systems has highlighted the pivotal role of the RFTS domain as the dynamics hub in both intra- and inter-molecular interactions. The site perturbation and network modeling approaches have revealed the different and more complex allosteric interaction landscape in both DNMT1 complexes, involving the events caused by mutational hotspots and post-translation modification sites through protein-protein interactions (PPIs). Furthermore, communication pathway analysis and pocket detection have provided new mechanistic insights into molecular mechanisms underlying quaternary structures of DNMT1 complexes, suggesting potential targeting pockets for PPI-based allosteric drug design.