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    • Fig C presents the secondary structure

    2022-01-20

    Fig. 1(C) presents the secondary structure arrangement of the PAS-A and PAS-B domains in both HIF-2α and ARNT. The common scaffold of the PAS domains is a β sheet with numerous flanking α helixes. The PAS-A domains of both HIF-2α and ARNT have A’α, Aβ, Bβ, Cα, Dα, Eα, Fα, Gβ, Hβ, and Iβ from the N to C termini. The PAS-B domains of both HIF-2α and ARNT are very similar to the PAS-A domains, except they lack an A’α helix before the β sheet, and Gβ is dissected into two short Gβ1 and Gβ2 strands. The recently determined HIF-2α–ARNT dimer structure (not covering their TADs) revealed the presence of six domain–domain interfaces (Fig. 1(B)): (1) HIF-2α’s bHLH with ARNT’s bHLH, (2) HIF-2α’s PAS-A with ARNT’s PAS-A, (3) HIF-2α’s PAS-B with ARNT’s PAS-A, (4) HIF-2α’s PAS-B with ARNT’s PAS-B, (5) HIF-2α’s PAS-A with HIF-2α’s PAS-B, and (6) HIF-2α’s bHLH with HIF-2α’s PAS-B. Furthermore, Wu et al. [22] mutated numerous amino Sabutoclax synthesis residues on different interfaces to examine their effects on HIF-2α–ARNT dimerization. Fig. 1(B) shows these mutated residues. The HIF-2α–ARNT complex is very sensitive to ARNT mutations on interfaces 1 and 2, whereas those on interfaces 3 and 4 are comparatively tolerable. Interestingly, mutations on HIF-2α PAS-A domain, such as R171A, V192D, and R171A/V192D, located on interface 5 to govern HIF-2α’s PAS-A and PAS-B contact, exert some allosteric effect to hamper HIF-2α–ARNT dimerization. However, F169D and H194A mutations, also in interface 5 and on HIF2α PAS-A domain, cause only minor interference and maintain HIF-2α–ARNT dimerization. Fig. 1(B) shows the locations of F169, R171, V192, and H194, which are numbered as sites 4, 5, 11, and 12, respectively. With this study, we aimed to differentiate the impact caused by F169D, R171A, V192D, R171A/V192D, and H194A mutations toward HIF-2α–ARNT dimerization. We first explored the HIF-2α–ARNT complex (PDB code: 4ZP4) to characterize the essential interactions between the two proteins by calculating the binding free energy of the two protein using Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method. Furthermore, we extracted the HIF-2α structure from the HIF-2α–ARNT complex to generate the initial structures of the wild-type HIF-2α and the aforementioned five mutants for molecular dynamics (MD) simulations to distinguish their conformational variances that maintain or destabilize HIF-2α–ARNT dimerization.
    Materials and methods
    Results and discussion
    Conclusion Because the HIF-2α–ARNT dimer is involved in cellular adaptations for oxygen stress related to tumor growth and progression and serves as an ideal cancer therapy target [39], [40], [41], [42], [43], small compound inhibitors, including OX3 and proflavine, have been developed to block HIF-2α–ARNT dimerization. According to the determined X-ray crystallography structures for OX3- and proflavine-bound HI-F-2α–ARNT dimers (PDB codes: 4ZQD and 4ZPH, respectively), the 0×3-binding pocket is situated inside the HIF-2α PAS-B domain [22], whereas the bound proflavine is surrounded by W318 (on the HIF-2α PAS-B domain), R266′, F303′, V304′, and V305′ (on the ARNT PAS-A domain) [22]. Notably, proflavine is designed to interrupt interface 3 and further interfere with HIF-2α–ARNT dimerization, which is consistent with our structural analysis results that demonstrated the pivotal role of E346–R266′ electrostatic interaction in interface 3. Furthermore, E346 can be used as one of the two standpoints for monitoring the fluctuating structural relevance between the PAS-A and PAS-B domains of wild-type and mutated HIF-2α systems.
    Acknowledgment The authors gratefully acknowledge the financial support provided by the Ministry of Science and Technology of Taiwan (MOST 104-2113-M-390-003).
    Introduction Ischemic stroke is one of leading causes of mortality and long-term disability in the world (Miller et al., 2017). Ischemia can lead to vascular cell damage, which induces dysregulation of vascular reactivity and endothelial cell apoptosis (Date et al., 2006). The vascular endothelium form the basis of BBB and play critical roles in maintaining the integrity of the BBB and brain homeostasis (Sandoval and Witt, 2008). The death of brain microvascular endothelial cells (BMECs) can lead to BBB disruption which is associated with a poor prognosis of ischemia stroke (Hawkins and Davis, 2005). Thus, protecting BMECs against I/R induced injury is an important strategy in ischemia management and may help to develop novel strategy against neuronal damages.