The human glucagon receptor (GCGR) is a class B G-protein-coupled receptor (GPCR). The GCGR can be activated by glucagon and regulates the release of glucose. The GCGR has been proposed to be an important drug target for type 2 diabetes. Based on the structural model of a full-length glucagon-bound GCGR (glu-GCGR), we performed accelerated molecular dynamics (aMD) simulations, potential of mean force (PMF) calculations, cross-correlation analysis and community network analysis to study the activation mechanism and the conformational dynamics during the activation process. The PMF map depicts three different conformational states of the GCGR: the inactive, intermediate and active states. The activation of the GCGR is characterized by the outward movement of the intracellular side of helix VI. In the active state of the GCGR, the Arg1732.46–Ser3506.41 and Glu2453.50–Thr3516.42 hydrogen bonds break, and the χ1 rotamer of Phe3225.54 changes from perpendicular to parallel to helix VI. The binding of the agonist glucagon decreases the correlated motions of the extracellular loops (ELCs) and the helices around the glucagon-binding site. During the activation of the GCGR, the connections between the intracellular sides of helices become weaker, and the connections between glucagon and ECLs and the extracellular sides of helices become stronger. These facilitate G-protein coupling on the intracellular side and glucagon binding on the extracellular side, and stabilize the GCGR in the active state. We expect that this study can provide useful information on the activation mechanism of the GCGR and facilitate the future design of GCGR inhibitors.

The human corticotropin-releasing factor receptor type 1 (CRF1R) is a class B G-protein-coupled receptor (GPCR), which mediates the response to stress and has been considered as a drug target for depression and anxiety. Based on the CRF1R-antagonist crystal structure, we study the binding mechanism of two distinct antagonists, CP-376395 and MTIP, and the dynamics behaviors of CRF1R induced by an antagonist binding. Key residues interacting with both antagonists and residues specifically binding to one of them are identified. Both antagonists interact with Asn283, Phe203, Met206, Leu280, Tyr316, Leu323, Leu287, Phe284, Val279, Leu319, Phe207, Gly210 and Phe362. CP-376395 specifically binds to Glu209 and Phe160, while MTIP specifically binds to Leu320, Leu213, Ile290, Phe162 and Val313. The total binding free energy of MTIP is lower than that of CP-376395; this is consistent with the experimental observation that MTIP shows higher binding affinity than CP-376395. The conformational dynamic behaviors of antagonist bound holo-CRF1R were found to be different from those of apo-CRF1R in three aspects: (i) the “ionic lock” between side chains of Arg151 in TM2 and Glu209 in TM3 was broken in apo-CRF1R, but was formed in holo-CRF1Rs; (ii) Phe203 in TM3 and Tyr327 in TM6 were in close proximity to each other in apo-CRF1R, while they were far apart resulting from the shift of TM6 in holo-CRF1Rs; and (iii) the “rotamer toggle switch”, Tyr327/Leu323/Phe284, adopted different rotameric conformations in apo-CRF1R and holo-CRF1Rs. We hope that our results could be helpful in further development of the drug design of CRF1R.

Apoferritin is a versatile platform for encapsulating a variety of materials, such as inorganic particles and metal complexes, in its nanocavity. However, loading apoferritin with rare earth metal complexes has been less studied, let alone investigating the binding effect of rare earth metal complexes on iron mineralization in apoferritin. In this work, a new cationic Dy(III) complex, [Dy(NO3)(H2O)(tmp)2](NO3)2 (tmp = trimethylolpropane), was synthesized and subsequently loaded in apoferritin. The binding of the Dy(III) complex to apoferritin inhibited iron mineralization, a plausible explanation being that the early recognition of Fe2+ at the ferroxidase sites was disrupted as a result of the binding of the Dy(III) complex.This paper reports the synthesis of a new cationic Dy(III) complex, its binding to apoferritin and the remarkable iron mineralization inhibition effect as a result of this binding.

•The mechanism of PAD4 inhibition by F-amidine was investigated using the QM/MM approach.•State I can transform to State N through a stepwise manner.•In the PAD4-F–amidine reactant complex, the active site Cys645 exists as a thiolate and His471 is protonated (i.e. State I).•The inhibition reaction proceeds through a three-membered sulfonium ring transition state, His471 acts as a proton donor and facilitates the inhibition reaction.Protein arginine deiminase 4 (PAD4) catalyzes the hydrolysis of a peptidylarginine residue to form a citrulline residue and ammonia during posttranslational modification. This process plays a pivotal role in rheumatoid arthritis (RA) and gene regulation. F-amidine belongs to a series of haloacetamidine compounds that are the most potent PAD4 inhibitors described to date. F-amidine acts as a mechanism-based inhibitor of PAD4, inactivating PAD4 by the covalent modification of the active site Cys645. In this manuscript, the fundamental mechanism of PAD4 inhibition by F-amidine is investigated using a QM/MM approach. Our simulations show that in the PAD4–F-amidine reactant complex, the active site Cys645 exists as a thiolate and His471 is protonated. This is consistent with the reverse protonation mechanism wherein the active site nucleophile, Cys645, in PAD4 exists as a thiolate in the active form of the enzyme. Inhibition of PAD4 by F-amidine is initiated by the nucleophilic addition of Sγ to the Cζ of F-amidine, leading to the formation of a tetrahedral intermediate. His471 serves as a proton donor, helping F to leave the fluoroacetamidine moiety of F-amidine; meanwhile, Sγ forms a three-membered ring with Cζ and Cη of F-amidine. Subsequently, the three-membered sulfonium ring collapses and rearranges to the final thioether product. His471 acts as a proton donor in the transition state and facilitates the inhibition reaction of PAD4.In the PAD4–F-amidine reactant complex, the active site Cys645 exists as a thiolate and His471 is protonated. The inhibition reaction proceeds through a three-membered sulfonium ring transition state, His471 acts as a proton donor and facilitates the inhibition.

The human glucagon receptor (GCGR) is a class B G-protein-coupled receptor (GPCR). The GCGR can be activated by glucagon and regulates the release of glucose. The GCGR has been proposed to be an important drug target for type 2 diabetes. Based on the structural model of a full-length glucagon-bound GCGR (glu-GCGR), we performed accelerated molecular dynamics (aMD) simulations, potential of mean force (PMF) calculations, cross-correlation analysis and community network analysis to study the activation mechanism and the conformational dynamics during the activation process. The PMF map depicts three different conformational states of the GCGR: the inactive, intermediate and active states. The activation of the GCGR is characterized by the outward movement of the intracellular side of helix VI. In the active state of the GCGR, the Arg1732.46–Ser3506.41 and Glu2453.50–Thr3516.42 hydrogen bonds break, and the χ1 rotamer of Phe3225.54 changes from perpendicular to parallel to helix VI. The binding of the agonist glucagon decreases the correlated motions of the extracellular loops (ELCs) and the helices around the glucagon-binding site. During the activation of the GCGR, the connections between the intracellular sides of helices become weaker, and the connections between glucagon and ECLs and the extracellular sides of helices become stronger. These facilitate G-protein coupling on the intracellular side and glucagon binding on the extracellular side, and stabilize the GCGR in the active state. We expect that this study can provide useful information on the activation mechanism of the GCGR and facilitate the future design of GCGR inhibitors.