No abstract

The β2-adrenergic receptor (β2AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein Gs and stimulates cAMP formation. Functional studies have shown that the β2AR also couples to inhibitory G protein Gi, activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE 2001, re15 (2001)]. A crystal structure of the β2AR-Gs complex revealed the interaction interface of β2AR-Gs and structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549–555 (2011)], yet, the dynamic process of the β2AR signaling through Gs and its preferential coupling to Gs over Gi is still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the β2AR in response to the full agonist BI-167107 and Gs and Gi1. These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the β2AR are qualitatively the same for Gs and Gi1, we detected distinct differences between the β2AR-Gs and the β2AR-Gi1 complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of Gs. These differences between the β2AR-Gs and β2AR-Gi1 complexes in ICL2 may be key determinants for G protein coupling selectivity.

In this study, we have used density functional theory to identify a new mechanism for the formation of carboxylate compounds from heteroarenes, such as benzoxazole, in the presence of copper catalysts. This new mechanism involves the formation of a carbene intermediate that is indirectly stabilized by the electron-releasing copper. This intermediate carbene can isomerize to the experimentally observed resting state of the catalytic cycle, but it is the intermediate carbene itself that has the greater reactivity toward CO 2 and that leads to the final carboxylate product via a lower-energy pathway. Our findings demonstrate the importance of considering metal-stabilized carbenes in such reactions. Our findings also suggest that this carbene intermediate can act as a nucleophile in other organometallic reactions.

Computation of phosphodiester hydrolysis in different models with one or two metal ions, representing typical active site architectures of nucleases, reveal an associative mechanism to be favorable in all of the cases studied in this work. Direct attack of the nucleophilic water molecule with proton transfer to the phosphate group is facilitated by an extra positive charge as provided by a metal ion located at the attack site or a positively charged histidine residue whereas no such contribution can be observed on leaving group departure. A major catalytic effect is found by proton transfer from the nucleophilic water molecule to a histidine-aspartate cluster. Attack of the thus generated hydroxide ion on the phosphate group is just sufficiently stabilized by the metal ions to allow subsequent P-O bond dissociation.

Apurinic/apyrimidinic endonuclease 1 (Ape1) is an important metal-dependent enzyme in the base excision repair mechanism, responsible for the backbone cleavage of abasic DNA through a phosphate hydrolysis reaction. Molecular dynamics simulations of Ape1 complexed to its substrate DNA performed for models containing 1 or 2 Mg -ions as cofactor located at different positions show a complex with 1 metal ion bound on the leaving group site of the scissile phosphate to be the most likely reaction-competent conformation. Active-site residue His309 is found to be protonated based on pKa calculations and the higher conformational stability of the Ape1-DNA substrate complex compared to scenarios with neutral His309. Simulations of the D210N mutant further support the prevalence of protonated His309 and strongly suggest Asp210 as the general base for proton acceptance by a nucleophilic water molecule.