Pathways shaping the mitochondrial inner membrane

Mitochondria are complex organelles with two membranes. Their architecture is determined by characteristic folds of the inner membrane, termed cristae. Recent studies in yeast and other organisms led to the identification of four major pathways that cooperate to shape cristae membranes. These include dimer formation of the mitochondrial ATP synthase, assembly of the mitochondrial contact site and cristae organizing system (MICOS), inner membrane remodelling by a dynamin-related GTPase (Mgm1/OPA1), and modulation of the mitochondrial lipid composition. In this review, we describe the function of the evolutionarily conserved machineries involved in mitochondrial cristae biogenesis with a focus on yeast and present current models to explain how their coordinated activities establish mitochondrial membrane architecture.

Exploring the signaling space of a GPCR using bivalent ligands with a rigid oligoproline backbone

G protein–coupled receptors (GPCRs) are one of the most important drug–target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated therapeutics. In this work, we have used rigid oligoproline backbones to generate bivalent ligands for the gastrin-releasing peptide receptor (GRPR) with a fixed distance between their recognition motifs. This allows the stabilization of GPCR dimers irrespective of their physiological occurrence and relevance, thus expanding the space for medicinal chemistry. Specifically, we observed that compounds presenting agonists or antagonists at 20- and 30-Å distance induce GRPR dimerization. Furthermore, we found that 1) compounds with two agonists at 20- and 30-Å distance that induce dimer formation show bias toward Gq efficacy, 2) dimers with 20- and 30-Å distance have different potencies toward β-arrestin-1 and β-arrestin-2, and 3) the divalent agonistic ligand with 10-Å distance specifically reduces Gq potency without affecting β-arrestin recruitment, pointing toward an allosteric effect. In summary, we show that rigid oligoproline backbones represent a tool to develop ligands with biased GPCR signaling.

The Molecular Basis for Erns Dimerization in Classical Swine Fever Virus

The pestivirus classical swine fever virus (CSFV) represents one of the most important pathogens of swine. Its virulence is dependent on the RNase activity of the essential structural glycoprotein Erns that uses an amphipathic helix as a membrane anchor and forms homodimers via disulfide bonds employing cysteine 171. Dimerization is not necessary for CSFV viability but for its virulence. Mutant Erns proteins lacking cysteine 171 are still able to interact transiently as shown in crosslink experiments. Deletion analysis did not reveal the presence of a primary sequence-defined contact surface essential for dimerization, but indicated a general importance of an intact ectodomain for efficient establishment of dimers. Pseudoreverted viruses reisolated in earlier experiments from pigs with mutations Cys171Ser/Ser209Cys exhibited partially restored virulence and restoration of the ability to form Erns homodimers. Dimer formation was also observed for experimentally mutated proteins, in which other amino acids at different positions of the membrane anchor region of Erns were replaced by cysteine. However, with one exception of two very closely located residues, the formation of disulfide-linked dimers was only observed for cysteine residues located at the same position of the helix.

Quantum chemical study of NO reduction mechanism on Ag /Al2O3 catalysts

It was shown that N2O content among NO reduction products increases with an increase of the silver concentration in the catalyst because the nature of the catalytic centers changes and leads to a subsequent change in the mechanism of the reaction. Two reaction mechanisms were proposed and studied by means of quantum chemistry: a two-stage mechanism that proceeds via NO dimer formation on catalysts with high (above 2 wt. %) silver concentration and a parallel mechanism with isocyanates involved on catalysts with low (below 2 wt. %) silver concentration. It was demonstrated that on catalysts with high silver concentration mechanism that involves stepwise NO reduction via N2O to N2 is realised. Moreover, the final stage is complicated by the fact that formed intermediates and N2O are likely to desorb from the catalyst surface. In the case of catalysts with low silver concentration, the formation of both products (N2O and N2) proceeds in parallel and the lower activation barriers of the reaction leading to N2, as well as the thermodynamic profitability of its formation, lead to the predominance of the target product. The competition between the proposed mechanisms was studied in the case of catalytic centers represented by silver dimers. It was shown that activation barriers of reaction proceeding via NO dimer formation are lower than the corresponding barriers of the reaction with isocyanates involved, which confirms the prevalent realisation of the first process and the predominance of N2O among the final products. The obtained results explain the experimental data and are significant for further modelling of the mechanism of nitrogen oxides catalytic reduction considering the Al2O3 support.

EndoBind detects endogenous protein-protein interactions in real time

We present two high-throughput compatible methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer formation over an extended duration. Using examples of the Nrf2-KEAP1 and the CRAF-KRAS-G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of endogenous protein-protein interactions in real time.

Free Energy Change during the Formation of Crystalline Contact between Lysozyme Monomers under Different Physical and Chemical Conditions

We use the MM/GBSA method to calculate the free energies of dimer formation by binding two monomers with different combinations of precipitant ions, both embedded in the structure of monomers and in the crystallization solution. It shows that the largest difference in free energy values corresponds to the most accurate dimer model, which considers all precipitant ions in their structure. In addition, it shows that in the absence of precipitant ions in the solution of lysozyme molecules, a monomer is a more energetically favorable state.