Biophysical Characterization of Pro-apoptotic BimBH3 Peptides Reveals an Unexpected Capacity for Self-Association

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome.This work was funded by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC-2033—Projektnummer 390677874, the DFG Priority Program SPP1601 “New Frontiers in Sensitivity in EPR Spectroscopy” (to E.B.), DFG BO 3000/5-1 (to E.B.), SFB958 – Z04 (to E.B.), DFG grant INST 130/972-1 FUGG (to E.B.). P.E.C. is supported by an Australian NHMRC fellowship (1079700

Bax Crystal Structures Reveal How BH3 Domains Activate Bax and Nucleate Its Oligomerization to Induce Apoptosis

SummaryIn stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2–α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other’s surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis

Silver coordination polymers with isonicotinic acid derived short polyethylene glycol – Synthesis, structures, anion effect and solution behavior

Silver coordination compounds and one-dimensional polymers have been studied, using a linear, flexible N-donor ligand. The solid state structures are well known and show panoply of different structures including a large number of polymorphism, pseudo-polymorphism and isomers. We have studied these structures under the influence of anions, solvents and crystallization conditions, studying also some solution effects. We here present an overview with highlights of some case studies of the synthesis, structures, anion and solvent effects as well as solution behavior during formation of silver coordination polymers. Finally, we will give an outlook on the potential applications of these materials

On the coordination behaviour of NO₃⁻ in coordination compounds with Ag⁺Part 1. Solubility effect on the formation of coordination polymer networks between AgNO₃ and <strong>L</strong>(<strong>L</strong>= ethanediyl bis(isonicotinate) as a function of solvent

The influence of the solubility of AgNO₃ in three solvent systems is studied for the reaction between AgNO₃ and the ligand L(= ethanediyl bis(isonicotinate)). Three solid state structures are obtained, differing in the relative ratio Ag : L in the first case, and in polymorphism in the second. The Ag–O(NO₃⁻) distance correlates strongly with the solubility of AgNO₃ in the used solvent. Solution studies prove indeed the existence of close ion contact pairs in the less good solvents, where as ion solvation is observed in good solvents for AgNO₃. The three different structures are compared to two solvated structures in which H₂O demonstrates coordination to the nitrate anion via H-bonding

From simple rings to one-dimensional channels with calix[8]arenes, water clusters, and alkali metal ions

The macrocycle 4-tert-butylcalix[8]arene (L) was reacted with alkali metal carbonates (Li₂CO₃, Na₂CO₃, K₂CO₃, Rb₂CO₃, and Cs₂CO₃) at the interface of a biphasic THF/water system. Needle-like crystals with a general formula [Ax(4-tert-butylcalix[8]arene-xH)(THF)y(H₂O)z] (with A=Li, Na, K, Rb, Cs, x=1, 2, y=4, 5, 8, and z=6, 7) were thereby obtained. The solid state structures were investigated by X-ray diffraction of single crystals and by TGA measurements. They do not appear to be maintained in solution

The many faces of aspartate kinases.

International audienceBased on recent X-ray structures and biochemical characterizations of aspartate kinases from different species, we show in this review how various organizations of a regulatory domain have contributed to the different mechanisms of control observed in aspartate kinases allowing simple to complex allosteric controls in branched pathways. The aim of this review is to show the relationships between domain organization, effector binding sites, mechanism of inhibition and regulatory function of an allosteric enzyme in a biosynthetic pathway

A new mode of dimerization of allosteric enzymes with ACT domains revealed by the crystal structure of the aspartate kinase from Cyanobacteria.

International audienceAspartate kinases (AKs) can be divided in two subhomology divisions, AKalpha and AKbeta, depending on the presence of an extra sequence of about 60 amino acids, which is found only in the N-terminus of all AKalpha's. To date, the structures of AKalpha failed to provide a role for this additional N-terminal sequence. In this study, the structure of the AKbeta from the Cyanobacteria Synechocystis reveals that this supplementary sequence is linked to the dimerization mode of AKs. Its absence in AKbeta leads to the dimerization by the catalytic domain instead of involving the ACT domains [Pfam 01842; small regulatory domains initially found in AK, chorismate mutase and TyrA (prephenate dehydrogenase)] as observed in AKalpha. Thus, the structural analysis of the Synechocystis AKbeta revealed a dimer with a novel architecture. The four ACT domains of each monomer interact together and do not make any contact with those of the second monomer. The enzyme is inhibited synergistically by threonine and lysine with the binding of threonine first. The interaction between ACT1 and ACT4 or between ACT2 and ACT3 generates a threonine binding site and a lysine binding site at each interface, making a total of eight regulatory sites per dimer and allowing a fine-tuning of the AK activity by the end products, threonine and lysine