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

BNP controls early load-dependent regulation of SERCA through calcineurin

Heart failure is characterised by reduced expression of sarcoplasmic reticulum calcium-ATPase (SERCA) and increased expression of B-type natriuretic peptide (BNP). The present study was performed to investigate causality of this inverse relationship under in vivo conditions in the transversal aortic constriction mouse model (TAC). Left ventricular SERCA-mRNA expression was significantly upregulated in TAC by 32% after 6 h, but not different from sham after 24 h. Serum proANP and BNP levels were increased in TAC after 24 h (BNP +274%, p < 0.01; proANP +60%, p < 0.05), but only proANP levels were increased after 6 h (+182%, p < 0.01). cGMP levels were only increased 24 h after TAC (+307%, p < 0.01), but not 6 h after TAC. BNP infusion inhibited the increase in SERCA expression 6 h after TAC. In BNP-receptor-knockout animals (GC-A), the expression of SERCA was still significantly increased 24 h after TAC at the mRNA level by 35% (p < 0.05), as well as at the protein level by 25% (p < 0.05). MCIP expression as an indicator of calcineurin activity was regulated in parallel to SERCA after 6 and 24 h. MCIP-mRNA was increased by 333% 6 h after TAC, but not significantly different from sham after 24 h. In the GC-A-KO mice, MCIP-mRNA was significantly increased in TAC compared to WT after 24 h. In mice with BNP infusion, MCIP was significantly lower 6 h after TAC compared to control animals. In conclusion, mechanical load leads to an upregulation of SERCA expression. This is followed by upregulation of natriuretic peptides with subsequent suppression of SERCA upregulation. Elevated natriuretic peptides may suppress SERCA expression by inhibition of calcineurin activity via activation of GC-A

Spectroscopically Orthogonal Spin Labels in Structural Biology at Physiological Temperatures

Electron paramagnetic resonance spectroscopy (EPR) is mostly used in structural biology in conjunction with pulsed dipolar spectroscopy (PDS) methods to monitor interspin distances in biomacromolecules at cryogenic temperatures both in vitro and in cells. In this context, spectroscopically orthogonal spin labels were shown to increase the information content that can be gained per sample. Here, we exploit the characteristic properties of gadolinium and nitroxide spin labels at physiological temperatures to study side chain dynamics via continuous wave (cw) EPR at X band, surface water dynamics via Overhauser dynamic nuclear polarization at X band and short-range distances via cw EPR at high fields. The presented approaches further increase the accessible information content on biomolecules tagged with orthogonal labels providing insights into molecular interactions and dynamic equilibria that are only revealed under physiological conditions