Solution structure and interaction with copper in vitro and in living cells of the first BIR domain of XIAP

Abstract

The X-chromosome linked inhibitor of apoptosis (XIAP) is a multidomain metalloprotein involved in caspase inhibition and in copper homeostasis. It contains three zinc-binding baculoviral IAP repeats (BIR) domains, which are responsible for caspase interaction. Recently, it has been suggested that the BIR domains can bind copper, however high resolution data on such interaction is missing. Here we characterize by NMR the structural properties of BIR1 in solution, and the effects of its interaction with copper both in vitro and in physiological environments. BIR1 is dimeric in solution, consistent with the X-ray structure. Cysteine 12, located in the unfolded N-terminal region, has a remarkably low redox potential, and is prone to oxidation even in reducing physiological environments. Interaction of BIR1 with copper(II) results in the oxidation of cysteine 12, with the formation of either an intermolecular disulfide bond between two BIR1 molecules or a mixed disulfide bond with glutathione, whereas the zinc binding site is not affected by the interaction.

In-cell destabilization of a homo-dimeric protein complex detected by DEER spectroscopy

“…Increasing the salt concentration in solution, to account for the in-cell ionic strength, did reduce the dimer stability. This is expected owing to screening effects that destabilize the salt bridge (96) holding the BIR1 dimer together (67). We note, however, that the effect of salt addition was not as strong as observed in the cell.…”

“…In addition to C12, BIR1 harbors three additional cysteine residues, C63, C66, and C90, that constitute a zinc binding site, which are critical for the stability of the BIR1 domain fold. Zinc removal in the presence of excess EDTA results in unfolding (67). Mass spectrometry confirmed that each protein construct was labeled at the expected, single site ( Fig.…”

“…Residues 1-19 of Nterminal BIR1 dimers are not observed in the X-ray structure (68, 69) ( Fig. 1a), and they are highly flexible as determined by NMR (67). The main interactions between BIR1 monomers are mediated by a salt bridge between D71 in one monomer and R72 in the other; breaking this salt bridge produces the monomeric protein in solution without major structural changes in domain architecture (67).…”

“…It harbors three zinc-binding BIR domains (BIR1-BIR3), the second and third of which are responsible for interactions with caspase (63)(64)(65)(66). The structures of the three BIR domains have been determined by NMR spectroscopy and X-ray crystallography (63)(64)(65)(66)(67)(68)(69) and BIR1 has been found to exist as a stable homodimer in vitro (63,65,66). Furthermore, interaction of BIR1 dimer with the TAK1-binding protein TAB1 was reported to be essential for activation of the NF-κB signaling pathway (69).…”

“…1a), and they are highly flexible as determined by NMR (67). The main interactions between BIR1 monomers are mediated by a salt bridge between D71 in one monomer and R72 in the other; breaking this salt bridge produces the monomeric protein in solution without major structural changes in domain architecture (67). In a previous study, we reported that in-cell NMR spectra of 15 N-labeled BIR1 either overexpressed in E. coli, injected into Xenopus laevis oocytes, or transduced into HKT293 cells showed no observable NMR signals (67), suggesting that cellular interactions broadened BIR1 resonances beyond detection.…”