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

Meng-Meng Hou,Jia-Liang Chen,Xiao Wang,Enrico Luchinat,Shen-Na Chen,Panagis Polykretis,Yansheng Ye,Hui-Hui Zuo,Xun-Cheng Su,Conggang Li,Yin Yang,Lucia Banci

doi:10.1038/s41598-017-16723-5

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.

Highlights

Backbone 15N-relaxation data of wild type BIR1 and D71N/R72E mutant were recorded at 298 K using standard experiments[24]
In this study, using high-resolution NMR spectroscopy, we show that the BIR1 domain of human XIAP forms a homodimer in solution, in accordance with the X-ray crystal structure
A TCVP motif was identified in the N-terminal unstructured segment, which is conserved among XIAP orthologues and likely plays an important role in the interaction with copper

Summary

Introduction

Backbone 15N-relaxation data of wild type BIR1 and D71N/R72E mutant were recorded at 298 K using standard experiments[24]. The triple-resonance NMR experiments were performed on protein samples at concentration of 1 mM 15N,13C-BIR1 (both for WT BIR1 and D71N/R72E mutant) in 20 mM Bis-Tris buffer at pH 6.5. The solution of 0.3 mM BIR1 (wild type or D71N/R72E) was dialyzed in 20 mM Tris buffer at room temperature, at pH 8.0, for 12 hours and the oxidation process was monitored by 15N-HSQC spectra.

Results

Conclusion