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

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.

The X-chromosome linked inhibitor of apoptosis (XIAP) is a direct inhibitor of caspases and has been regarded as a potential target for therapy of cancer1,2. XIAP is a 497-residue

cytoplasmic zinc-binding protein containing three baculoviral IAP repeats (BIR) domains at the N-terminal region, followed by an ubiquitin-associated domain (UBA) and a Really Interesting

New Gene (RING) domain at the C-terminus. Each BIR domain contains a CCHC zinc binding motif, while the RING domain contains a CCCHCCCC motif that binds two zinc ions. XIAP was first

recognized as an inhibitor of apoptosis due to its specific interactions with caspases 3 and 7, mediated by BIR2 domain, and caspase 9, mediated by BIR33,4,5,6. BIR1 was found to be involved

in the interaction with TAB1 in the NF-κB pathway7. Recently, additional roles of XIAP in receptor signaling, cell division, ubiquitin ligation and copper homeostasis have been reported8.

In human cells, the concentration of copper is tightly controlled by copper chaperones and imbalances of copper concentration results in diseases like Menkes or Wilson’s disease9. Recent

studies have shown that XIAP is involved in cellular copper homeostasis and the direct interaction of XIAP with copper ions was reported10,11,12. It was also suggested that XIAP binds copper

via the coordination of cysteines and it undergoes conformation changes upon copper binding11, resulting in decreased stability of XIAP. A later study showed that several cysteine residues

in the BIR2 and BIR3 domains can coordinate copper(I), both at the zinc binding site and at additional surface sites12. Compared with BIR2 and BIR3, BIR1 was less studied, even though it has

been reported by X-ray crystallography that BIR1 forms dimeric complex either in its free state or complexed with TAB17. BIR1 forms a stable homodimer in the crystal structure, in which the

BIR1 monomers are held together by electrostatic and hydrophobic interactions. However, the structural characterization of BIR1 in solution has not been reported and the function of the

N-terminal residues (1–19), which were not observed in the crystal structure, has not been elucidated.

In the present study, the structural properties of BIR1 were characterized using high-resolution NMR spectroscopy in vitro and in different physiological environments, namely in the

cytoplasm of E. coli, X. laevis oocytes and cultured human cells. The effect of copper addition in both oxidation and reduced states on BIR1 was then characterized. Wild-type BIR1 exists as

a stable homodimer in solution, in line with the existing X-ray data. The dimerization can be impaired by two point mutations at the dimer interface, D71N/R72E, resulting in a well-folded

monomeric protein. In-cell NMR experiments show that BIR1 interacts with cellular constituents in the cytoplasm of different cells, causing the loss of NMR signals from the well-structured

residues. The N-terminal residues 1–19, which are absent in the crystal structure, are unstructured. No interaction between BIR1 and copper(I) was observed both in vitro and in-cell.

Remarkably cysteine 12, which resides in a well conserved TCVP motif in the unstructured N-terminal tail among XIAP homologues, presents a very low redox potential (~−300 mV), and is found

to react with copper(II) leading to the formation of either a disulfide-linked protein dimer or an adduct with glutathione in vitro and in-cell lysate.

The apo form of BIR1 (i.e. BIR1 lacking the zinc ion) is unfolded in solution as determined by 15N-HSQC spectra since all the cross-peaks show narrow dispersions and sharp linewidths, and

hence in the present study all the protein samples were prepared by denature-refolding process in the presence of zinc ion (see experimental section). Wild-type BIR1 (WT BIR1) shows well

dispersed resonances in the 15N-HSQC spectra, which are characteristic of a well-folded protein but characterized by quite large linewidths (Fig. 1A). 15N heteronuclear relaxation rates R1

and R2 of backbone amide nitrogen indicated different behaviors for various stretches of the protein, with the N- and C-termini characterized by high R1 and low R2, indicative of fast

motions on the ns-ps time scale and the central part with the opposite pattern (Figure S1). Overall, the linewidths of most cross-peaks are larger than those expected from a monomeric

polypeptide chain, suggesting that the protein exists as an oligomer or experiences conformation exchange in solution. The averaged R2/R1 ratios in the well-structured segments indicated a

rotational correlation time (τc) of 17.5 ns, consistent with a dimeric protein of 23 kDa. The large R2 values hampered the spectral assignment procedure based on standard triple resonance

experiments. Therefore, we sought to design a mutant that would be monomeric in solution, in order to facilitate the NMR assignment. We then transferred the assignment of the mutant to the

15N-HSQC spectra of WT BIR1.

In the crystal structures of homodimeric BIR113, D71 and R72 of one subunit form salt bridges with R82 and D77, respectively, of the other subunit. Additional hydrogen bonds are also present

between T60-K85 and D71-K85. As the salt bridges involving D71 and R72 likely play an essential role in stabilizing the dimeric complex, the double-point mutant D71N/R72E was investigated.

Compared to WT BIR1, D71N/R72E BIR1 gave rise to significantly narrower NMR signals (Fig. 1B), which permitted the assignment of most backbone resonances with the triple resonance NMR

spectra HNCA, CBCA(CO)NH and NOESY-15N-HSQC. The cross-peaks in 15N-HSQC spectrum corresponding to residues of T2, F3, E20, F23, V24 and G56 were not assigned due to line broadening effects

(Fig. 1). The 15N heteronuclear relaxation values R1 and R2 of backbone amide nitrogens have a similar pattern to those of WT BIR1 over the various sequence segments. The R2/R1 ratio gives a

τc of 7.3 ns, consistent with the monomeric state.

NOESY-15N-HSQC spectra recorded for WT BIR1 and D71N/R72E indicated that both two protein constructs share similar secondary structural elements to the dimeric structure as determined by

X-ray crystallography.13 The backbone assignment of D71N/R72E BIR1 was therefore transferred to WT BIR1 and cross-checked with 3D HNCA, CBCA(CO)NH and 15N-NOESY-HSQC spectra of WT BIR1.

Residues E22, E25, A69, E72, Y75, G76, D77, K85, V86, F92, I93 and N94 could not be identified in the 15N-HSQC spectrum of WT BIR1, most likely due to line broadening effects.

Chemical shift differences between the two forms were observed, as expected, for the residues vicinal to the homo-dimeric interface (Fig. 2A). Most unassigned residues in WT BIR1 are located

at the dimer interface (Fig. 2B), suggesting that the dimeric BIR1 complex in solution experiences conformational exchange that broadens a number of residues in the 15N-HSQC spectra. The

N-terminal residues 1–19, which are not observed in the X-ray structure7,13, generally produce narrower cross-peaks in the 15N-HSQC spectrum. Interestingly, the N-terminal residue T11 and

C-terminal residues F96 and Y97 also show significant chemical shift difference between the dimeric and monomeric forms, suggesting that a dynamic contact between these residues may occur in

the dimeric form in solution. The secondary structure prediction based on the backbone chemical shifts, obtained using TALOS+14, suggested that the stretches containing residues 3–16 and

97–105 are unstructured.

Chemical shift perturbations of D71N/R72E mutant on BIR1. (A) Plot of chemical shift perturbations caused by D71N/R72E mutation on BIR1 with the function of amino acid sequence. The

chemical-shift differences between WT and D71N/R72E BIR1 were calculated as δ = ((ΔδH)2 + (ΔδN/10)2)1/2. (B) Chemical shift differences between WT BIR1 and D71N/R72E mutant plotted on the

dimeric structure of BIR1 (PDB code: 2QRA)13, of which the Cα atoms are shown in red spheres with δ > 0.2 ppm, yellow spheres with 0.1