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. 2019 May 15;9(1):7428.
doi: 10.1038/s41598-019-42875-7.

Third BIR domain of XIAP binds to both Cu(II) and Cu(I) in multiple sites and with diverse affinities characterized at atomic resolution

Shen-Na Chen  1 , Tian Fang  1 , Jing-Yang Kong  1 , Bin-Bin Pan  1 , Xun-Cheng Su  2
Affiliations

Third BIR domain of XIAP binds to both Cu(II) and Cu(I) in multiple sites and with diverse affinities characterized at atomic resolution

Shen-Na Chen et al. Sci Rep. .

Abstract

The X-chromosome linked inhibitor of apoptosis, XIAP, is mainly known as the inhibitor of caspases by direct interaction with caspases with its baculoviral IAP repeat (BIR) domains. XIAP has three BIR domains and each BIR domain contains a zinc binding site, normally known as zinc finger motif. Recent studies showed that XIAP is involved in copper homeostasis in cells and the BIR domains bind copper ion. However, structural details of the second and third BIR domain, BIR2 and BIR3, in XIAP, with copper as well as the binding modes are not known. In the present work we characterize the structural properties of BIR3 in solution by high resolution NMR and other biophysical techniques. The interaction of BIR3 with copper both in vitro and in cell lysates was analyzed. Our results show that BIR3 is able to form stable complexes both with Cu(II) and Cu(I), whereas zinc binding site is not affected and zinc retains tightly bound in the zinc finger during these interactions. Surprisingly, BIR3 has multiple binding sites for Cu(II) and Cu(I) but with varied binding affinities. In addition, the solvent exposed Cys351 is readily oxidized by Cu(II) resulting an intermolecular disulfide bond either between two BIR3 molecules or a mixed disulfide bond with glutathione in cell lysates.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Amino acid sequence of XIAP. The individual domains containing BIR domain, UBA domain and RING domain, are marked and each domain is separated by vertical blue lines. Cys and His in zinc fingers are colored yellow, and other Cys in blue and His in magenta, respectively.
Figure 2
Figure 2
15N-HSQC spectra of BIR3 in solution. The NMR spectrum was recorded for 0.1 mM wild type BIR3 (241–356) in 20 mM Bis-Tris buffer at pH 6.5 and 298 K with a proton frequency of 600 MHz. The cross-peaks with assignment were labelled.
Figure 3
Figure 3
Interaction of BIR3 with Cu(II) analyzed by SEC and MALDI-TOF spectrometry. (A) Results of SEC experiments recorded for the mixture of wild type BIR3 before and after addition of Cu(II): 0.1 mM BIR3 (black); mixture of 0.1 mM BIR3 and 0.1 mM CuSO4 (red); mixture of 0.1 mM BIR3 and 0.1 mM CuSO4 after treatment with 0.6 mM DTT (blue). (B) Results of SEC experiments recorded for the mixture of BIR3 C351S mutant before and after addition of Cu(II): 0.1 mM BIR3 C351S (back); mixture of 0.1 mM BIR3 C351S and 0.1 mM CuSO4 (red). (C) MALDI-TOF mass spectrometry of the SEC fraction recorded for the reaction mixture of BIR3 and CuSO4. Top: free BIR3 as reference; middle: fraction with larger molecular weight (first fraction in A); bottom: fraction with similar weight of BIR3 (second fraction in A). (D) SDS-PAGE results run for the different protein samples from left to right lane. Lane 1: molecular marker; 2: free BIR3; 3: BIR3 treated with Cu(II) (also in Fig. S3); 4: fraction with large molecular weight from SEC experiment for the reaction mixture of BIR3 and Cu(II).
Figure 4
Figure 4
Superimposition of 15N-HSQC spectra recorded for 15N-labeled Cys BIR3 H302A/H343A/H346A in the absence (blue) and presence of Cu(II) or Cu(II) and GSH (red) in vitro and in E. coli lysates. (A) NMR spectra were recorded in 20 mM Bis-Tris buffer, pH 6.5. From left to right: 0.1 mM BIR3 and 0.1 mM CuSO4 (red), 0.8 mM GSH was added into the mixture of 0.1 mM BIR3 and 0.1 mM CuSO4 and the reaction mixture was incubated for 24 h (red). (B) NMR spectra were recorded in E. coli cell lysates. From left to right: 0.1 mM BIR3 and 0.6 mM CuSO4 (red); 0.1 mM BIR3 and 1.5 mM CuSO4 (red). It was noted that a new cross-peak of C351 was produced that was due to the disulfide bond formation between BIR3 C351 and GSH.
Figure 5
Figure 5
Interaction of BIR3 and its mutant with Cu(II) evaluated by 15N-HSQC spectra. (A–C) Superimposition of 15N-HSQC spectra recorded for 0.1 mM BIR3 protein before (blue) and after addition of 0.1 mM CuSO4 (red). (A) BIR3 C351S; (B) H346A/C351S; (C) H343A/H346A/C351S. (D) Plot of cross-peak attenuation in the 15N-HSQC of BIR3 mutant after addition of Cu(II) as shown in (A–C), I/I0, with the function of amino acid sequence, where I and I0 are the cross-peak intensities recorded for BIR3 mutant after and before addition of Cu(II), respectively. (E) Structural comparison of solution NMR structure colored in grey (PDB code: 1G3F) and crystal structure colored in cyan (PDB code: 3HL5), of which the backbone Cα atoms were labeled with red spheres for the residues I/I0 < 0.5. The NMR spectra were recorded in 20 mM Bis-Tris buffer, pH 6.5, at 298 K.
Figure 6
Figure 6
Interaction of 0.5 mM BIR3 or its C351S mutant with Cu(I). (A) SEC experiment performed for the reaction mixture of free BIR3 (black), BIR3 and 1 eq. Cu(I) (red), the mixture of BIR3 and 1 eq. Cu(I) treated with 4 eq. BCS (magenta), BIR3 and 1 eq. Cu(II)-VC (blue), respectively. (B) SEC experiment performed for the reaction mixture of free BIR3 C351S mutant (black), BIR3 C351S and 1 eq. Cu(I) (red), the mixture of BIR3 C351S and 1 eq. Cu(II)-VC (blue). (C) SDS-PAGE results run for the fractions from the SEC experiment of 0.5 mM BIR3 and 0.5 mM [Cu(CH3CN)4][PF6] from the left to right (also in Fig. S3): Lanes 1–4 lanes are the fractions of i to iv, respectively; 5: free BIR3; 6: mixture of BIR3 and 1 eq. Cu(I); 7: molecular weight marker.
Figure 7
Figure 7
Interaction of BIR3 mutant with Cu(I) by 15N-HSQC experiment. Superimposition of 15N-HSQC spectra recorded for 0.15 mM BIR3 mutant in the absence (red) and presence of 1 eq [Cu(CH3CN)4][PF6](blue) in 20 mM Bis-Tris buffer, pH 6.5. (A) BIR3 C351S; (B) BIR3 H343A/H346A/C351S; (C) H302A/C351S. (D) Plot of cross-peak intensity ratio of I/I0 with the function of amino acid, where I and I0 are the cross-peak intensities in 15N-HSQC spectra recorded for BIR3 in the presence and absence of [Cu(CH3CN)4][PF6], respectively, as shown in A to C.
Figure 8
Figure 8
Interaction of BIR3 mutant with Cu(II) by 15N-HSQC experiment. Plot of cross-peak intensity ratio of I/I0 with the function of amino acid sequence, where I and I0 is cross-peak intensity ratio in the 15N-HSQC spectra recorded for 0.1 mM BIR3 mutant in the presence and absence of 1 eq. Cu(II)-VC, respectively.
Figure 9
Figure 9
Structural representative of multiple binding sites in BIR3 for copper ion (PDB code: 3HL5). Zinc ion is labeled as black sphere in the zinc finger motif, and the flexible C351 that can be oxidized by Cu(II) and also binds to Cu(I) is absent in the crystal structure.
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