Uranyl-cytochrome b₅ interaction regulated by surface mutations and cytochrome c

Uranyl-cytochrome b 5 interaction regulated by surface mutations and cytochrome c 1 first-author 1 1 2 1 2 corresp Corresponding author, linlinying@hotmail.com Corresponding author, linlinying@hotmail.com linlinying@hotmail.com School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China Understanding uranium-protein interaction is important for revealing the mechanism of uranyl ion (UO 2 2+ ) toxicity. In this study, we investigated the interaction between UO 2 2+ and a quadruple mutant of cytochrome b 5 (E44/48/56A/D60A cyt b 5 , namely 4A cyt b 5 ) by spectroscopic approaches. The four mutated negatively-charged surface residues of cyt b 5 have been considered to be the interactive sites with cytochrome c (cyt c ). Also, we studied the interaction between UO 2 2+ and the protein-protein complex of 4A cyt b 5 -cyt c . The results were compared to the interaction between UO 2 2+ and cyt b 5 , and the interaction between cyt c and cyt b 5 -cyt c complex, from previous studies. It was found that the interaction of UO 2 2+ -cyt b 5 , i.e., uranyl ion binding to cyt b 5 surface at Glu37 and Glu43 as previously proposed by molecular modeling, is regulated by both surface mutations of cyt b 5 and its interacting protein partner cyt c . These provide valuable information on metal-protein-protein interactions and clues for understanding the mechanism of uranyl toxicity. Uranium Cytochrome Heme protein Protein-protein interaction Fluorescence I. INTRODUCTION 1 Developing nuclear energy is of social and economic importance, while this arises a major concern over environmental pollution by long-lived radioactive wastes, such as uranyl ion (UO 2 2+ ), the most stable form of uranium under physiological conditions [ 1 1 ]. In addition to its radiation hazard, UO 2 2+ is of high toxicity because it interacts with both DNA [ 2 2 ] and proteins [ 3 3 - 6 6 3-6 ] and disrupts their biological functions. To date, plentiful proteins have been found to be the targets of UO 2 2+ , such as transferrin, ferritin and albumin [ 3 3 - 6 6 3-6 ]. A protein data bank (PDB) survey shows that UO 2 2+ binds to proteins mainly through carboxylic acid groups such as those of aspartate (Asp) and glutamate (Glu), as well as other coordinating amino acids such as histidine (His) and tyrosine (Tyr) [ 7 7 ]. In previous papers [ 8 8 , 9 9 ], we studied the structural and functional consequences of UO 2 2+ binding to cytochrome b 5 (cyt b 5 ), cytochrome c (cyt c ), and the protein-protein complex of cyt b 5 -cyt c , by both experimental and theoretical approaches. Cyt b 5 is a small membrane heme protein, which is characterized by a highly negatively charged surface in presence of a series of acidic residues surrounding the heme group, known as an acidic cluster ( Fig. 1 Fig. 1 ) [ 10 10 ]. It has been shown that the acidic cluster participates in formation of cyt b 5 -cyt c complex [ 11 11 - 17 17 11-17 ], and is crucial in mediating cyt c signaling in apoptosis [ 18 18 ]. With a positive charge, UO 2 2+ has a strong tendency to be absorbed at negatively charged surface of membrane [ 19 19 ], where it has a large possibility of interacting with membrane proteins such as cyt b 5 . In previous study [ 8 8 ], we proposed a UO 2 2+ binding site for cyt b 5 at surface residues of Glu37 and Glu43 ( Fig. 1 Fig. 1 ), based on molecular modeling and dynamics simulation. To further probe the role of acidic cluster in uranyl-cyt b 5 interaction and to reveal the effect of surface mutations of cyt b 5 on UO 2 2+ binding, we herein choose a quadruple mutant of cyt b 5 as a target, in which four acidic residues were replaced with alanine, E44/48/56A/D60A cyt b 5 , namely 4A cyt b 5 . This mutant was designed previously for studying the interface between cyt b 5 and cyt c , where the replaced residues were considered to be interactive sites [ 16 16 ]. It keeps the uranyl-binding site of Glu37 and Glu43 but has a low binding affinity for cyt c , and therefore is an ideal model protein for investigating the regulation effect of cyt c by a comparison with that of cyt b 5 -cyt c complex. II. MATERIALS AND METHODS 1 4A cyt b 5 was expressed and purified as described previously [ 16 16 ]. Horse heart cyt c (Type VI) was purchased from Sigma Chemical Co. Uranyl nitrate and other chemicals were commercial products and of analytical grade. Double distilled water was used throughout the experiments. Fluorescence spectra of 4A cyt b 5 and 4A cyt b 5 -cyt c complex (4 μm in 50 mM Tris.HCl buffer, pH 7.0) with titration of UO 2 2+ up to 10 equivalents, were collected at 25 ℃ on a LS45 fluorescence spectrometer (Perkin Elmer, USA). The excitation wavelength was 295 nm and emission spectra were recorded from 310 nm to 410 nm. The apparent dissociation constant ( K D ) was calculated from double reciprocal plot by fitting to Eq. (1) [ 20 20 ], <math display="block" id="M1"><mrow><mn>1</mn><mo>/</mo><mi>Δ</mi><mi>F</mi><mo>=</mo><mo stretchy="false">(</mo><msub><mi>K</mi><mtext>D</mtext></msub><mo>/</mo><mi>Δ</mi><msub><mi>F</mi><mrow><mtext>inf</mtext></mrow></msub><mo stretchy="false">)</mo><mo stretchy="false">(</mo><mn>1</mn><mo>/</mo><mo stretchy="false">[</mo><msubsup><mrow><mtext>UO</mtext></mrow><mn>2</mn><mrow><msup><mrow/><mrow><mn>2</mn><mo>+</mo></mrow></msup></mrow></msubsup><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>+</mo><mn>1</mn><mo>/</mo><mi>Δ</mi><msub><mi>F</mi><mrow><mtext>inf</mtext></mrow></msub><mn>,</mn></mrow></math> where Δ F is the difference between the maximum fluorescence in the absence and presence of UO 2 2+ and Δ F inf is the fluorescence change for the complete binding of UO 2 2+ . The number of UO 2 2+ binding site was calculated from Δ F of fluorescence spectra using the Hill plot [ 21 21 ], i.e. the yielded slope. <math display="block" id="M2"><mrow><mi>log</mi><mo stretchy="false">[</mo><mi>Δ</mi><mi>F</mi><mo>/</mo><mo stretchy="false">(</mo><mi>Δ</mi><msub><mi>F</mi><mrow><mtext>inf</mtext></mrow></msub><mo>−</mo><mi>Δ</mi><mi>F</mi><mo stretchy="false">)</mo><mo stretchy="false">]</mo><mo>=</mo><mi>s</mi><mi>l</mi><mi>o</mi><mi>p</mi><mi>e</mi><mtext> </mtext><mi>log</mi><mo stretchy="false">[</mo><msubsup><mrow><mtext>UO</mtext></mrow><mn>2</mn><mrow><msup><mrow/><mrow><mn>2</mn><mo>+</mo></mrow></msup></mrow></msubsup><mo stretchy="false">]</mo></mrow></math> Circular dichroism (CD) spectra of 4A cyt b 5 and 4A cyt b 5 -cyt c (20 μm in 50 mM Tris.HCl buffer, pH 7.0), in the absence or presence of 5 equivalents of UO 2 2+ , were collected at 25 ℃ from 300 nm to 600 nm (1.0 cm path length), with a Jasco J720 spectrometer (Japan). Dilution effect of addition of UO 2 2+ was corrected by adding the same volume of buffer solution into the protein solution. III. RESULTS AND DISCUSSION 1 The single tryptophan residue (Trp22) in cyt b 5 ( Fig. 1 Fig. 1 ) serves as a convenient and sensitive reporter for studying the interaction of UO 2 2+ -cyt b 5 by fluorescence spectroscopy. Figure 2(a) Figure 2(a) shows that the fluorescence intensity of 4A cyt b 5 decreases gradually upon titration of UO 2 2+ ions, suggesting a fluorescence quenching as observed for uranyl titration of transferrin, ferritin and albumin [ 3 3 - 6 6 3-6 ]. This observation is similar to that of wild-type (WT) cyt b 5 [ 9 9 ]. When the changes of the maximu m fluorescence were fitted to a double reciprocal plot ( Fig. 2(a) Fig. 2(a) , right up) and a Hill plot ( Fig. 2(a) Fig. 2(a) , right down), its binding affinity ( K D = 40 μm) is 4 times lower than that of cyt b 5 ( Table 1 Table 1 ), though 4A cyt b 5 remains a single binding site of UO 2 2+ (slope = 1.1). It is likely due to a conformational change of residues Glu37 and Glu43 as a result of the four mutations in the acidic cluster, especially for Glu44 close to the proposed uranyl-binding site, as observed in the crystal structure of 4A cyt b 5 (PDB entry 1M2M) [ 16 16 ]. These observations indicate that the negative charges on the acidic cluster of cyt b 5 play important roles in uranyl-protein interaction. 1 1 Uranyl-complex 1 1 K D (μM) 1 1 Refs. 1 1 UO 2 2+ -cyt b 5 1 1 10 1 1 [ 9 9 ] 1 1 UO 2 2+ -4A cyt b 5 1 1 40 1 1 This work 1 1 UO 2 2+ -cyt c 1 1 87 1 1 [ 9 9 ] 1 1 UO 2 2+ -cyt b 5 -cyt c 1 1 30 1 1 [ 9 9 ] 1 1 UO 2 2+ -4A cyt b 5 -cyt c 1 1 15 1 1 This work On the other hand, in titration of 4A cyt b 5 -cyt c complex with UO 2 2+ ( Fig. 2(b) Fig. 2(b) ), the single uranyl-binding site on 4A cyt b 5 surface in the complex has a higher affinity of UO 2 2+ ( K D = 15 μm) than that on the surface of isolated 4A cyt b 5 ( K D = 40 μm). This observation suggests that the conformation of surface residues, Glu37 and Glu43, are tuned to be suitable for uranyl-binding as a result of cyt c interacting with 4A cyt b 5 , though the binding constant decreases from cyt b 5 -cyt c (2.2×10 4 M -1 ) to 4 A cyt b 5 -cyt c (5.5×10 3 M -1 ), as determined by NMR technique in a previous study [ 15 15 ]. In Table 1 Table 1 , the uranyl-binding factor ( K D =15 μm) of 4A cyt b 5 -cyt c complex is two times lower than that of cyt b 5 -cyt c complex (30 μm), suggesting that a different protein-protein interface was adopted for 4 A cyt b 5 -cyt c complex as a result of mutation of four acidic residues on the same heme-exposed edge side of cyt b 5 ( Fig. 1 Fig. 1 ). In an earlier study, Huang and co-workers [ 17 17 ] showed that the charge neutralization of 4 A cyt b 5 greatly increased the relative contribution of the heme propionate and other charged residues surrounding the heme edge to contact with cyt c . Thus, our observations indicate that UO 2 2+ -cyt b 5 interaction is regulated by both surface mutations of cyt b 5 and its partner cyt c . The structural perturbations of 4A cyt b 5 and its complex with cyt c upon uranyl binding were studied by circular dichroism (CD) spectroscopy ( Fig. 3 Fig. 3 ). It shows that UO 2 2+ -4A cyt b 5 complex has a slightly decreased negative Cotton effect at 419 nm, whereas the Cotton effect in 300–350 nm is more positive than that of 4A cyt b 5 ( Fig. 3(a) Fig. 3(a) ), which indicates that, similar to the observation for UO 2 2+ -cyt b 5 [ 9 9 ], UO 2 2+ binding to the surface of 4A cyt b 5 slightly alters the conformation of heme-binding domain and aromatic amino acids such as Phe35 and Trp22 in the protein hydrophobic core. The CD spectra also show that upon uranyl binding, the 4A cyt b 5 -cyt c complex has an altered positive Cotton effect and a decreased negative Cotton effect compared to that in the absence of UO 2 2+ ions, with decreased intensity in 300–350 nm ( Fig. 3(b) Fig. 3(b) ). These spectral changes are similar to the case of UO 2 2+ binding to cyt b 5 -cyt c complex, whereas both cases are less obvious than that for UO 2 2+ binding to cyt c [ 9 9 ]. These observations suggest that UO 2 2+ ions alter the heme active site in both 4A cyt b 5 -cyt c and cyt b 5 -cyt c complexes, which are regulated by cyt c , likely through forming dynamic protein-protein complexes and competing with UO 2 2+ ions. IV. CONCLUSION 1 In summary, we investigated the interactions of UO 2 2+ -4A cyt b 5 and UO 2 2+ -4A cyt b 5 -cyt c complex, and compared to that of UO 2 2+ -cyt b 5 , UO 2 2+ -cyt c and UO 2 2+ -cyt b 5 -cyt c from our previous studies [ 8 8 , 9 9 ]. With four acidic residues on protein surface replaced by alanine, 4A cyt b 5 exhibits a lower affinity for UO 2 2+ compared to that for WT cyt b 5 , though it remains the proposed uranyl-binding site at Glu37 and Glu43, which suggests a key role of the acidic cluster of cyt b 5 in tuning uranyl-protein interaction. On the other hand, UO 2 2+ binds more tightly to 4A cyt b 5 -cyt c complex than to the cyt b 5 -cyt c complex, presumably due to the conformational changes of Glu37 and Glu43 by a protein-protein interface different from that of cyt b 5 -cyt c complex. These observations indicate that UO 2 2+ -cyt b 5 interaction is regulated by both the surface acidic cluster of cyt b 5 and its interacting protein partner cyt c , which provides valuable information on metal-protein-protein interactions, and clues for understanding the mechanism of uranyl toxicity. References A E V Gorden , J Xu , K N Raymond , et al . 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DOI: 10.1042/bj2790245 http://doi.org/10.1042/bj2790245 Spectroscopic and binding studies on the stereoselective interaction of tyrosine with horseradish peroxidase and lactoperoxidase We gratefully thank Prof. HUANG Zhong-Xian at Fudan University, Shanghai, China, for providing cytochrome b 5 gene, Prof. TAN Xiang-Shi and Dr. LI Wei at Fudan University, Shanghai, China, for collecting CD spectra. 10.13538/j.1001-8042/nst.26.050303 Supported by National Natural Science Foundation of China (Nos. 21101091 and 11275090) 2014-12-05 2015-01-19 2015-10-20 2015-10-20 2021-04-11 2015 Nuclear Science and Techniques 5 26