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The Structure of Human EXD2 Reveals a Chimeric 3 to 5 Exonuclease

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The Structure of Human EXD2 Reveals a Chimeric 3 to 5 Exonuclease Nucleic Acids Research, 2019 1 doi: 10.1093/nar/gkz454 The structure of human EXD2 reveals a chimeric 3 to 5 exonuclease domain that discriminates substrates via metal coordination Downloaded from https://academic.oup.com/nar/advance-article-abstract/doi/10.1093/nar/gkz454/5498624 by University of Cambridge user on 03 June 2019 Jumi Park1,7,†, Song-Yi Lee2,3,†, Hanbin Jeong1,7, Myeong-Gyun Kang2, Lindsey Van Haute4, Michal Minczuk4, Jeong Kon Seo5, Youngsoo Jun6,7, Kyungjae Myung1,8, Hyun-Woo Rhee3,* and Changwook Lee 1,7,8,* 1Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, 50 UNIST-gil, Ulsan 44919, Republic of Korea, 2Department of Chemistry, Ulsan National Institute of Science and Technology, 50 UNIST-gil, Ulsan 44919, Republic of Korea, 3Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea, 4MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge CB2 0XY, UK, 5UNIST Central Research Facilities (UCRF), Ulsan National Institute of Science and Technology, 50 UNIST-gil, Ulsan 44919, Republic of Korea, 6School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea, 7Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea and 8Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea Received April 17, 2019; Revised May 07, 2019; Editorial Decision May 09, 2019; Accepted May 10, 2019 ABSTRACT INTRODUCTION EXD2 (3-5 exonuclease domain-containing protein EXD2 (3 -5 exonuclease domain-containing protein 2) was 2) is an essential protein with a conserved DEDDy identified as a new component that plays an essential role superfamily 3-5 exonuclease domain. Recent re- in mediating the repair of DNA double-strand breaks (1). search suggests that EXD2 has two potential func- It was suggested that EXD2 might be required to facili- tions: as a component of the DNA double-strand tate DNA end resection during homologous recombination by functionally interacting with the MRE11-RAD50-NBS1 break repair machinery and as a ribonuclease for (MRN) complex. Primary structure analysis revealed a 3 to the regulation of mitochondrial translation. Herein, 5 exonuclease domain belonging to the DnaQ (DEDDy) electron microscope imaging analysis and proxim- family sharing high sequence similarity with the exonucle- ity labeling revealed that EXD2 is anchored to the ase domain of Werner syndrome protein (WRN), an en- mitochondrial outer membrane through a conserved zyme involved in maintaining genome stability, and recom- N-terminal transmembrane domain, while the C- binant EXD2 exhibits 3 to 5 exonuclease activity against terminal region is cytosolic. Crystal structures of the DNA in vitro (1). exonuclease domain in complex with Mn2+/Mg2+ re- However, in contrast to previous results, database analy- vealed a domain-swapped dimer in which the central sis using The Human Protein Atlas (www.proteinatlas.org) ␣5−␣7 helices are mutually crossed over, resulting (2), a public resource of immunofluorescence images for the in chimeric active sites. Additionally, the C-terminal human proteome, raised the possibility that EXD2 might be localized to mitochondria in various mammalian cell segments absent in other DnaQ family exonucleases lines such as A-431, U-2 OS and U-251 MG. Supporting enclose the central chimeric active sites. Combined this possibility, Hensen et al. recently reported that EXD2 structural and biochemical analyses demonstrated is predominantly associated with the mitochondrial outer that the unusual dimeric organization stabilizes the membrane (3). Meanwhile, Stracker and colleagues sug- active site, facilitates discrimination between DNA gested that EXD2 is localized to the inner mitochondrial and RNA substrates based on divalent cation coor- membrane (IMM) or matrix (4). According to the report, dination and generates a positively charged groove EXD2 is a mitochondrial ribonuclease that plays impor- that binds substrates. tant roles in mitochondrial functions such as respiration, *To whom correspondence should be addressed. Tel: +82 52 217 2534; Fax: +82 52 217 2639; Email: [email protected] Correspondence may also be addressed to Hyun-Woo Rhee. Email: [email protected] †The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Nucleic Acids Research, 2019 ATP production and mitochondrial translation. The sub- Fluorescence microscopy imaging location of EXD2 within mitochondria was examined us- At 18−24 h after transfection in HeLa cells, the medium ing proteinase K digestion of purified mitochondria in com- of the EXD2-V5-APEX2-transfected sample was changed bination with electron microscopy (EM) imaging follow- to fresh growth medium containing 500 ␮M biotin-phenol. ing staining with immuno-gold (4). However, such conven- Downloaded from https://academic.oup.com/nar/advance-article-abstract/doi/10.1093/nar/gkz454/5498624 by University of Cambridge user on 03 June 2019 This culture was incubated for 30 min and labeled accord- tional approaches based on protease accessibility of the ing to a previously published protocol (10). Next, H O sub-mitochondrial proteome in purified mitochondria can 2 2 was added to a final concentration of 1 mM, and the plate lead to mis-interpretation if the target protein is unexpect- was gently agitated for 1 min at room temperature. The re- edly resistant to proteases, as demonstrated for NDUFB10 action was quenched by washing twice with Quencher so- (5). Furthermore, immuno-gold EM imaging requires cel- lution consisting of Dulbecco’s phosphate-buffered saline lular permeabilization, which occasionally degrades cellu- (DPBS) containing 5 mM Trolox, 10 mM sodium azide lar ultrastructure (6), and treatment with immuno-gold may and 10 mM sodium ascorbate. The biotin labeling step was cause unintended extraction or relocalization of proteins omitted for Non-APEX2 samples. Biotin-labeled and non- (7). labeled cells were fixed with 4% paraformaldehyde. Cells Interestingly, EXD2 exhibited exonuclease substrate dis- were then washed with DPBS and permeabilized with cold crimination activity depending on the metal cofactors coor- ◦ methanol at −20 C for 5 min. Cells were washed again with dinated (4). In the presence of MnCl , EXD2 displays com- 2 DPBS and blocked for 1 h with blocking solution consisting parable activity toward DNA and RNA substrates, but ac- of 2% bovine serum albumin (BSA) in TRIS-buffered saline tivity toward DNA substrates is significantly reduced when with Tween 20 (TBST) at room temperature. MgCl is used as a cofactor. This is an interesting result be- 2 To detect APEX2-fusion expression, cells were incubated cause substrate discrimination via divalent cations bound with both mouse anti-V5 antibody (Invitrogen, cat. no. to exonucleases is a novel feature shown only for EXD2. R960-25, 1:5000 dilution) and rabbit anti-TOM20 (Santa Even in WRN exonuclease, which shares high sequence sim- Cruz, cat. no. sc-11415, 1:200 dilution) as a mitochondrial ilarity with EXD2, metal cofactor-driven substrate discrim- marker for 1 h at room temperature. To detect Flag-tagged ination has not been observed (8,9). Moreover, given that EXD2 expression, cells were incubated with both mouse EXD2 acts as a ribonuclease in mitochondria rather than anti-Flag antibody (Sigma Aldrich, cat. no. F1804, 1:3000 the nucleus, this could be advantageous and allow EXD2 to dilution) and rabbit anti-TOM20 for 1 h at room tem- modulate its activity against DNA or RNA (4). However, perature. After washing with TBST, cells were simultane- it remains unknown exactly how the exonuclease activity ously incubated with secondary Alexa Fluor 568 goat anti- of EXD2 toward DNA substrates is specifically limited in mouse IgG (Invitrogen, cat. no. A-11004, 1:1000 dilution) the presence of the Mg2+ cation, considered the likely phys- and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, cat. iological metal ion within cells. Therefore, molecular-level no. A-11008, 1:1000 dilution) for 30 min at room tempera- studies on EXD2 are needed to explore these distinctive fea- ture. SA-Alexa Fluor 647 IgG (Invitrogen, cat. no. S32357, tures. 1:1000 dilution) was incubated with biotin-labeled samples In the present study, we demonstrated that EXD2 is an in dialyzed blocking solution. Cells were then washed with outer mitochondrial membrane (OMM) protein anchored TBST and maintained in DPBS on ice for imaging using an to the membrane through the N-terminal transmembrane FV1000SPD instrument (Olympus) at the UNIST Optical (TM) domain, while most of the functional region, includ- Biomed Imaging Center (UOBC) in the Ulsan National In- ing the exonuclease domain, faces the cytosol. Furthermore, stitute of Science and Technology (UNIST), Korea. we determined crystal structures of the exonuclease domain of EXD2 in complex with Mn2+/Mg2+ and (d)GMP,reveal- ing a novel molecular mechanism by which EXD2 can
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