Structure and function of the N‐terminal domain of the human mitochondrial calcium uniporter
2015; Springer Nature; Volume: 16; Issue: 10 Linguagem: Inglês
10.15252/embr.201540436
ISSN1469-3178
AutoresYoungjin Lee, Choon Kee Min, Tae‐Gyun Kim, Hong Ki Song, Yunki Lim, Dong‐Wook Kim, Kahee Shin, Moonkyung Kang, Jung Youn Kang, Hyung‐Seop Youn, Jung‐Gyu Lee, Jun Yop An, Kyoung Ryoung Park, Jia Jia Lim, Ji Hun Kim, Ji Hye Kim, Zee‐Yong Park, Yeon‐Soo Kim, Jimin Wang, Do Han Kim, Soo Hyun Eom,
Tópico(s)Adipose Tissue and Metabolism
ResumoArticle4 September 2015Open Access Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter Youngjin Lee Youngjin Lee School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Choon Kee Min Choon Kee Min School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Tae Gyun Kim Tae Gyun Kim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Hong Ki Song Hong Ki Song School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Yunki Lim Yunki Lim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Dongwook Kim Dongwook Kim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Kahee Shin Kahee Shin School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Moonkyung Kang Moonkyung Kang Graduate School of New Drug Discovery & Development, Chungnam National University, Daejon, Korea Search for more papers by this author Jung Youn Kang Jung Youn Kang School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Hyung-Seop Youn Hyung-Seop Youn School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Jung-Gyu Lee Jung-Gyu Lee School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Jun Yop An Jun Yop An School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Kyoung Ryoung Park Kyoung Ryoung Park School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Jia Jia Lim Jia Jia Lim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Ji Hun Kim Ji Hun Kim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Ji Hye Kim Ji Hye Kim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Zee Yong Park Zee Yong Park School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Yeon-Soo Kim Yeon-Soo Kim Graduate School of New Drug Discovery & Development, Chungnam National University, Daejon, Korea Search for more papers by this author Jimin Wang Jimin Wang Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Department of Molecular Biochemistry and Biophysics, Yale University, New Haven, CT, USA Search for more papers by this author Do Han Kim Corresponding Author Do Han Kim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Soo Hyun Eom Corresponding Author Soo Hyun Eom School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Department of Chemistry, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Youngjin Lee Youngjin Lee School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Choon Kee Min Choon Kee Min School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Tae Gyun Kim Tae Gyun Kim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Hong Ki Song Hong Ki Song School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Yunki Lim Yunki Lim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Dongwook Kim Dongwook Kim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Kahee Shin Kahee Shin School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Moonkyung Kang Moonkyung Kang Graduate School of New Drug Discovery & Development, Chungnam National University, Daejon, Korea Search for more papers by this author Jung Youn Kang Jung Youn Kang School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Hyung-Seop Youn Hyung-Seop Youn School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Jung-Gyu Lee Jung-Gyu Lee School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Jun Yop An Jun Yop An School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Kyoung Ryoung Park Kyoung Ryoung Park School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Jia Jia Lim Jia Jia Lim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Ji Hun Kim Ji Hun Kim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Ji Hye Kim Ji Hye Kim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Zee Yong Park Zee Yong Park School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Yeon-Soo Kim Yeon-Soo Kim Graduate School of New Drug Discovery & Development, Chungnam National University, Daejon, Korea Search for more papers by this author Jimin Wang Jimin Wang Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Department of Molecular Biochemistry and Biophysics, Yale University, New Haven, CT, USA Search for more papers by this author Do Han Kim Corresponding Author Do Han Kim School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Soo Hyun Eom Corresponding Author Soo Hyun Eom School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Department of Chemistry, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Search for more papers by this author Author Information Youngjin Lee1,2,‡, Choon Kee Min1,3,7,‡, Tae Gyun Kim1,2, Hong Ki Song1,3, Yunki Lim1,3, Dongwook Kim1,3, Kahee Shin1,3, Moonkyung Kang4, Jung Youn Kang1,2, Hyung-Seop Youn1,2, Jung-Gyu Lee1,2, Jun Yop An1,2, Kyoung Ryoung Park1,2, Jia Jia Lim1,2, Ji Hun Kim1,2, Ji Hye Kim1,2, Zee Yong Park1, Yeon-Soo Kim4, Jimin Wang2,5, Do Han Kim 1,3 and Soo Hyun Eom 1,2,6 1School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea 2Steitz Center for Structural Biology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea 3Systems Biology Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea 4Graduate School of New Drug Discovery & Development, Chungnam National University, Daejon, Korea 5Department of Molecular Biochemistry and Biophysics, Yale University, New Haven, CT, USA 6Department of Chemistry, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea 7Present address: New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Korea ‡These authors contributed equally to this work *Corresponding author. Tel: +82 62 715 2485; Fax: +82 62 715 3411; E-mail: [email protected] *Corresponding author. Tel: +82 62 715 2493; Fax: +82 62 715 2521; E-mail: [email protected] EMBO Reports (2015)16:1318-1333https://doi.org/10.15252/embr.201540436 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca2+ uptake in a stable MCU knockdown HeLa cell line and exerted dominant-negative effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation. Synopsis This study reports the crystal structures of the N-terminal domain (NTD) of wild-type and a S92A mutant form of MCU. Functional analyses suggest that the N-terminal domain of MCU is essential for the modulation of MCU activity. Crystal structures of the NTD of WT and a S92A mutant form of MCU were determined. The structure of MCU NTD contains a novel fold important for post-translational modifications. Functional analysis suggests that MCU NTD is essential for the modulation of MCU activity. Introduction Energized mitochondria take up large quantities of Ca2+ through the putative tetrameric mitochondrial calcium uniporter (MCU) with a highly selective channel driven by a large electrochemical potential across the inner mitochondrial membrane (IMM) 12. MCU is associated with many regulatory proteins such as mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 (MICU1 and MICU2), an MCU paralog (MCUb), and essential MCU regulator (EMRE), forming the mitochondrial calcium uniporter complex (uniplex) 34567. MICU1 and MICU2 contain two conserved EF-hand Ca2+-binding domains that regulate the activity of MCU 34. EMRE is a single-pass membrane protein with a highly conserved aspartate-rich tail and is required for interaction between MCU and MICU1/MICU2 8. Evidence also exists for a possible interaction between MCU and MCUR1. Overexpression of MCUR1 in HeLa cells increases mitochondrial Ca2+ uptake, while its knockdown suppresses it 9. Moreover, MCUR1 has recently been reported as a regulator for cytochrome c oxidase assembly 10. MCU consists of two conserved transmembrane helices (TMs) connected by a 9-aa linker with a four-residue "DIME" motif flanked by an N-terminal domain (NTD) and C-terminal domain located within the mitochondrial matrix 111213. Computational modelling as well as biochemical experiments suggests that MCU tetramerization forms a highly Ca2+-selective eight TM channel inside the IMM 51112. The two negatively charged residues "D" and "E" in the "DIME" motif are essential for MCU function, presumably providing Ca2+-binding site(s) 1112. Balanced mitochondrial [Ca2+] is critical for the regulation of mitochondrial functions such as fission–fusion and ATP production 14. Uncontrolled mitochondrial Ca2+ overload caused by oncogenes and tumour suppressors can lead to the opening of the mitochondrial permeability transition pore (mPTP) with disruption of mitochondrial membrane potential 15. Excess Ca2+ entry in mitochondria has been associated with apoptosis and necrosis in many pathological states 16. Overexpression or silencing of MCU causes muscular diseases 17. Furthermore, knockdown of MCU results in energetic and developmental defects in Trypanosoma brucei 18 and zebrafish 19. MCU knockdown can also cause embryonic lethality in a pure C57/BL/6 inbred mouse strain 20, although mild phenotypic changes have been reported in MCU knockout mouse models 2122. More research is needed to know the structural basis for the diverse functions of MCU. In this study, we present the crystal structures of MCU NTD in a novel fold. Our biochemical and functional characterization indicated that MCU NTD is essential for MCU activity, and NTD deletion or S92A mutation impair the function of MCU. Results Overall structure of MCU NTD and NTD-E To elucidate the structural basis for MCU functions, we designed a set of human MCU truncation experiments for crystallographic and biochemical studies. We determined the first structure, at a 1.80 Å resolution, of the highly conserved NTD of MCU, corresponding to residues 75–165, encoded by exons 3 and 4, fused with the bacteriophage T4 lysozyme at the N-terminal end of the MCU NTD (Figs 1 and EV1A, Appendix Fig S1, Table 1). The T4 lysozyme fusion was used to enhance solubility and to phase a new crystal structure using molecular replacement. We also determined the structure of an extended version of the MCU NTD (MCU NTD-E), corresponding to residues 75–185, without the T4 lysozyme fusion at a 1.50 Å resolution (Figs 1 and EV1B, Table 1). The structure of MCU NTD-E was determined by molecular replacement using the MCU NTD structure as a template. Figure 1. Overall structure of MCU NTD Schematic diagram of the MCU sequence. MCU is composed of an N-terminal transit signal peptide (S), N-terminal domain (NTD), two transmembrane domains (TM1 and TM2), a "DIME" motif and two coiled-coils (CC). K180 is ubiquitination or biotinylation site 2526. Topology diagram of MCU in the IMM. Crystal structure of MCU NTD (orange) and MCU NTD-E (20-a.a. extension in magenta) is shown. Overall structure of MCU NTD and MCU NTD-E. MCU NTD is composed of two helices (α1 and α2), six β-strands (β1–β6), and two conserved loops (L2 and L4). MCU NTD-E has an additional α-helix (α3) and C-terminal tail (magenta). Top view of L2 and L4 loops (site A in C), showing the hydrogen bonding and hydrophobic interaction. Residues are shown in stick, one water molecule (W1) as red dots. Dashed lines (red) denote hydrogen bonds. The putative phosphorylation site, S92, is described in stick in the L2 loop. Highly conserved L2 and L4 loops in MCU NTD by ConSurf analysis 61. Residues represented in the L2 and L4 loops are coloured according to conservation analysis by ConSurf, using 250 MCU NTD homologues selected from the UniRef90 database. C-terminal hydrophobic helical regions (α2 and α3; site B in C). α2- and α3-helices of the leucine-rich (LR) region are shown in orange and magenta, respectively. Tail region (site C in C), showing the ubiquitination or biotinylation site K180 within MCU NTD-E. Download figure Download PowerPoint Table 1. Data collection and refinement statistics T4 lysozyme-MCU NTD T4 lysozyme-MCU NTD S92A MCU NTD-E PDB ID: 4XSJ PDB ID: 5BZ6 PDB ID: 4XTB Data collection Space groupa P65 P65 P65 X-ray sourceb and detector PAL-5C ADSC Q315r PAL-5C ADSC Q315r PAL-7A ADSC Q270 Wavelength (Å) 0.9795 0.9795 0.9793 Unit cell: a, b, c (Å) 98.1, 98.1, 62.4 97.8, 97.8, 61.5 55.5, 55.5, 68.9 α, β, γ (°) 90.0, 90.0, 120.0 90.0, 90.0, 120.0 90.0, 90.0, 120.0 Resolution range (Å)c 50–1.80 (1.83–1.80) 50–2.75 (2.80–2.75) 50−1.50 (1.53−1.50) Rmerged 6.8 (54.6) 12.3 (56.3) 4.7 (51.5) I/σI 18.2 (3.2) 6.8 (3.3) 11.9 (3.6) Completeness (%) 99.5 (98.3) 99.5 (100.0) 99.4 (100.0) Redundancy 8.0 (6.3) 4.7 (5.3) 5.7 (5.6) Refinement Resolution range (Å)c 48.5–1.80 34.9–2.75 48.0–1.50 No. reflections 29581 8147 17594 Rworke (%)/Rfree (%) 12.7/19.0 16.4/23.5 14.0/17.6 No. atoms Protein 2008 2007 863 Ligand − − 13f Ion () 15 30 − Water 431 41 143 B-factors (Å2) Protein 27.0 33.7 18.2 Ligand − − 30.3 Ion () 40.6 56.7 − Water 47.4 29.9 39.5 Model statistics rmsd bond length (Å) 0.010 0.014 0.010 rmsd bond angles (°) 1.33 1.67 1.64 Ramachandran plot (%) favoured/allowed/disallowed 98.8/1.2/0 97.2/2.8/0 99.1/0.9/0 a P65-related MCU NTD/MCU NTD interactions are conserved in these three P65 crystal forms. b Beamline 5C and 7A at Pohang Accelerator Laboratory (PAL) in South Korea. c Values in parentheses are for highest-resolution shell. d Rmerge = ∑h ∑i │I(h)i−‹I(h)›│/∑h ∑iI(h)i, where I(h) is the intensity of reflection of h, ∑h is the sum over all reflections and ∑i is the sum over i measurements of reflection h. e Rwork = ∑hkl ¦¦Fo¦-¦Fc¦¦/∑hkl¦Fo¦; 5% of the reflections were excluded for the Rfree calculation. f An unidentified electron density was observed and modeled with a tetraethylene glycol molecule. Click here to expand this figure. Figure EV1. Detailed structure of MCU NTD Structure of MCU NTD fused with T4 lysozyme at its N-terminus. MCU NTD is shown in orange, while bacteriophage T4 lysozyme is represented in green. Superposition of the MCU NTD and MCU NTD-E structures. Superposition of the two structures revealed a root mean square deviation (RMSD) of 0.29 Å for 90 Cα atoms. Two MCU NTDs are shown in cyan for T4 lysozyme-MCU NTD and in orange for MCU NTD-E. Extended C-terminal residues in MCU NTD-E comprising residues 166–182 are shown in magenta. Stereoview of the hydrophobic interior of MCU NTD-E. The hydrophobic residues are shown as a grey surface. Download figure Download PowerPoint The structure of MCU NTD consists of one α-helix and six β-strands (Fig 1C) that form a central core, two highly conserved loops (L2 and L4) (Fig 1D and E) and one leucine-rich short α-helix (α2) (Fig 1F). Hydrophobic residues in the α2-helix (140GIDLLL145) stabilize the hydrophobic interior of MCU NTD through interactions among V108, I127 and F149 (Fig EV1C). MCU NTD-E has an additional α-helix (α3) and a C-terminal tail (Fig 1C, F and G). The L2 and L4 loops are stabilized by hydrogen bonds and hydrophobic interaction formed by highly conserved L90, S92, R93, E95, E118, D119, I122 and one water molecule (W1) (Fig 1D and E). S92 located in the L2 loop forms a hydrogen bond with D119 located in L4 loop, stabilizing the local structure in these loops (Fig 1D). S92 is predicted as a potential CaMKII phosphorylation site 23, and we could expect that S92 phosphorylation induces conformational changes by breaking the hydrogen bonds in these loops and modulates MCU function 2324. The C-terminal tail in MCU NTD-E forms an extended coil structure and contains K180, a known ubiquitination and biotinylation site (Fig 1G) 2526. We hypothesize that ubiquitination of K180, which is located close to the MCU NTD core, is involved not only in ubiquitin-dependent proteasomal degradation, but also in the regulation of MCU function by inducing structural changes in MCU. Of interest, we observed an unidentified electron density, which is predicted as a linear lipid-like molecule with 13–16 carbon atoms and modelled with a tetraethylene glycol molecule (Appendix Fig S2). The lipid-like molecule interacts with the residues in the L1 loop, two helices (α2 and α3) and C-terminus tail (Appendix Fig S2B). Identification of MCU domain-like fold We identified MCU NTD as a novel fold and named it "MCU domain-like fold" superfamily based on the analysis of the Structural Classification of Proteins 2 (SCOP2) 27 (Fig 2). In a search for structures similar to MCU NTD using the Dali program 28 and CATH database 29, the ubiquitin-like (Ub) β-grasp fold (β-GF) ranked the highest in the Dali search (Z-score > 5.3) (Appendix Table S1), whereas the immunoglobulin (Ig)-like fold was the highest rank in the CATH database (SSAP score > 70) (Appendix Table S2). However, MCU NTD fold is different from β-GF and Ig-like fold. The MCU NTD core domain contains six β-strands and one α-helix in the following order: β4-β5-β6-α1-β1-β2-β3, in which each of the three β-strands forms two β-sheets (A- and B-sides) (Fig 2A and B). In contrast, the Ub core domain contains four β-strands and one α-helix in the following order: β3-β4-α1-β1-β2 (Fig 2C and D). The connection between β1-β2-α1 is highly conserved in all β-GFs 30. However, in MCU NTD, β3 is inserted between β2 and α1, and one additional β-strand is inserted after α1 (Fig 2B). Although the two domains could be superposed with an RMSD of 2.73 Å based on their α1 and α1ʹ-helices (Fig 2G), the directionality of the β-strands is different. Furthermore, when the domains were superposed based on the four β-strands, the α1- and α1ʹ-helices of MCU NTD and Ub, respectively, are located in the opposite side (Fig 2H). On the other hand, the β2-macroglobulin core domain, one of Ig-like fold, consists of a 7- to 9-strand sandwich structure, including a Greek-key motif 31 (Fig 2E and F). Although the MCU NTD and β2-macroglobulin could be superposed with an RMSD of 4.9 Å, the orientation of the two β-sheets in the A-side is rotated by 42 degrees (Fig 2I). Figure 2. Topology comparison of MCU NTD, ubiquitin-like β-GF and immunoglobulin-like fold A–F. Ribbon diagrams of MCU NTD (A), ubiquitin-like β-GF (Ub) (PDB code 1UBQ) (C) and Ig-like fold (β2-microglobulin were selected for comparison, chain F of PDB code 1IM3) (E). Topology diagrams of MCU NTD (B), Ub (D) and β2-macroglobulin (F). β-strands are represented by arrows, α-helices by cylinders and loops by lines. Each of the three β-strands of MCU NTD forms two β-sheets (A- and B-sides). G. Stereo view of the superposition of MCU NTD (orange) and Ub (cyan) based on α1- and α1ʹ-helices. H. Superposition of MCU NTD (orange) and Ub (cyan) based on the four β-strands (red dashed lines) aligned between (B) and (D). I. Superposition of MCU NTD (orange) and β2-microglobulin (blue) based on the central six β-strands (blue dashed lines) aligned between (B) and (F). Download figure Download PowerPoint Potential protein–protein interaction interfaces of MCU NTD and NTD-E In the structure of MCU NTD-E, hydrophobic residues in the α2-helix (L143, L144 and L146) together with L167 and L168 in the α3-helix (166DLLSHENA173) form a hydrophobic surface surrounded by hydrophilic residues (namely D142, D166 and E171) (Fig 1F). This surface and C-terminus tail (174ATLNNVKTL182) were predicted as potential protein–protein interaction (PPI) interfaces by the InterProSurf analysis 32 (Fig EV2A and C) and the consensus Protein–Protein Interaction Site Predictor (cons-PPISP) server 33 (Fig EV2B and C). In the crystals of T4 lysozyme-MCU NTD and MCU NTD-E, MCU NTDs form helical oligomers around the 65 screw axis in a similar manner (Fig EV3A and B). In the crystal of MCU NTD-E, oligomers are stabilized by additional interactions through an extended C-terminal tail in a manner resembling domain swapping (Fig EV3B–D), and the interface in MCU NTD and the C-terminal tail between subunits in the oligomer is consistent with the predicted PPI surface (Fig EV3E). Click here to expand this figure. Figure EV2. Prediction of the surface involved in protein–protein interaction (PPI) The potential PPI interface is shown as a green surface, as predicted by the InterProSurf server 32. The residues listed by the InterProSurf analysis show ˜70% accuracy for the prediction of the PPI interface, based on the accessible surface area (ASA). Each cluster of selected surface residues was ranked according to its scoring function. The potential PPI interface is shown as an orange surface, as predicted by the consensus Protein–Protein Interaction Site Predictor (cons-PPISP) server 33. The residues listed by cons-PPISP analysis show 80% accuracy with 51% coverage through sequence profiles and solvent accessibility. List of residues shown in (A, B). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. T4 lysozyme-MCU NTD and MCU NTD-E crystallographic packing A, B. Oligomers in crystal lattice of T4 lysozyme-MCU NTD (A) and MCU NTD-E (B). MCU NTDs form helical oligomers around the 65 screw axis in a similar manner. Continuous domain swapping by C-terminal tail forms oligomer along c-axis in the crystal lattice of MCU NTD-E (B). C. Ribbon diagram of MCU NTD-E oligomer in the crystal. A protomer (in orange) interacts with adjacent molecules through MCU NTD and C-terminus tail. D. Zoomed-in view of the interfaces. E. Crystal packing and PPI interfaces. Interface involved in the crystal packing matches with predicted PPI surface (InterProSurf and cons-PPISP) (Fig EV2). Download figure Download PowerPoint We also identified MCU NTD-Es oligomerized in solution by glutaraldehyde cross-linking assay in phosphate-buffered saline (PBS) and crystallization conditions (Appendix Fig S3). Thus, the results suggest that MCU NTD is involved in the oligomerization and interactions with the uniplex, although MCU NTD is not essential for the formation of the uniporter complex (see below). Overexpression of MCUΔNTD exerts a dominant-negative function without alteration of the uniplex assembly MCU forms homo-oligomers as well as hetero-oligomers (with MCUb) 5 and interacts with various regulatory proteins, including MICU1 and MICU2, in the intermembrane space forming a uniplex 83435. To investigate the functional role of the MCU NTD, we deleted this domain by generating an MCU mutant lacking residue
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