Crystal structure of Mdm12 reveals the architecture and dynamic organization of the ERMES complex
2016; Springer Nature; Volume: 17; Issue: 12 Linguagem: Inglês
10.15252/embr.201642706
ISSN1469-3178
AutoresHanbin Jeong, Jumi Park, Changwook Lee,
Tópico(s)Protein Tyrosine Phosphatases
ResumoArticle7 November 2016free access Source DataTransparent process Crystal structure of Mdm12 reveals the architecture and dynamic organization of the ERMES complex Hanbin Jeong Hanbin Jeong orcid.org/0000-0002-3878-1813 Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju, Korea Search for more papers by this author Jumi Park Jumi Park Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju, Korea Search for more papers by this author Changwook Lee Corresponding Author Changwook Lee [email protected] orcid.org/0000-0002-3016-9478 Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju, Korea Center for Genome Integrity, Institute for Basic Science (IBS), Ulsan, Korea Search for more papers by this author Hanbin Jeong Hanbin Jeong orcid.org/0000-0002-3878-1813 Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju, Korea Search for more papers by this author Jumi Park Jumi Park Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju, Korea Search for more papers by this author Changwook Lee Corresponding Author Changwook Lee [email protected] orcid.org/0000-0002-3016-9478 Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju, Korea Center for Genome Integrity, Institute for Basic Science (IBS), Ulsan, Korea Search for more papers by this author Author Information Hanbin Jeong1,2,‡, Jumi Park1,2,‡ and Changwook Lee *,1,2,3 1Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea 2Cell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju, Korea 3Center for Genome Integrity, Institute for Basic Science (IBS), Ulsan, Korea ‡These authors contributed equally to this work *Corresponding author. Tel: +82 52 217 2534; Fax: +82 52 217 2639; E-mail: [email protected] EMBO Reports (2016)17:1857-1871https://doi.org/10.15252/embr.201642706 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 endoplasmic reticulum–mitochondria encounter structure (ERMES) is a protein complex that plays a tethering role in physically connecting ER and mitochondria membranes. The ERMES complex is composed of Mdm12, Mmm1, and Mdm34, which have a SMP domain in common, and Mdm10. Here, we report the crystal structure of S. cerevisiae Mdm12. The Mdm12 forms a dimeric SMP structure through domain swapping of the β1-strand comprising residues 1–7. Biochemical experiments reveal a phospholipid-binding site located along a hydrophobic channel of the Mdm12 structure and that Mdm12 might have a binding preference for glycerophospholipids harboring a positively charged head group. Strikingly, both full-length Mdm12 and Mdm12 truncated to exclude the disordered region (residues 74–114) display the same organization in the asymmetric unit, although they crystallize as a tetramer and hexamer, respectively. Taken together, these studies provide a novel understanding of the overall organization of SMP domains in the ERMES complex, indicating that Mdm12 interacts with Mdm34 through head-to-head contact, and with Mmm1 through tail-to-tail contact of SMP domains. Synopsis The crystal structure of Mdm12, a cytosolic component of the ER–mitochondria encounter structure (ERMES) complex, reveals the structural basis for phospholipid binding and selectivity of Mdm12, and the organization of SMP domains in the ERMES complex. The crystal structure of Mdm12 reveals a highly conserved β1-strand that is critical for oligomerization. The SMP domain of Mdm12 selectively binds phospholipids with a positively charged head group via a negatively charged surface platform. Biochemical analysis suggests that Mdm12 provides two distinct contact sites to connect Mmm1 and Mdm34. Introduction Eukaryotic cells are composed of membrane-bound subcellular compartments that play distinct and essential roles for cell survival. The compartments not only work independently, but also they actively cooperate to achieve their ultimate roles. Apart from communication among subcompartments achieved through vesicular trafficking, direct contact sites of subcompartment membranes have been discovered through electron microscopy (EM) 123. Such membrane contact sites (MCSs) are involved in essential processes for cell survival, such as subcellular communications, ion homeostasis, metabolic pathways, and lipid biosynthesis 12345. Among several MCSs, ER–mitochondria direct contact sites have been extensively studied in terms of physical tethering of two membranes and their physiological relevancies, such as lipid trafficking and Ca2+ exchange 6789. The endoplasmic reticulum–mitochondria encounter structure (ERMES) components were first identified as molecular tethering factors in the formation of ER–mitochondrial junctions using synthetic biology screens in S. cerevisiae 10. The ERMES complex consists of four proteins with different subcellular localizations. Mdm12 (mitochondrial distribution and morphology protein 12) is a soluble protein present in the cytosol, while Mmm1 (maintenance of mitochondrial morphology protein 1) and Mdm34/Mdm10 are integral membrane proteins that are anchored in the ER and mitochondrial outer membranes, respectively. Additionally, Gem1 (GTPase EF-hand protein of mitochondrial 1), a Ca2+-binding Miro GTPase, associates with ERMES and regulates the number, size, and functions of these complexes in yeast 1112. In addition to its primary role in maintaining a close proximity (10–30 nm) between two membranes independently of fusion or fission, the ERMES complex also has been known to function in lipid trafficking to cooperatively synthesize phosphatidylcholine (PC) from phosphatidylserine (PS) in ER and mitochondria junctions 10131415. However, there is a conflicting report that ERMES and Gem1 do not directly affect PS trafficking 16. Recently, a couple of redundant pathways for lipid trafficking involved in the maintenance of mitochondrial lipid homeostasis have been reported. For example, the EMC (ER–membrane protein complex) located in the ER tethers a phosphatidylethanolamine (PE) to mitochondria by interacting with a TOM (translocase of the outer membrane) 17. The vCLAMP (vacuole and mitochondria patch) is another alternative pathway for transferring lipids to the mitochondria 1819. Composite defects in these pathways result in severe disruption of mitochondrial lipid homeostasis. In addition to lipid trafficking, the Mdm12–Mmm1 complex plays an important role in β-barrel assembly of mitochondrial outer membrane proteins, and in the maintenance of mitochondrial morphology and mtDNA 620. Furthermore, the ERMES complex has been repeatedly implicated in essential activities for cell survival such as mitophagy, inheritance, mtDNA inheritance, and mitochondrial dynamics 122122232425. Primary structure analyses of ERMES components reveal that Mdm12, Mmm1, and Mdm34 share a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain, although their sequences are not closely related to each other 13. In particular, full-length Mdm12 contains SMP domains across its entire sequence, while SMP domains in Mmm1 and Mdm34 account for half of the C-terminus and N-terminus, respectively. The remaining halves of the Mmm1 and Mdm34 protein sequences are predicted to be unstructured and not conserved among species, and the C-terminus of Mdm34 is known to be anchored into the outer mitochondrial membrane 26. Structural studies demonstrated that the SMP domain adopts a dimer configuration rather than existing solely as a monomer 272829. The association of SMP domains might act as the driving force in the assembly of ERMES components and maintain intact membrane proximity. Biochemistry experiments combined with a negative-staining EM structure revealed that Mdm12–Mmm1 forms a hetero-tetramer through the direct association of SMP domains, generating an arch-shaped structure with dimensions of ~210 × 45 × 35 Å 30. However, despite its importance in ER–mitochondria contact, no high-resolution structures of the ERMES complex are available. Therefore, the molecular details of how the SMP domains in the ERMES complex are organized to tether two organelles, and how ERMES recognizes certain lipids and facilitates their trafficking, remain unknown. In this study, we determined the crystal structures of full-length Mdm12 and ΔMdm12 (Δ74–114) and elucidated the molecular details of the contact regions for self-association of SMP domains and of lipid coordination in Mdm12. Furthermore, we suggest that two interfaces between SMP domains, head-to-head and tail-to-tail, provide a mechanistic understanding of the assembly and organization of the ERMES tetrameric complex at a molecular level. Results The oligomeric state of full-length Mdm12 and Mmm1 We prepared the Mdm12 protein from S. cerevisiae by expression in E. coli bacterial cells. Interestingly, the S. cerevisiae Mdm12 migrated differently on size-exclusion columns, depending on the presence or absence of N-terminus hexa-histidine (His6) tag plus TEV cleavage site (ENLYFQS) for full-length Mdm12 proteins. Full-length Mdm12 without His6 eluted from the column at a volume corresponding to approximately the mass of the Mdm12 dimer. On the other hand, His6–Mdm12 eluted from the column at a mass corresponding to the Mdm12 monomer (Fig 1A and B). The TEV cleavage site existing between His6 tag and Mdm12 was not vulnerable to proteases, suggesting that the N-terminus including the TEV cleavage site of Mdm12 was somehow masked by the protein itself. To further investigate the oligomeric state of Mdm12 and measure the molecular weights in solution, we conducted analytical ultracentrifugation with native Mdm12 and His6-Mdm12 proteins. Consistent with gel-filtration chromatography, Mdm12 and His6-Mdm12 were measured as 58.3 kDa (dimer) and 34.5 kDa (monomer), respectively (Figs 1C and EV1). From this observation, we propose that the N-terminus of Mdm12 could be critically involved in self-association and that the extra amino acid sequences consisting of the His6 tag and TEV cleavage sequence might disturb the dimerization of the protein. Figure 1. Mdm12 and Mmm1 organization Schematic diagrams showing the domain structures of Mdm12 and Mmm1 used in this study. Size-exclusion chromatography (SEC) experiments of Mdm12, tMmm1, and the Mdm12–tMmm1 complex comparing the molecular size of these proteins in solution. The proteins indicated were injected into a Superdex 200 column (GE Healthcare) with a buffer containing 25 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 5 mM DTT. The standard molecular masses for the SEC experiments (top) are shown for relative molecular weight comparison (blue dextran, void; ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 44 kDa; and carbonic anhydrase, 29 kDa). Graph indicating the molecular weights of Mdm12, His6–Mdm12, and the Mdm12–tMmm1 complex in solution as measured by analytical ultracentrifugation. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Analytical ultracentrifugation experiments Sedimentation equilibrium fitting results following analytical ultracentrifugation of wild-type Mdm12 (left), N-terminus hexahistidine-tagged Mdm12 (His6–Mdm12, middle), and the Mdm12–Mmm1 complex (right). The lower panel depicts the fitted overlay (red line) to the experimental data (blue circles). The upper panel depicts the residuals. Sedimentation velocity analytical ultracentrifugation profiles of wild-type Mdm12. Self-oligomerization of wild-type Mdm12 was analyzed at various concentrations (0.5, 1, and 2 mg/ml) at 20,124 g. Peak sedimentation coefficient values of 2.40 S and 3.17 S correspond to monomer and dimer, respectively. Download figure Download PowerPoint Mmm1 from S. cerevisiae was eluted in the void volume fraction during gel-filtration column chromatography, indicating that by itself Mmm1 is aggregated in solution (Fig 1B). However, when we co-expressed Mmm1 with Mdm12 in BL21 (DE3) bacterial cells, the complex displayed a monodisperse profile on the gel-filtration column, with an estimated molecular weight of around 200 kDa, suggesting that the Mdm12–Mmm1 complex exists as a hetero-tetramer in solution. This result was confirmed by analytical ultracentrifugation (Fig 1C, M.W. 122.7 kDa) and is consistent with previous data 30. Crystal structure determination for S. cerevisiae Mdm12 Full-length Mdm12 proteins from S. cerevisiae were crystallized under various conditions. The best crystals grew in a P21212 space group and diffracted to 3.1 Å resolution at a synchrotron source. The initial electron density map was calculated to 3.5 Å resolution from Se-Met-derivatized crystals using a single-wavelength anomalous diffraction (SAD) experiment, and the structure was phase extended and refined to 3.1 Å resolution with native crystal with Rwork/Rfree values of 21.2/26.5%. Statistics for data collection and refinement are presented in Table 1. Table 1. Data collection and refinement statistics Mdm12 native Mdm12 Se-SAD ΔMdm12 native Dataset PDB accession # 5GYD 5GYK X-ray source Beamline 5C, PAL Beamline 5C, PAL Beamline 5C, PAL Temperature (K) 100 100 100 Space group P21212 P21212 P212121 Cell parameters a, b, c (Å) 142.592, 219.073, 73.097 142.592, 219.017, 73.268 109.239, 148.243, 212.394 Data processing Wavelength (Å) 0.97933 0.97928 0.97957 Resolution (Å) 35.0–3.10 (3.15–3.10) 50.0-3.50 (3.55–3.50) 50.0-3.60 (3.66–3.60) Rmerge (%)a 11.0 (84.5) 14.3 (65.8) 14.2 (67.9) CC1/2 0.995 (0.626) 0.994 (0.841) 0.995 (0.648) I/σ 19.9 (2.22) 20.6 (3.98) 11.1 (2.21) Completeness (%) 99.6 (100.0) 99.8 (100.0) 99.7 (100.0) Redundancy 5.3 (5.3) 6.4 (6.6) 3.6 (3.7) Measured reflections 221,431 190,622 146,540 Unique reflections 41,953 29,933 40,722 Refinement statistics Data range (Å) 35.0–3.10 50.0–3.60 Reflections 41909 40625 Non-hydrogen atoms 7409 10865 R.m.s. ∆ bonds (Å)b 0.009 0.007 R.m.s. ∆ angles (°)b 1.490 1.369 R-factor (%)c 21.2 22.5 Rfree (%)c,d 26.5 26.7 Ramachandran plot, residues in Most favored regions (%) 91.9 88.3 Additional allowed regions (%) 7.6 10.2 Generously allowed regions (%) 0.5 1.5 Disallowed regions (%) 0 0 Highest resolution shell is shown in parenthesis. a Rmerge = 100 × ∑h∑i | Ii(h) − |/∑h , where Ii(h) is the ith measurement and is the weighted mean of all measurements of I(h) for Miller indices h. b Root-mean-squared deviation (r.m.s. ∆) from target geometries. c R-factor = 100 × ∑|FP – FP(calc)|/∑ FP. d Rfree was calculated with 5% of the data. Overall structure of Mdm12 The molecular models of Mdm12 are presented in Fig 2A–D. As observed by size-exclusion chromatography, the full-length Mdm12 forms dimers in the crystals with the asymmetric unit containing two Mdm12 dimers (four Mdm12 monomers in total) related to twofold symmetry. The four Mdm12 molecules are almost identical with a RMSD of < 0.3 Å. The crystal structure reveals that the Mdm12 dimer adopts an elongated tubular structure with dimensions of 40 Å × 60 Å × 110 Å (Fig 2A). The Mdm12 monomer consists of three structural elements: (i) β1-dimerization center; (ii) β-barrel with incomplete and highly twisted β-strands and three α-helices, which are comparably organized as shown in most synaptotagmin (SMP) domain-containing proteins 272829; and (iii) proline rich region, which protrudes from the SMP domain from the middle of the last strand of the β-barrel (Figs 2B and EV2). The truncated cone-shaped structure of the Mdm12 monomer forms an extensive hydrophobic channel through the elongated cavity, which was reported to provide a binding channel for particular fatty acids (discussed below) in previous studies 272829. Two Mdm12 molecules are arranged in a twofold symmetry and associate with each other through domain swapping of the N-terminus β-strand (β1) comprising residues 1–7 as detailed below. Overall, the Mdm12 dimer structure resembles that of members of the TULIP family such as E-SYT2 (extended synaptotagmin 2, RMSD: 5.71), CETP (cholesteryl ester transfer protein, RMSD: 4.47), and BPI (bactericidal/permeability-increasing protein, RMSD: 4.26) despite the absence of any significant sequence similarity among them 272829. Notably, BPI and CETP exist as monomers containing two separate SMP domains that show no significant sequence conservation between them. Figure 2. Overall structure of Mdm12 A. Ribbon diagram of the yeast Mdm12 dimer. The crystal structure of full-length Mdm12 was determined by SAD and refined with native data to 3.1 Å resolution. Lipids bound to Mdm12 are drawn with black stick models. B. Schematic diagram indicating the secondary structure elements and their organization in Mdm12. Three structural elements of Mdm12 are highlighted in different colored boxes. C, D. Surface representations of the Mdm12 dimer are shown in different orientations. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Sequence conservation of Mdm12Sequence alignment of Mdm12 orthologs in fungi. The secondary structure elements are indicated above the sequences with helices, strands, loops, and disordered regions represented by arrows, cylinders, solid lines, and dashed lines, respectively. The absolutely conserved and highly similar sequences are highlighted in red and yellow, respectively. Download figure Download PowerPoint No electron density was observed for residues 74–114 of Mdm12, suggesting that this region might be highly flexible. Furthermore, these residues are not conserved in several Mdm12 orthologs. We obtained another orthorhombic crystal from the construct excluding the disordered region (Δ74–114, referred to as ΔMdm12 hereafter) in full-length Mdm12. The crystals of ΔMdm12 grew in a P212121 space group and diffracted to 3.6 Å resolution. The structure of ΔMdm12 was solved by molecular replacement using the full-length Mdm12 structure as the search model and refined to 3.6 Å resolution. ΔMdm12 also crystallized as a dimer, and the structures and twofold arrangement of Mdm12 and ΔMdm12 are almost identical with a RMSD of 0.5 Å. The highly conserved β1-strand of Mdm12 forms the dimeric interface for self-association The N-terminus of Mdm12 is highly conserved among Mdm12 orthologs (Fig 3A). In the Mdm12 dimer, residues 1–7 from one monomer fold into a β-strand that inserts itself between β1 and β2 from the second monomer, running antiparallel with β1 and parallel with β2 in the twofold center of the Mdm12 dimer. They are systematically associated with each other by forming a hydrogen bond network among main chains of the protein between β1 (residues 4–7) and β2 (residues 53–56) from counter molecules, and two β1 (residues 1–6) strands from two molecules (Fig 3A). The buried surface area caused by the dimerization of Mdm12 is around 1,400 Å2. E-SYT2 makes a twofold dimerization interface between two separate SMP domains using a highly conserved helix (residues 167–180) located at the beginning of each SMP domain (Fig 3C). The dimeric interface of Mdm12 closely resembles the twofold-like interface of CETP and BPI involving two SMP domains, an interface consisting of the central β-sheets comprising six antiparallel β-strands (Fig 3C). However, it is a distinctive feature of Mdm12 that the dimer is formed through domain swapping of the central β-strand located between the two SMP domains. Figure 3. Dimer interface of Mdm12 Ribbon diagram showing the twofold dimerization interface of Mdm12. Oxygen and nitrogen atoms are shown in red and blue, respectively. The orange dotted lines indicate intermolecular hydrogen bonds between two protomers of Mdm12. The sequence alignment of yeast Mdm12 orthologs is shown to highlight the sequence conservation in the N-terminus β1-strand. Ten orthologs are aligned from residues 1–11. Absolute and highly conserved residues are indicated in red and orange, respectively. The molecular weight of the Mdm12 (I5P) mutant was measured by size-exclusion chromatography (below) and ultracentrifugation (top) as in Fig 1B and C. Ribbon diagram showing the structures of the SMP domain in E-SYT2, CETP, and BPI for the comparison of dimeric interfaces among SMP domains. Note that CETP and BPI are not dimers but monomers containing two tandem SMP domains. Download figure Download PowerPoint To further investigate whether the role of the β1-strand in the dimerization of Mdm12 as observed in the crystal structure also applied to Mdm12 in solution, we generated a point mutant (I5P) aimed at disrupting the β1-strand structure. In the dimer, the main chain of I5 forms H-bonds with the main chain of M1 from the second Mdm12 molecule, and its side chain makes van der Waals interactions with the hydrophobic side chains of M1, F3, W7, and I54 in the second molecule. As expected, both gel-filtration and analytical ultracentrifugation experiments revealed that the I5P mutant could not form a homo-dimer (Fig 3B), supporting the critical involvement of the highly conserved β1-strand in Mdm12 homo-dimerization in solution. The SMP domain of Mdm12 binds phospholipid Initial electron density maps clearly displayed a lipid-like molecule inside the hydrophobic channel of the Mdm12 monomer (Fig 4A and B). We were unable to identify the bound phospholipid using only electron density maps because of (i) the mid-range resolution (~3.1 Å) of this structure and (ii) the disordered electron density corresponding to the head group of phospholipid. However, it was previously reported that the recombinant Mdm12 proteins expressed in bacteria bind PE (~80%) and PG (~15%) species 30. Therefore, we inferred that the diacyl glycerophospholipid bound to Mdm12 might be a PE or PG. To identify the phospholipids present in the Mdm12 structure, we performed denaturing quantitative APCI-MS using purified Mdm12 expressed in E. coli. The major phospholipid bound to Mdm12 was observed to have an m/z of 704.5 (Fig EV3B), which identified the molecule as PE, consistent with a previous lipidomic analysis in which PE (33:1) with an m/z of 704.5 was the predominant phospholipid co-purified with Mdm12 expressed in bacteria 30. We built a PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) into the diacyl-like ligand density (Fig 4A), and the lipid-bound Mdm12 structure was well refined with native diffraction data. Ligand positioning is almost identical among three Mdm12 molecules within the asymmetric unit, except in one Mdm12 molecule, where the hydrocarbon chain of lipid is displaced and the head region is disordered (Fig EV3A). This displacement might be the result of crystal packing because the hydrophobic cavity of this molecule was slightly shrunk through the formation of close contacts with the symmetry-related molecules in the crystal. Figure 4. Mdm12 binds lipid through the SMP domain Simulated annealing omit map (Fo-Fc, contoured at 1.5σ) showing the molecule bound to Mdm12 (left). The final model for the bound PE is shown as in stick representation. The electron density (2Fo-Fc) calculated in the final model is shown with the stick model of PE in the right (3.1 Å resolution, contoured at 0.8σ). Surface representation of the Mdm12 dimer. Hydrophobic amino acids lining the Mdm12 channel are indicated by a blue mesh. Lipids built in Mdm12 are in space-filling representation. Ribbon diagram showing lipid coordination by Mdm12. Mdm12 residues and lipid fatty acids are colored in green and yellow, respectively. Mdm12 binds NBD-PE. Wild-type and monomeric (I5P mutant) Mdm12 were incubated with NBD-PE and separated from free NBD-PE in native PAGE. Coomassie staining (left) and fluorescent (right) detection indicates that Mdm12 directly interacts with NBD-PE in vitro. Quantitative data showing binding affinities for NBD-PE by Mdm12. The binding affinities of Mdm12 (monomer/dimer shown in native PAGE and I5P mutant) for NBD-PE was measured with a NBD-PE concentration-dependent manner. All experiments were carried out three times, and the means ± SD are given. Mdm12 mutants (L256W, I262W, and L256W/I262W double mutants) were incubated with NBD-PE and subjected to native PAGE. Because wild-type Mdm12 separates as both monomer and dimer on native PAGE, the purely monomeric form (I5P) of Mdm12 was used as the wild type for clarity. The graph in the right indicates the quantities measured in the experiments. The bar shows the relative amounts of the band ratio (fluorescence/Coomassie). Values represent the means and SD from three independent experiments. Source data are available online for this figure. Source Data for Figure 4 [embr201642706-sup-0002-SDataFig4.pptx] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Structural comparison of lipids bound to Mdm12 in the crystallographic asymmetric unit and lipids identified from APCI-MS analysis Ribbon diagram showing the overlay of the lipids bound to the SMP domains of the four Mdm12 molecules in the asymmetric unit. Four Mdm12 molecules and the hydrocarbon chains of bound lipids are identically colored in pink, green, cyan, and yellow. Oxygen and nitrogen atoms in lipids are colored in red and blue, respectively. The α1-helices and bound lipids in the three Mdm12 molecules (pink, cyan, yellow) precisely align with each other. However, the α1-axis of one Mdm12 (green) molecule is tilted around 9 degrees owing to crystal packing. The displaced α1-helix induces a break in coordination of the lipid hydrocarbon chain, and the head group of the lipid is disordered in the structure. Right figure shows only the lipids bound to Mdm12 for clarity. Quantitative profiling of phospholipids bound to Mdm12 purified from E. coil using APCI-MS (see Materials and Methods section for details). The most abundant species bound to Mdm12 had a mass of 704.5 Da and was identified as PE (33:1), consistent with a previous report 30. Download figure Download PowerPoint Based on our crystal structure, the head group of phospholipid is exposed into the solvent and makes no direct contacts with neighboring residues of Mdm12, indicating that Mdm12 might have no clear selectivity for specific phospholipids. However, the fatty acyl chain of PE was tightly coordinated by the hydrophobic side chains of neighboring amino acids including I20, F45, L47, L177, F179, F251, L256, I262, and L264 (Fig 4C). We tested the ability of phospholipids to bind directly to the SMP domain of Mdm12 in vitro. We used the fluorescently labeled PE (7-nitro-benz-2-oxa-1,3-diazol-4-yl-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, referred to as NBD-PE) and full-length Mdm12 purified from E. coli to measure their binding, as previously described 2930. Mdm12 proteins incubated with NBD-PE were run onto native PAGE to remove unbound NBD-PE, and NBD-PE-bound Mdm12 was quantified with fluorescence detection. Figure 4D and E shows that Mdm12 binds NBD-PE in a concentration-dependent manner. Unexpectedly, while around half of the full-length Mdm12 appeared as a dimer (46% of total Mdm12), the other half ran as a monomer (54%) in the native PAGE, as compared with Mdm12 (I5P) that migrated only as a monomer. The monomer and dimer distribution of Mdm12 observed in native PAGE was not correlated with NBD-PE incorporation (Figs 4D and EV4B). More surprisingly, monomeric Mdm12 had a higher affinity for NBD-PE than did dimeric Mdm12. Interestingly, the I5P mutant showed the highest affinity for NBD-PE, suggesting that the N-terminal β1-strand of Mdm12 might be involved in regulating lipid trafficking, including access. Indeed, the structure shows the lipid-binding region, including the head group, to be very close to the dimerization interface. The dimerization of Mdm12 could thus sterically occlude lipid access, and the perturbation of the β1-strand by mutation therefore increased the affinity for NBD-PE (Fig EV4). Click here to expand this figure. Figure EV4. Mdm12 preferentially binds phospholipids with a positively charged head group at the dimerization interface A. Figure highlights that the lipid-binding site of Mdm12 is proximal to the dimerization interface. Views are along the twofold rotation axis. The bound lipids are shown as spheres. The hydrocarbon, oxygen, and nitrogen are colored in black, red, and blue, respectivel
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