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Structural and Functional Analysis of Mid51, a Dynamin Receptor Required for Mitochondrial Fission

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Structural and Functional Analysis of Mid51, a Dynamin Receptor Required for Mitochondrial Fission Published Online: 10 February, 2014 | Supp Info: http://doi.org/10.1083/jcb.201311014 JCB: Report Downloaded from jcb.rupress.org on September 13, 2018 Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission Viviane Richter,1,2 Catherine S. Palmer,1,2 Laura D. Osellame,1 Abeer P. Singh,1,2 Kirstin Elgass,1,2 David A. Stroud,1 Hiromi Sesaki,3 Marc Kvansakul,1 and Michael T. Ryan1,2 1Department of Biochemistry and 2Australian Research Council Centre of Excellence in Coherent X-Ray Science, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia 3Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 itochondrial fission is important for organelle adopts a nucleotidyltransferase fold. Although MiD51 transport, inheritance, and turnover, and alter- lacks catalytic residues for transferase activity, it specifi- M ations in fission are seen in neurological dis- cally binds guanosine diphosphate and adenosine di- ease. In mammals, mitochondrial fission is executed by phosphate. MiD51 mutants unable to bind nucleotides dynamin-related protein 1 (Drp1), a cytosolic guanosine were still able to recruit Drp1. Disruption of an additional triphosphatase that polymerizes and constricts the organ- region in MiD51 that is not part of the nucleotidyltransfer- elle. Recruitment of Drp1 to mitochondria involves recep- ase fold blocked Drp1 recruitment and assembly of MiD51 tors including Mff, MiD49, and MiD51. MiD49/51 form into foci. MiD51 foci are also dependent on the presence foci at mitochondrial constriction sites and coassemble of Drp1, and after scission they are distributed to daugh- with Drp1 to drive fission. Here, we solved the crystal ter organelles, supporting the involvement of MiD51 in the structure of the cytosolic domain of human MiD51, which fission apparatus. Introduction Mitochondria are highly dynamic organelles and undergo endoplasmic reticulum crosses (Friedman et al., 2011). There, continuous fission and fusion events, a balance of which is it polymerizes into spirals and through GTP-dependent con- vital for their function and cellular distribution (Westermann, formational changes it constricts the organelle leading to 2010). Mutations in genes associated with mitochondrial dy- membrane scission (Lackner et al., 2009). The crystal struc- namics have been linked with peripheral and optic neuropa- tures of dynamin and Drp1 revealed interfaces involved in thies (Alexander et al., 2000; Delettre et al., 2000; Züchner protein oligomerization along with a mechanism for polymer et al., 2004; Davies et al., 2007) and fatal developmental ab- constriction (Faelber et al., 2011; Ford et al., 2011; Fröhlich THE JOURNAL OF CELL BIOLOGY normalities (Ishihara et al., 2009), whereas defects in mito- et al., 2013). Unlike dynamin-1, Drp1 lacks a pleckstrin ho- chondrial dynamics have been implicated in Parkinson’s, mology domain and is recruited to mitochondria through outer Alzheimer’s, and Huntington’s disease (Wang et al., 2008; membrane receptors Fis1 (Mozdy et al., 2000; Yoon et al., Arduíno et al., 2011; Reddy et al., 2011). The primary regu- 2003; Stojanovski et al., 2004), Mff (Gandre-Babbe and lator of fission, controlled through both posttranslational van der Bliek, 2008; Otera et al., 2010), along with chordate- modifications and interactions with mitochondrial adaptor specific MiD49 and MiD51 (Palmer et al., 2011; Zhao et al., proteins, is the dynamin family member Drp1 (Dnm1 in yeast; 2011). Individually, Mff, MiD49, and MiD51 have been shown Bui and Shaw, 2013; Elgass et al., 2013). Cytosolic Drp1 to be sufficient to recruit Drp1 to drive fission (Koirala et al., is recruited to mitochondrial constrictions, often where the 2013; Losón et al., 2013; Palmer et al., 2013), whereas the role of Fis1 as a Drp1 receptor has been questioned (Otera et al., 2010). Recently, addition of stoichiometric amounts of MiD49 was found to increase Drp1-mediated constrictions Correspondence to Marc Kvansakul: [email protected]; or Michael T. Ryan: [email protected] © 2014 Richter et al. This article is distributed under the terms of an Attribution– Abbreviations used in this paper: 4-OHT, 4-hydroxytamoxifen; cGAS, cyclic Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- GMP–AMP synthase; DRR, Drp1 recruitment region; ITC, isothermal titra- lication date (see http://www.rupress.org/terms). After six months it is available under a tion calorimetry; MEF, mouse embryonic fibroblast; RMSD, root mean square Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, deviation. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 204 No. 4 477–486 www.jcb.org/cgi/doi/10.1083/jcb.201311014 JCB 477 Figure 1. Crystal structure of a MiD51 cyto- solic domain sufficient for Drp1 recruitment. (A) MiD51-GFP and MiD51DR-GFP were ex- pressed in wild-type MEFs and MEFs lacking endogenous MiD51 (MiD51TALEN). Cells were subsequently immunostained for Drp1 and the mitochondrial marker protein Tom20 and visu- alized by fluorescence microscopy. (right) Lin- escans demonstrating colocalization of Drp1 with MiD51-GFP and MiD51DR-GFP. Bars, 20 µm. (B) Structure of MiD51N118 displaying the nucleo- tidyltransferase domain in blue and DRR do- main in green. (C) Surface representation of MiD51N118; view and coloring as in B. (D) Topol- ogy of MiD51 and illustration of mutant con- structs used in this study. TM, transmembrane domain; DR, disordered region; NT, nucleotidyl- transferase domain. of liposomes from 31 to 15 nm, a point at which membrane Results and discussion scission is possible (Koirala et al., 2013). This indicates that MiD49/51 may be actively involved in facilitating Drp1 MiD51 belongs to the scission similar to the unrelated yeast adaptors Mdv1/Caf4 nucleotidyltransferase fold superfamily that are absent in higher eukaryotes (Bui and Shaw, 2013). of proteins MiD49/51 lack significant sequence similarity to any other To search for a construct amenable to crystallization, we performed proteins and they do not possess characteristic sequence do- limited proteolysis on mouse MiD49 lacking its transmembrane mains/motifs. Here we determine the crystal structure of the domain. We identified a large ( 40 kD) trypsin-resistant frag- cytosolic domain of MiD51 and identify a critical region in ment of MiD49 that lacks a predicted disordered region of the MiD51 important for Drp1 recruitment and assembly of the protein (Fig. S1 A). When human MiD51 lacking a homologous fission apparatus. disordered region (residues 50–123) but still containing its 478 JCB • VOLUME 204 • NUMBER 4 • 2014 Table 1. Data collection and refinement statistics for crystals of the non-nucleotide–bound MiD51, the Xenon derivative used for SAD phasing, and various MiD51–nucleotide complexes Native MiD51 Xe derivative MiD51–GDP MiD51–ADP MiD51PEYFP–GDP MiD51PEYFP–ADP Data collection Wavelength (Å) 0.9537 1.4586 0.9537 0.9537 1.4586 1.4586 Space group P1 P1 P1 P1 P43212 P43212 Cell dimensions a, b, c (Å) 72.72, 78.73, 73.14, 78.27, 72.34, 79.09, 72.57, 79.26, 57.72, 57.72, 57.53, 57.53, 79.36 80.42 80.05 79.38 255.40 253.75 , , (°) 66.26, 84.93, 65.17, 83.79, 65.81, 84.36, 65.44, 84.18, 90.00, 90.00, 90.00, 90.00, 64.07 62.39 64.08 63.35 90.00 90.00 Resolution (Å)a 40.67–2.12 45.80–2.60 40.00–2.30 50.00–2.30 50.00–2.55 50.00–2.55 (2.23–2.12) (2.74–2.60) (2.42–2.30) (2.42–2.30) (2.69–2.55) (2.69–2.55) a Rsym or Rmerge 0.066 (0.500) 0.059 (0.324) 0.078 (0.447) 0.082 (0.489) 0.086 (0.499) 0.083 (0.466) I/Ia 11.0 (2.8) 16.6 (3.6) 9.4 (2.2) 10.8 (2.7) 31.2 (7.9) 28.7 (7.7) Completeness (%)a 97.3 (97.3) 84.4 (44.2) 98.1 (97.6) 97.8 (97.3) 100.0 (100.0) 100.0 (100.0) Redundancya 2.7 (2.8) 3.5 (3.4) 2.5 (2.5) 3.5 (3.5) 25.9 (27.5) 22.3 (23.4) Refinement Resolution (Å)a 39.01–2.12 – 39.05–2.30 39.19–2.30 42.82–2.55 42.62–2.55 (2.20–2.12) (2.33–2.30) (2.38–2.30) (2.71–2.55) (2.64–2.55) No. of reflections 79,549 – 63,108 62,135 15,007 14,827 Rwork/Rfree 0.2228/0.2626 – 0.1913/0.2375 0.2161/0.2477 0.2085/0.2452 0.1965/0.2414 No. of atoms Protein 10,892 – 10,872 10,652 2,674 2,650 Water 527 – 356 341 114 94 B factors Protein 48.81 – 48.03 45.70 56.05 52.13 Water 46.81 – 44.59 37.42 52.84 53.43 RMSDs Bond lengths (Å) 0.004 – 0.005 0.006 0.003 0.005 Bond angles (°) 0.802 – 0.90 1.028 0.761 1.005 A single crystal was used for collection of each dataset. aValues in parentheses are for highest-resolution shell. N-terminal transmembrane anchor (termed MiD51DR) was <0.1 considered not biologically relevant. A notable feature transiently expressed in mouse embryonic fibroblasts (MEFs), of this interface were pairs of equally charged acidic and basic it was still able to recruit cytosolic Drp1 to mitochondria like residues, which would result in charge repulsion between two full-length MiD51 (Fig. 1 A). We also confirmed this by ex- MiD51 monomers, suggesting that this interface is unlikely to pressing MiD51DR in MEFs lacking endogenous MiD51 after have biological relevance.
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