Novel Structure of the N Terminus in Yeast Fis1 Correlates with a Specialized Function in Mitochondrial Fission
2005; Elsevier BV; Volume: 280; Issue: 22 Linguagem: Inglês
10.1074/jbc.m414092200
ISSN1083-351X
AutoresMotoshi Suzuki, Albert Neutzner, Nico Tjandra, Richard J. Youle,
Tópico(s)RNA and protein synthesis mechanisms
ResumoMitochondrial fission is facilitated by a multiprotein complex assembled at the division site. The required components of the fission machinery in Saccharomyces cerevisiae include Dnm1, Fis1, and Mdv1. In the present study, we determined the protein structure of yeast Fis1 using NMR spectroscopy. Although the six α-helices, as well as their folding, in the yeast Fis1 structure are similar to those of the tetratricopeptide repeat (TPR) domains of the human Fis1 structure, the two structures differ in their N termini. The N-terminal tail of human Fis1 is flexible and unstructured, whereas a major segment of the longer N terminus of yeast Fis1 is fixed to the concave face formed by the six α-helices in the TPR domains. To investigate the role of the fixed N terminus, exogenous Fis1 was expressed in yeast lacking the endogenous protein. Expression of yeast Fis1 protein rescued mitochondrial fission in Δfis1 yeast only when the N-terminal TPR binding segment was left intact. The presence of this segment is also correlated to the recruitment of Mdv1 to mitochondria. The conformation of the N-terminal segment embedded in the TPR pocket indicates an intra-molecular regulation of Fis1 bioactivity. Although the TPR-like helix bundle of Fis1 mediates the interaction with Dnm1 and Mdv1, the N terminus of Fis1 is a prerequisite to recruit Mdv1 to facilitate mitochondrial fission. Mitochondrial fission is facilitated by a multiprotein complex assembled at the division site. The required components of the fission machinery in Saccharomyces cerevisiae include Dnm1, Fis1, and Mdv1. In the present study, we determined the protein structure of yeast Fis1 using NMR spectroscopy. Although the six α-helices, as well as their folding, in the yeast Fis1 structure are similar to those of the tetratricopeptide repeat (TPR) domains of the human Fis1 structure, the two structures differ in their N termini. The N-terminal tail of human Fis1 is flexible and unstructured, whereas a major segment of the longer N terminus of yeast Fis1 is fixed to the concave face formed by the six α-helices in the TPR domains. To investigate the role of the fixed N terminus, exogenous Fis1 was expressed in yeast lacking the endogenous protein. Expression of yeast Fis1 protein rescued mitochondrial fission in Δfis1 yeast only when the N-terminal TPR binding segment was left intact. The presence of this segment is also correlated to the recruitment of Mdv1 to mitochondria. The conformation of the N-terminal segment embedded in the TPR pocket indicates an intra-molecular regulation of Fis1 bioactivity. Although the TPR-like helix bundle of Fis1 mediates the interaction with Dnm1 and Mdv1, the N terminus of Fis1 is a prerequisite to recruit Mdv1 to facilitate mitochondrial fission. Mitochondria are dynamic organelles that change their morphology by fusion and fission. Such processes, apparently counteracting each other, are facilitated by two independent molecular machineries. Mitochondrial outer membrane fusion is regulated by the integral membrane proteins Mfn1 and Mfn2 in the case of mammals (1Santel A. Fuller M.T. J. Cell Sci. 2001; 114: 867-874Crossref PubMed Google Scholar) or Fzo1 in the case of yeast (2Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (424) Google Scholar, 3Rapaport D. Brunner M. Neupert W. Westermann B. J. Biol. Chem. 1998; 273: 20150-20155Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). These proteins span the mitochondrial outer membranes twice, exposing an N-terminal GTPase domain and a C-terminal coiled-coil domain to the cytosol. A recent study of mouse Mfn1 revealed that the C-terminal coiled-coil domain points outward from mitochondrial membranes to form homodimers in an antiparallel fashion, which is implicated in tethering of two mitochondria together at a distance of about 10 nm (4Koshiba T. Detmer S.A. Kaiser J.T. Chen H. McCaffery J.M. Chan D.C. Science. 2004; 305: 858-862Crossref PubMed Scopus (651) Google Scholar). Subsequent inner membrane fusion is mediated by a protein called OPA1 in the case of mammals (5Delettre C. Lenaers G. Griffoin J.M. Gigarel N. Lorenzo C. Belenguer P. Pelloquin L. Grosgeorge J. Turc-Carel C. Perret E. Astarie-Dequeker C. Lasquellec L. Arnaud B. Ducommun B. Kaplan J. Hamel C.P. Nat. Genet. 2000; 26: 207-210Crossref PubMed Scopus (1144) Google Scholar, 6Alexander C. Votruba M. Pesch U.E. Thiselton D.L. Mayer S. Moore A. Rodriguez M. Kellner U. Leo-Kottler B. Auburger G. Bhattacharya S.S. Wissinger B. Nat. Genet. 2000; 26: 211-215Crossref PubMed Scopus (1052) Google Scholar) or Mgm1 in yeast (7Shepard K.A. Yaffe M.P. J. Cell Biol. 1999; 144: 711-720Crossref PubMed Scopus (146) Google Scholar, 8Wong E.D. Wagner J.A. Gorsich S.W. McCaffery J.M. Shaw J.M. Nunnari J. J. Cell Biol. 2000; 151: 341-352Crossref PubMed Scopus (267) Google Scholar), which localizes to the inner membrane with the GTPase domain facing the intermembrane space. Mitochondrial fission is mediated by a dynamin-related protein, Drp1, identified as Dnm1 in yeast (9Otsuga D. Keegan B.R. Brisch E. Thatcher J.W. Hermann G.J. Bleazard W. Shaw J.M. J. Cell Biol. 1998; 143: 333-349Crossref PubMed Scopus (332) Google Scholar, 10Bleazard W. McCaffery J.M. King E.J. Bale S. Mozdy A. Tieu Q. Nunnari J. Shaw J.M. Nat. Cell Biol. 1999; 1: 298-304Crossref PubMed Scopus (586) Google Scholar, 11Sesaki H. Jensen R.E. J. Cell Biol. 1999; 147: 699-706Crossref PubMed Scopus (428) Google Scholar). Drp1 and Dnm1 localize in the cytosol as well as in foci at the dividing site of the mitochondrial outer membrane surface during mitochondrial fission (9Otsuga D. Keegan B.R. Brisch E. Thatcher J.W. Hermann G.J. Bleazard W. Shaw J.M. J. Cell Biol. 1998; 143: 333-349Crossref PubMed Scopus (332) Google Scholar, 12Smirnova E. Griparic L. Shurland D.L. van der Bliek A.M. Mol. Biol. Cell. 2001; 12: 2245-2256Crossref PubMed Scopus (1311) Google Scholar). In contrast to proteins that are involved in fusion, Drp1 and Dnm1 do not span membrane bilayers. Drp1- and Dnm1-mediated mitochondrial fission is achieved with other accompanying proteins. Genetic studies of Saccharomyces cerevisiae identified Mdv1 and Fis1 as proteins involved in the Dnm1-dependent fission process (13Mozdy A.D. McCaffery J.M. Shaw J.M. J. Cell Biol. 2000; 151: 367-379Crossref PubMed Scopus (534) Google Scholar, 14Tieu Q. Nunnari J. J. Cell Biol. 2000; 151: 353-365Crossref PubMed Scopus (285) Google Scholar). Mdv1 is a peripheral membrane protein that localizes with Dnm1 at punctate structures along the mitochondrial outer membranes to regulate Dnm1. Fis1 is an integral membrane protein that is uniformly distributed along the mitochondrial outer membranes that is required for immobilization of Dnm1 and Mdv1. A high molecular weight complex of Dnm1, Mdv1, and Fis1 constructs the punctate structures at the dividing site and facilitates constriction and division of mitochondrial membrane (13Mozdy A.D. McCaffery J.M. Shaw J.M. J. Cell Biol. 2000; 151: 367-379Crossref PubMed Scopus (534) Google Scholar, 14Tieu Q. Nunnari J. J. Cell Biol. 2000; 151: 353-365Crossref PubMed Scopus (285) Google Scholar). Mdv1 orthologs have not been identified in nematode, fruit fly, or vertebrates, and it is not known whether another protein takes its place or whether the molecular machinery of mitochondrial fission in the various species is different. In contrast, human and mouse orthologs of Fis1 have been identified, suggesting that the role of Fis1 in mitochondrial fission is conserved among lower and higher eukaryotes. Although increased levels of human Fis1 in mammalian cells may (15Frieden M. James D. Castelbou C. Danckaert A. Martinou J.C. Demaurex N. J. Biol. Chem. 2004; 279: 22704-22714Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 16James D.I. Parone P.A. Mattenberger Y. Martinou J.C. J. Biol. Chem. 2003; 278: 36373-36379Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar, 17Stojanovski D. Koutsopoulos O.S. Okamoto K. Ryan M.T. J. Cell Sci. 2004; 117: 1201-1210Crossref PubMed Scopus (256) Google Scholar, 18Yoon Y. Krueger E.W. Oswald B.J. McNiven M.A. Mol. Cell. Biol. 2003; 23: 5409-5420Crossref PubMed Scopus (629) Google Scholar) or may not (19Suzuki M. Jeong S.Y. Karbowski M. Youle R.J. Tjandra N. J. Mol. Biol. 2003; 334: 445-458Crossref PubMed Scopus (126) Google Scholar) accelerate mitochondrial fission, a reduction in human Fis1 level results in notable extensions in the length of mitochondria (17Stojanovski D. Koutsopoulos O.S. Okamoto K. Ryan M.T. J. Cell Sci. 2004; 117: 1201-1210Crossref PubMed Scopus (256) Google Scholar, 18Yoon Y. Krueger E.W. Oswald B.J. McNiven M.A. Mol. Cell. Biol. 2003; 23: 5409-5420Crossref PubMed Scopus (629) Google Scholar, 20Lee Y.J. Jeong S.Y. Karbowski M. Smith C.L. Youle R.J. Mol. Biol. Cell. 2004; 15: 5001-5011Crossref PubMed Scopus (839) Google Scholar), indicating that, as in yeast, Fis1 is required for mitochondrial fission in mammals. However, the regulation of Drp1- and Fis1-mediated mitochondrial fission remains unclear in both yeast and mammals. Comparing orthologs is one way to help understand the general scheme of protein function as well as their molecular evolution. In previous studies (19Suzuki M. Jeong S.Y. Karbowski M. Youle R.J. Tjandra N. J. Mol. Biol. 2003; 334: 445-458Crossref PubMed Scopus (126) Google Scholar, 21Dohm J.A. Lee S.J. Hardwick J.M. Hill R.B. Gittis A.G. Proteins. 2004; 54: 153-156Crossref PubMed Scopus (61) Google Scholar), it was reported that human Fis1 assembles into a novel tetratricopeptide repeat (TPR) 1The abbreviations used are: TPR, tetratricopeptide repeat; NOE, nuclear Overhauser effects; NOESY, NOE spectroscopy; PDB, Protein Data Bank.1The abbreviations used are: TPR, tetratricopeptide repeat; NOE, nuclear Overhauser effects; NOESY, NOE spectroscopy; PDB, Protein Data Bank.-like helix bundle, and it was suggested, based on similarity to other TPR domain proteins, that its concave surface may provide a means to recruit other proteins such as Drp1. Here we present the three-dimensional protein structure of yeast Fis1 and show that, in contrast to human Fis1, an extended N-terminal domain binds to the concave surface of the TPR motif. We also show that this self-interacting region of the N terminus that is absent in human Fis1 is required for yeast Fis1 bioactivity. Recombinant Protein—The protein corresponding to residues 1–138 of yeast Fis1 was prepared for NMR studies. The residues 139–152, half of the membrane-spanning domain and residues that face the mitochondrial intermembrane space, were excluded to solubilize the recombinant protein. The cDNA of Fis1 was cloned from S. cerevisiae genomic DNA library (American Type Culture Collection). NdeI and XhoI sites were introduced next to the initiation and stop codons of the cDNA, respectively, and inserted into pET-17b (Novagen Inc.). Then, the fragment corresponding to nucleotides 1–413 was excised using NdeI and RsaI. pET21b was treated with XhoI followed by Mung bean nuclease and then with NdeI. The excised DNA and the digested pET21b vector were ligated. The resulting plasmid encodes residues Met1–Val138 of Fis1 linked to the C-terminal hexa histidine tag and thus was termed pET21-yFis1-His6. Escherichia coli BL21(DE3) (Novagen Inc.) harboring the plasmid was cultured in Martek-9N or Martek-9CN (Spectra Stable Isotope) to produce uniformly 15N-labeled and uniformly 15N-, 13C-labeled protein, respectively. The recombinant protein was isolated from the cytosol by metal chelate affinity chromatography on a Ni2+-resin column (Novagen Inc.) using 20 mm Tris-HCl, pH 7.9, 500 mm NaCl, 0 or 100 mm EDTA and then further purified by collecting the flow-through from an ion-exchange chromatography on a mono-Q column (Amersham Biosciences) using 20 mm Tris-HCl, pH 8.9, without NaCl. No detergents were used in any step of the protein purification. All NMR samples contained 0.5–1.0 mm protein in 10 mm Tris acetate, pH 5.5, in 90% H2O/10% D2O or 100% D2O. The recombinant protein of human Fis1 for the study of backbone dynamics was prepared as described previously (19Suzuki M. Jeong S.Y. Karbowski M. Youle R.J. Tjandra N. J. Mol. Biol. 2003; 334: 445-458Crossref PubMed Scopus (126) Google Scholar). NMR Spectroscopy—All NMR spectra were acquired at 32 °C on Bruker 600 or 800 MHz NMR spectrometers. The spectra were processed using the NMRPipe (22Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11450) Google Scholar) and analyzed with PIPP (23Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (800) Google Scholar). The following experiments were used for assignments of 1H, 13C, and 15N resonances: CBCA(CO)NH (24Grzesiek S. Bax A. J. Am. Chem. Soc. 1992; 114: 6291-6293Crossref Scopus (923) Google Scholar), HNCACB (25Wittekind M. Mueller L. J. Magn. Reson. Ser. B. 1993; 101: 201-205Crossref Scopus (849) Google Scholar), HBHA(CO)NH (26Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Crossref Scopus (790) Google Scholar), HNCO (27Kay L.E. Xu G.Y. Yamazaki T. J. Magn. Reson. Ser. A. 1994; 109: 129-133Crossref Scopus (417) Google Scholar), and three-dimensional HCCH-TOCSY (28Bax A. Clore G.M. Gronenborn A.M. J. Magn. Reson. 1990; 88: 425-431Google Scholar). For stereospecific assignment for methyls of leucines and valines, a 1H-13C CT-HSQC experiment (29Vuister G.W. Bax A. J. Magn. Reson. 1992; 98: 428-435Google Scholar) was carried out using the protein obtained from a bacterial culture using a minimal medium containing 10% [U-13C]glucose-90% [U-12C]glucose (30Neri D. Szyperski T. Otting G. Senn H. Wuthrich K. Biochemistry. 1989; 28: 7510-7516Crossref PubMed Scopus (563) Google Scholar). Proton homonuclear nuclear Overhauser effects (NOEs) were obtained using three-dimensional 15N-edited NOESY (26Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Crossref Scopus (790) Google Scholar), four-dimensional 15N/13C-edited NOESY (31Kay L.E. Clore G.M. Bax A. Gronenborn A.M. Science. 1990; 249: 411-414Crossref PubMed Scopus (283) Google Scholar), and four-dimensional 13C/13C-edited NOESY (32Clore G.M. Kay L.E. Bax A. Gronenborn A.M. Biochemistry. 1991; 30: 12-18Crossref PubMed Scopus (168) Google Scholar) experiments. Residual dipolar couplings for N-H and Cα-Hα were calculated from the difference in corresponding scalar couplings measured in the presence and absence of Pf1 phage (11 mg/ml in 150 mm NaCl) (33Hansen M.R. Mueller L. Pardi A. Nat. Struct. Biol. 1998; 5: 1065-1074Crossref PubMed Scopus (687) Google Scholar). A two-dimensional IPAP 15N-1H HSQC experiment (34Ottiger M. Delaglio F. Bax A. J. Magn. Reson. 1998; 131: 373-378Crossref PubMed Scopus (837) Google Scholar) was used to obtain the one-bond N-H scalar couplings. A CT-(H)CA(CO)NH experiment (35Tjandra N. Bax A. J. Am. Chem. Soc. 1997; 119: 9576-9577Crossref Scopus (130) Google Scholar) was used to obtain the one-bond Cα-Hα scalar couplings. Relaxation values (15N T1 and 15N T1ρ) for backbone amides were calculated from the peak intensities measured using conventional pulse programs (36Barbato G. Ikura M. Kay L.E. Pastor R.W. Bax A. Biochemistry. 1992; 31: 5269-5278Crossref PubMed Scopus (889) Google Scholar). Steady state heteronuclear NOE for the backbone amides was derived from the ratio of peak intensities of experiments performed with and without 1H-presaturation using a reported pulse program and corrected to compensate for errors caused by incomplete 1H magnetization recovery (37Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12593-12594Crossref Scopus (1009) Google Scholar). The measurements were repeated twice. Structure Calculation—Peak intensities from NOESY experiments were translated into a continuous distribution of proton-proton distances. Generic hydrogen bond distance restraints were employed for α-helical regions that were determined based on secondary 13Cα chemical shifts and medium range NOE patterns. The TALOS program (38Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2732) Google Scholar) predicted the backbone dihedral angles (φ, Ψ) from 13Cα, 13Cβ, 13C′, 1Hα, and 15NH chemical shifts. Statistically significant angles were used as structural restraints with at least 20° margins. Residual dipolar coupling restraints were separated into mobile and rigid regions determined based on the backbone dynamic data. Structures of yeast Fis1 were calculated by a distance geometry and simulated annealing protocol (39Kuszewski J. Nilges M. Brunger A.T. J. Biomol. NMR. 1992; 2: 33-56Crossref PubMed Scopus (210) Google Scholar) with the incorporation of dipolar coupling restraints (40Tjandra N. Omichinski J.G. Gronenborn A.M. Clore G.M. Bax A. Nat. Struct. Biol. 1997; 4: 732-738Crossref PubMed Scopus (469) Google Scholar) using the program XPLOR-NIH (41Schwieters C.D. Kuszewski J.J. Tjandra N. Clore G.M. J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1854) Google Scholar). Structure calculations employed 2235 inter-residue and 803 intra-residue proton-proton distance restraints, 124 hydrogen bond distance restraints, 92 φ and 92 Ψ angle restraints, and 90 N-H and 41 Cα-Hα dipolar couplings. Fis1 Function in Yeast—Yeast vector pH62 was kindly provided by Dr. Reed B. Wickner (NIDDK, National Institutes of Health). pH62 is a derivative of pRS315 and contains CEN replicon, LEU2 marker, and ADH1 promoter. The DNA fragment containing Fis1 cDNA was excised from the pET17-yFis1 plasmid using XbaI and XhoI and then inserted into pH62 at XbaI and XhoI sites (pH62-FIS1). Plasmids for deletion mutants were constructed in the same way. The deletion mutant DM1 lacks the N-terminal 14 residues, DM2 lacks the N-terminal 5 residues, and DM3 lacks Phe6–Tyr14 (see Fig. 7A). All yeast strains are derived from strain yAN001 (met15 leu2 his3 ura3). Cells were transformed using the lithium acetate method (42Gietz D. St Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2883) Google Scholar) and were grown in synthetic glucose medium missing the appropriate nutrients to select for the plasmids. Mitochondria were visualized using a mitochondria-targeted GFP (pVT100U-mitoGFP; kindly provided by Dr. Benedikt Westermann, Universität Bayreuth, Germany). Exponentially growing cells of strains yAN008, yAN009, yAN010, yAN011, yAN012, and yAN013 (Table I) were embedded in 0.2% agarose, and confocal pictures were taken using a LSM 510 Meta (Zeiss). Mitochondria were classified in two different phenotypes. More than 200 cells were counted to obtain the percentage of the phenotypes.Table IYeast strainsStrainGenotypeyAN002fis1::G418yAN003fis1::G418 pH62-FIS1yAN004fis1::G418 pH62yAN005fis1::G418 pH62-FIS1-DM1yAN006fis1::G418 pH62-FIS1-DM2yAN007fis1::G418 pH62-FIS1-DM3yAN008fis1::G418 pVT100U-mitoGFPyAN009fis1::G418 pH62-FIS1 pVT100U-mitoGFPyAN010fis1::G418 pH62 pVT100U-mitoGFPyAN011fis1::G418 pH62-FIS1-DM1 pVT100U-mitoGFPyAN012fis1::G418 pH62-FIS1-DM2 pVT100U-mitoGFPyAN013fis1::G418 pH62-FIS1-DM3 pVT100U-mitoGFPyAN014fis1::G418 pGALL-GFP-MDV1yAN015fis1::G418 pH62-FIS1 pGALL-GFP-MDV1yAN016fis1::G418 pH62 pGALL-GFP-MDV1yAN017fis1::G418 pH62-FIS1-DM1 pGALL-GFP-MDV1yAN018fis1::G418 pH62-FIS1-DM2 pGALL-GFP-MDV1yAN019fis1::G418 pH62-FIS1-DM3 pGALL-GFP-MDV1 Open table in a new tab Detection of Exogenous Fis1 Expression in Δfis1 Yeast—Cells of strains yAN003, yAN004, yAN005, yAN006, and yAN007 (Table I) were grown overnight in synthetic medium (SD-Leu) to logarithmic phase and harvested by centrifugation. After washing once with ice-cold water, the cells were resuspended in ice-cold lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 50 mm NaF, 5 mm EDTA, 0.1% IGEPAL CA-630, 1% Triton X-100) supplemented with protein inhibitor mixture (Sigma) and mixed with acid-washed glass beads (Sigma). The cells were broken by shaking in a mixer mill (Retsch) for 5 min, and then debris was removed by 3 min of centrifugation at 4 °C. Protein lysates were analyzed by Western blot using rabbit anti-Fis1 antibody (kindly provided by Dr. Jodi Nunnari, University of California, Davis), mouse anti-porin antibody (Molecular Probes), and horseradish peroxidase-coupled secondary antibodies (Amersham Biosciences). Subcellular Localization of Mdv1—The Δfis1 yeast harboring pH62-FIS1 constructs was transformed with pGALL-GFP-MDV1 (kindly provided by Dr. Jodi Nunnari, University of California, Davis). The strains yAN015, yAN016, yAN017, yAN018, and yAN019 (Table I) were grown overnight in synthetic medium (SRaf-Leu-URA) containing 2% raffinose to allow galactose induction and lacking leucine and uracil to select for plasmids. Production of GFP-Mdv1 was induced by treatment with 2% galactose for 2 h. Cells were fixed and analyzed by confocal microscopy. Location of GFP-Mdv1 was classified in two categories: diffuse in cytosol or localized to mitochondria. More than 200 cells were analyzed to score the localization of GFP-Mdv1. Structure Description of Yeast Fis1—The NMR derived structures of yeast Fis1 are presented in Fig. 1. The core domain of Fis1 consists of six α-helices. The six α-helices are determined based on a combination of NMR data (Fig. 2), including midrange NOE patterns and secondary chemical shifts. We define helix α1 for residues Pro19–Ser31, α2 for Ile39–Lys51, α3 for Val55–Lys70, α4 for Arg76–Lys89, α5 for Tyr93–Glu105, and α6 for Lys111–Glu127. The first 6 residues of the N-terminal tail do not adopt an ordered conformation. Their 13Cα and 13Cβ chemical shifts are close to their random coil values, and no 1H-1H NOE of midrange or long range can be observed. The region corresponding to residues Leu10–Tyr14 shows characteristics of an α-helix, such as positive 13Cα secondary chemical shifts and NOEs between Hα(i) and HN(i + 3) and between Hα(i) and Hβ(i + 3). However, as NOEs between Hα(i) and HN(i + 4) are absent, this region is not defined to be an α-helix. The C-terminal 17 residues of the recombinant protein, which include the hydrophobic region and the artificial His tag, show a disordered conformation in solution. Their secondary chemical shifts for 13Cα and 13Cβ are close to zero, and no long range 1H-1H NOE are observed.Fig. 2Data obtained using NMR spectroscopy to establish the secondary structure of yeast Fis1. Sequential and medium range NOE connectivities characteristic of α-helices as well as 13Cα and 13Cβ secondary shifts are presented along with the protein sequence. The height of bars for the NOE connectivities presentation indicates the NOE intensity. The absence of typical NOEs within the α-helical regions mostly means that data were not included due to the difficulty of picking overlapped peaks. The height of the bars for the secondary shifts presentation are the difference from the random coil values. The location of the α-helices defined from the NMR data is indicated at the bottom.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As seen in the ensemble of the 20 lowest energy structures (Fig. 1A), the region corresponding to residues Thr9–Glu127 converged into a well defined conformation. The atomic root mean square deviation about the mean of coordinates for six α-helices of the 20 structures was 0.4 ± 0.1 Å for backbone heavy atoms and 1.0 ± 0.1 Å for all heavy atoms. A data base search performed using the DALI program for structural similarities (43Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3556) Google Scholar) revealed, at the top of the list, 1IYG, the structure of mouse Fis1 as a representative structure of Fis1 orthologs. Currently, there are three PDB depositions for Fis1 orthologs: 1PC2 is the solution structure of human Fis1 (19Suzuki M. Jeong S.Y. Karbowski M. Youle R.J. Tjandra N. J. Mol. Biol. 2003; 334: 445-458Crossref PubMed Scopus (126) Google Scholar), 1NZN is the crystal structure of human Fis1 (21Dohm J.A. Lee S.J. Hardwick J.M. Hill R.B. Gittis A.G. Proteins. 2004; 54: 153-156Crossref PubMed Scopus (61) Google Scholar), and 1IYG is the solution structure of mouse Fis1. The core domain of human, mouse, and yeast Fis1 contains six α-helices with the same folds. Due to the high degree of structural similarity between yeast and mouse Fis1, the rest of the list from the DALI search is the same as the list that we obtained previously using the human Fis1 structure, 1PC2, as a query (19Suzuki M. Jeong S.Y. Karbowski M. Youle R.J. Tjandra N. J. Mol. Biol. 2003; 334: 445-458Crossref PubMed Scopus (126) Google Scholar). Namely, the overall fold of the six α-helices in yeast Fis1 is similar to the fold of helices composed of the TPR motif, although, like other Fis1 sequences, no significant sequence similarity to the typical TPR motif is found within the TPR-like core domain of yeast Fis1. The typical TPR motif contains degenerate 34-amino-acid sequences with 8 loosely conserved consensus residues (X3(WLF)X2(LIM)(GAS)X2(YLF)X8(ASE)X3(FYL)X2(ASL)X4(PKE)X2) and usually presents in a tandem array of multiple copies (44Blatch G.L. Lassle M. BioEssays. 1999; 21: 932-939Crossref PubMed Scopus (954) Google Scholar). The TPR motif is found in a number of functionally different proteins. The TPR-containing domains facilitate specific protein-protein interactions at the concave surfaces, although the common features of the interaction partners have not been defined. The structural analogy of yeast Fis1 to the typical TPR-containing proteins, although the protein sequences are discrete, suggests that yeast Fis1 may bind to other proteins at its concave protein surface. Structural Comparison between Yeast and Human Fis1—To evaluate the structural conservation between yeast and human Fis1, we compared these two structures (Fig. 3). The core domain of both yeast and human Fis1 consists of six α-helices. The length of the α1-helix in yeast Fis1 is shorter than that in human Fis1 (Fig. 3A). For the other five α-helices, the lengths of corresponding helices in yeast and human Fis1 are the same. Moreover, the folds of six α-helices are similar (Fig. 3, B and C), as indicated by the data base search described in the previous section. The pairwise root mean square difference, calculated using the backbone atoms (N, Cα, C′, and O) of the corresponding six helices for yeast and human Fis1, is 1.8 Å. Among the six α-helices from the two structures, the α1-helix shows a major difference in its location; it is shifted along its axis and results in the largest deviation in the pairwise comparison. We previously found that human Fis1 contains two TPR-like motifs, where the α2-loop-α3 hairpin forms one TPR-like motif and the α4-loop-α5 hairpin forms the other. The two loops in the yeast Fis1 structure (one between α2 and α3 and the other between α4 and α5) show the same conformation as those in the human Fis1 structure. The pairwise comparison of overall folds between yeast and human Fis1 using DALI results in the Z-score of 12. A major difference between the yeast and human Fis1 structures is the N terminus prior to the α1-helix. The N-terminal tail of yeast Fis1 is located at the concave side of the helix bundle (Fig. 1), whereas that of human Fis1 is not. A difference can also be seen in the backbone dynamics of the region (Fig. 4). In the human Fis1 structure, the entire N-terminal tail is flexible (Fig. 4A). In contrast, the N terminus of the yeast Fis1, which is longer than that of human Fis1 by 8 residues (Fig. 3A), is made of two parts with different characteristics (Fig. 4B). The first portion from the N-terminal end (Met1–Phe6) is flexible, and the second portion (Trp7–Tyr18) is rigid. It is not clear where the boundary between the flexible and rigid regions is. The backbone dynamics of Trp7 cannot be measured because the cross-peak for the amide 15N and 1H chemical shifts of Trp7 overlaps with those of Val133 and Val134. However, the aromatic side chain of Trp7 appears to be stabilized by hydrophobic interaction as 1H-1H NOEs to Tyr81 within α4, Leu103 within α5, and Val113 within α6 are observed. The rigid portion (residues Trp7–Tyr18) is bound to the concave side that is composed of the six helices. Hydrophobic interactions between Trp7, Pro8, Leu10, Ala13 within this loop and hydrophobic residues at the concave surface of the TPR-like domain stabilize the loop (Fig. 5). In addition, two negatively charged residues, Asp12 and Glu15, are proximate of positively charged Lys51 (Fig. 5), indicating that electrostatic interactions assist in the stabilization of the loop. Lack of these interactions in human Fis1 relegates its N-terminal tail out of the concave surface of the TPR-like domain (Fig. 6A).Fig. 5The position of the N-terminal rigid loop region of yeast Fis1. A close-up view of the N-terminal loop and the concave surface of the TPR-like domain is shown. The protein is viewed from an orientation similar to that in Fig. 1. The electrostatic potential surface of the TPR-like domain is color-coded in red and blue to denote negative and positive residues, respectively. The side chains of the residues Trp7–Leu17 are represented by balls and sticks. The region in yellow is highly mobile.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6N termini of the recombinant proteins. A, the N terminus of the recombinant human Fis1 (PDB accession number = 1PC2) has a native sequence, which shows a
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