Unique Dimeric Structure of BNip3 Transmembrane Domain Suggests Membrane Permeabilization as a Cell Death Trigger*

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.

[1]  B. O’Rourke.  Mitochondrial ion channels. , 2007, Annual review of physiology.

[2]  M. Deckert,et al.  CD47 and the 19 kDa Interacting Protein-3 (BNIP3) in T Cell Apoptosis* , 2003, Journal of Biological Chemistry.

[3]  Peter Güntert,et al.  Automated NMR protein structure calculation , 2003 .

[4]  C. Thompson,et al.  The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability , 2000, Cell Death and Differentiation.

[5]  J C Reed,et al.  Mitochondria and apoptosis. , 1998, Science.

[6]  Brent D. Davis,et al.  Proapoptotic triterpene electrophiles (avicins) form channels in membranes: cholesterol dependence. , 2005, Biophysical journal.

[7]  D. Nolde,et al.  Processing of heteronuclear NMR relaxation data with the new software DASHA , 1995 .

[8]  V. Skulachev.  Bioenergetic aspects of apoptosis, necrosis and mitoptosis , 2006, Apoptosis.

[9]  Lianfa Shi,et al.  Nix and Nip3 Form a Subfamily of Pro-apoptotic Mitochondrial Proteins* , 1999, The Journal of Biological Chemistry.

[10]  R. Dwek,et al.  The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[11]  V. Sklenar.  Suppression of Radiation Damping in Multidimensional NMR Experiments Using Magnetic Field Gradients , 1995 .

[12]  Jack Greenblatt,et al.  Methods for Measurement of Intermolecular NOEs by Multinuclear NMR Spectroscopy: Application to a Bacteriophage λ N-Peptide/boxB RNA Complex , 1997 .

[13]  Mitochondrial hyperpolarization: a checkpoint of T-cell life, death and autoimmunity. , 2004, Trends in immunology.

[14]  K. Mayo,et al.  Motional Model Analyses of Protein and Peptide Dynamics Using 13C and 15N NMR Relaxation , 1997 .

[15]  Kurt Wüthrich,et al.  Lipid–protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  K. Webster,et al.  BNip3 and signal-specific programmed death in the heart. , 2005, Journal of molecular and cellular cardiology.

[17]  T. DeCoursey.  Voltage-gated proton channels and other proton transfer pathways. , 2003, Physiological reviews.

[18]  Christian Griesinger,et al.  Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients , 1999 .

[19]  E. Baker,et al.  Hydrogen bonding in globular proteins. , 1984, Progress in biophysics and molecular biology.

[20]  J. Lemasters,et al.  Voltage-dependent anion channel (VDAC) as mitochondrial governator--thinking outside the box. , 2006, Biochimica et biophysica acta.

[21]  K. MacKenzie,et al.  Sequence-specific Dimerization of the Transmembrane Domain of the “BH3-only” Protein BNIP3 in Membranes and Detergent* , 2003, Journal of Biological Chemistry.

[22]  J. Thornton,et al.  AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR , 1996, Journal of biomolecular NMR.

[23]  P. Pinton,et al.  pH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant. , 2005, Biochemical and biophysical research communications.

[24]  K. Mackenzie.  Folding and Stability of α-Helical Integral Membrane Proteins , 2006 .

[25]  Erika Davies,et al.  Ca2+-dependent Control of the Permeability Properties of the Mitochondrial Outer Membrane and Voltage-dependent Anion-selective Channel (VDAC)* , 2006, Journal of Biological Chemistry.

[26]  E. Carmeliet.  Cardiac ionic currents and acute ischemia: from channels to arrhythmias. , 1999, Physiological reviews.

[27]  W. DeGrado,et al.  Helix-packing motifs in membrane proteins , 2006, Proceedings of the National Academy of Sciences.

[28]  I. Arkin,et al.  How pH opens a H+ channel: the gating mechanism of influenza A M2. , 2005, Structure.

[29]  John Calvin Reed,et al.  BNIP3 Heterodimerizes with Bcl-2/Bcl-XL and Induces Cell Death Independent of a Bcl-2 Homology 3 (BH3) Domain at Both Mitochondrial and Nonmitochondrial Sites* , 2000, The Journal of Biological Chemistry.

[30]  Y. Tsujimoto.  Cell death regulation by the Bcl‐2 protein family in the mitochondria , 2003 .

[31]  Berk Hess,et al.  GROMACS 3.0: a package for molecular simulation and trajectory analysis , 2001 .

[32]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[33]  K. Webster,et al.  Acidosis regulates the stability, hydrophobicity, and activity of the BH3-only protein Bnip3. , 2006, Antioxidants & redox signaling.

[34]  M. Gouy,et al.  Phylogenomics of life-or-death switches in multicellular animals: Bcl-2, BH3-Only, and BNip families of apoptotic regulators. , 2005, Molecular biology and evolution.

[35]  Y. Tsujimoto,et al.  Proapoptotic BH3-only Bcl-2 family members induce cytochrome c release, but not mitochondrial membrane potential loss, and do not directly modulate voltage-dependent anion channel activity. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[36]  J. Alimonti,et al.  BNIP3 and Genetic Control of Necrosis-Like Cell Death through the Mitochondrial Permeability Transition Pore , 2000, Molecular and Cellular Biology.

[37]  R. Youle,et al.  Control of mitochondrial permeability by Bcl-2 family members. , 2004, Biochimica et biophysica acta.

[38]  A. Andersson,et al.  Magnetic resonance investigations of lipid motion in isotropic bicelles. , 2005, Langmuir : the ACS journal of surfaces and colloids.

[39]  K. MacKenzie,et al.  Sequence dependence of BNIP3 transmembrane domain dimerization implicates side-chain hydrogen bonding and a tandem GxxxG motif in specific helix-helix interactions. , 2006, Journal of molecular biology.

[40]  K. Webster,et al.  A unique pathway of cardiac myocyte death caused by hypoxia–acidosis , 2004, Journal of Experimental Biology.

[41]  A. Arseniev,et al.  From Structure and Dynamics of Protein L7/L12 to Molecular Switching in Ribosome*[boxs] , 2004, Journal of Biological Chemistry.

[42]  John Calvin Reed,et al.  Mitochondria-dependent apoptosis and cellular pH regulation , 2000, Cell Death and Differentiation.

[43]  H. Kalbitzer,et al.  Protein NMR Spectroscopy. Principles and Practice , 1997 .

[44]  A. Palmer,et al.  Protein NMR Spectroscopy: principles and practice, 2nd ed. , 2006 .

[45]  A. Bax,et al.  Protein backbone angle restraints from searching a database for chemical shift and sequence homology , 1999, Journal of biomolecular NMR.