BNips: a group of pro-apoptotic proteins in the Bcl-2 family

BNip (formerly known as Nip) proteins, including homologues isolated from human, mouse and Caenorhabditis. elegans, are a relatively new subgroup of the Bcl-2 family. These proteins are classified into this family based on limited sequence homology with the Bcl-2 homology domain 3 and carboxyl terminal transmembrane domain. BNip proteins were first discovered based on their interaction with the adenovirus E1B 19 kDa/Bcl-2 family protein and since then, their roles in cell death pathways have been actively studied. However, the precise mechanisms by which the BNip proteins induce apoptosis and/or necrosis remain to be determined. To advance our knowledge, we have provided a summary and review of current literature regarding BNip proteins including comparative sequence analysis, mutational mapping of the functional domains, and cell death mechanisms involving disruption of mitochondrial homeostasis. Since BNip proteins are expressed at high levels in the heart as compared to other organs, their roles in cardiomyocyte injury during hypoxia or viral infection is a focus of this review. Finally, we discuss potential directions for further study on this increasingly important group of pro-apoptotic proteins.     

Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state

Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed alpha-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations.     

Conformational changes in BID, a pro-apoptotic BCL-2 family member, upon membrane binding. A site-directed spin labeling study

The BCL-2 family proteins constitute a critical control point in apoptosis. BCL-2 family proteins display structural homology to channel-forming bacterial toxins, such as colicins, transmembrane domain of diphtheria toxin, and the N-terminal domain of delta-endotoxin. By analogy, it has been hypothesized the BCL-2 family proteins would unfold and insert into the lipid bilayer upon membrane association. We applied the site-directed spin labeling method of electron paramagnetic resonance spectroscopy to the pro-apoptotic member BID. Here we show that helices 6-8 maintain an alpha-helical conformation in membranes with a lipid composition resembling mitochondrial outer membrane contact sites. However, unlike colicins and the transmembrane domain of diphtheria toxin, these helices of BID are bound to the lipid bilayer without adopting a transmembrane orientation. Our study presents a more detailed model for the reorganization of the structure of tBID on membranes.     

During apoptosis bcl-2 changes membrane topology at both the endoplasmic reticulum and mitochondria

In healthy cells the antiapoptotic protein Bcl-2 adopts a topology typical of tail-anchored proteins with only the hydrophobic carboxyl terminus inserted into the membrane, as shown by labeling cell lysates with a membrane-impermeant sulfhydryl-specific reagent. Induction of apoptosis in cells triggered a change in the conformation of Bcl-2 such that cysteine 158 near the base of helix 5 inserted into the lipid bilayer of both endoplasmic reticulum and mitochondria where it was protected from labeling. Addition of a peptide corresponding to the BH3 domain of the proapoptotic protein Bim to cell lysates triggered a similar conformational change in Bcl-2, demonstrating that preexisting, membrane-bound Bcl-2 proteins change topology.     

Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR

NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ∼75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ∼13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment.     

Structure, dynamics, and membrane topology of stannin: a mediator of neuronal cell apoptosis induced by trimethyltin chloride

Organotin compounds or alkyltins are ubiquitous environmental toxins that have been implicated in cellular death. Unlike other xenobiotic compounds, such as organomercurials and organoleads, alkyltins activate apoptotic cascades at low concentrations. Trimethyltin (TMT) chloride is amongst the most toxic organotin compounds, and is known to selectively inflict injury to specific regions of the brain. Stannin (SNN), an 88-residue mitochondrial membrane protein, has been identified as the specific marker for neuronal cell apoptosis induced by TMT intoxication. This high specificity of TMT makes SNN an ideal model system for understanding the mechanism of organotin neurotoxicity at a molecular level. Here, we report the three-dimensional structure and dynamics of SNN in detergent micelles, and its topological orientation in lipid bilayers as determined by solution and solid-state NMR spectroscopy. We found that SNN is a monotopic membrane protein composed of three domains: a single transmembrane helix (residues 10-33) that transverses the lipid bilayer at approximately a 20 degrees angle with respect to the membrane normal; a 28 residue unstructured linker, which includes a conserved CXC metal-binding motif and a putative 14-3-3zeta binding domain; and a distorted cytoplasmic helix (residues 61-79) that is partially absorbed into the plane of the lipid bilayer with a tilt angle of approximately 80 degrees from the membrane normal. The structure and architecture of SNN within the lipid environment provides insight about how this protein transmits toxic insults caused by TMT across the membrane.     

Influence of the extracellular domain size on the dynamic behavior of membrane proteins

The dynamic behavior of plasma membrane proteins mediates various cellular processes such as cellular motility, communication, and signaling. It is widely accepted that the dynamics of the membrane proteins is determined either by the interactions of the transmembrane domain with the surrounding lipids or by the interactions of the intracellular domain with cytosolic components such as cortical actin. Although initiation of different cellular signaling events at the plasma membrane has been attributed to the extracellular domain (ECD) properties recently, the impact of ECDs on the dynamic behavior of membrane proteins is rather unexplored. Here, we investigate how ECD properties influence protein dynamics in the lipid bilayer by reconstituting ECDs of different sizes or glycosylation in model membrane systems and analyzing ECD-driven protein sorting in lipid domains as well as protein mobility. Our data show that increasing the ECD mass or glycosylation leads to a decrease in ordered domain partitioning and diffusivity. Our data reconcile different mechanisms proposed for the initiation of cellular signaling by linking the ECD size of membrane proteins with their localization and diffusion dynamics in the plasma membrane.     

Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR

NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ∼75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ∼13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment.     

A multidomain outer membrane protein from Pasteurella multocida: modelling and simulation studies of PmOmpA

PmOmpA is a two-domain outer membrane protein from Pasteurella multocida. The N-terminal domain of PmOmpA is a homologue of the transmembrane beta-barrel domain of OmpA from Escherichia coli, whilst the C-terminal domain of PmOmpA is a homologue of the extra-membrane Neisseria meningitidis RmpM C-terminal domain. This enables a model of a complete two domain PmOmpA to be constructed and its conformational dynamics explored via MD simulations of the protein embedded within two different phospholipid bilayers (DMPC and DMPE). The conformational stability of the transmembrane beta-barrel is similar to that of a homology model of OprF from Pseudomonas aeruginosa in bilayer simulations. There is a degree of water penetration into the interior of the beta-barrel, suggestive of a possible transmembrane pore. Although the PmOmpA model is stable over 20 ns simulations, retaining its secondary structure and fold integrity throughout, substantial flexibility is observed in a short linker region between the N- and the C-terminal domains. At low ionic strength, the C-terminal domain moves to interact electrostatically with the lipid bilayer headgroups. This study demonstrates that computational approaches may be applied to more complex, multi-domain outer membrane proteins, rather than just to transmembrane beta-barrels, opening the possibility of in silico proteomics approaches to such proteins.     

Praf2 is a novel Bcl-xL/Bcl-2 interacting protein with the ability to modulate survival of cancer cells

Increased expression of Bcl-xL in cancer has been shown to confer resistance to a broad range of apoptotic stimuli and to modulate a number of other aspects of cellular physiology, including energy metabolism, cell cycle, autophagy, mitochondrial fission/fusion and cellular adhesion. However, only few of these activities have a mechanistic explanation. Here we used Tandem Affinity purification to identify novel Bcl-xL interacting proteins that could explain the pleiotropic effects of Bcl-xL overexpression. Among the several proteins co-purifying with Bcl-xL, we focused on Praf2, a protein with a predicted role in trafficking. The interaction of Praf2 with Bcl-xL was found to be dependent on the transmembrane domain of Bcl-xL. We found that Bcl-2 also interacts with Praf2 and that Bcl-xL and Bcl-2 can interact also with Arl6IP5, an homologue of Praf2. Interestingly, overexpression of Praf2 results in the translocation of Bax to mitochondria and the induction of apoptotic cell death. Praf2 dependent cell death is prevented by the co-transfection of Bcl-xL but not by its transmembrane domain deleted mutant. Accordingly, knock-down of Praf2 increases clonogenicity of U2OS cells following etoposide treatment by reducing cell death. In conclusion a screen for Bcl-xL-interacting membrane proteins let us identify a novel proapoptotic protein whose activity is strongly counteracted exclusively by membrane targeted Bcl-xL.     

BNip3 regulates mitochondrial function and lipid metabolism in the liver

BNip3 localizes to the outer mitochondrial membrane, where it functions in mitophagy and mitochondrial dynamics. While the BNip3 protein is constitutively expressed in adult liver from fed mice, we have shown that its expression is superinduced by fasting of mice, consistent with a role in responses to nutrient deprivation. Loss of BNip3 resulted in increased lipid synthesis in the liver that was associated with elevated ATP levels, reduced AMP-regulated kinase (AMPK) activity, and increased expression of lipogenic enzymes. Conversely, there was reduced β-oxidation of fatty acids in BNip3 null liver and also defective glucose output under fasting conditions. These metabolic defects in BNip3 null liver were linked to increased mitochondrial mass and increased hepatocellular respiration in the presence of glucose. However, despite elevated mitochondrial mass, an increased proportion of mitochondria exhibited loss of mitochondrial membrane potential, abnormal structure, and reduced oxygen consumption. Elevated reactive oxygen species, inflammation, and features of steatohepatitis were also observed in the livers of BNip3 null mice. These results identify a role for BNip3 in limiting mitochondrial mass and maintaining mitochondrial integrity in the liver that has consequences for lipid metabolism and disease.     

Conformational Changes in Talin on Binding to Anionic Phospholipid Membranes Facilitate Signaling by Integrin Transmembrane Helices

Integrins are heterodimeric (αβ) cell surface receptors that are activated to a high affinity state by the formation of a complex involving the α/β integrin transmembrane helix dimer, the head domain of talin (a cytoplasmic protein that links integrins to actin), and the membrane. The talin head domain contains four sub-domains (F0, F1, F2 and F3) with a long cationic loop inserted in the F1 domain. Here, we model the binding and interactions of the complete talin head domain with a phospholipid bilayer, using multiscale molecular dynamics simulations. The role of the inserted F1 loop, which is missing from the crystal structure of the talin head, PDB:3IVF, is explored. The results show that the talin head domain binds to the membrane predominantly via cationic regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel V-shaped conformation which optimizes its interactions with the membrane. Simulations of the complex of talin with the integrin α/β TM helix dimer in a membrane, show how this complex promotes a rearrangement, and eventual dissociation of, the integrin α and β transmembrane helices. A model for the talin-mediated integrin activation is proposed which describes how the mutual interplay of interactions between transmembrane helices, the cytoplasmic talin protein, and the lipid bilayer promotes integrin inside-out activation.     

Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins

Cell membranes undergo continuous curvature changes as a result of membrane trafficking and cell motility. Deformations are achieved both by forces extrinsic to the membrane as well as by structural modifications in the bilayer or at the bilayer surface that favor the acquisition of curvature. We report here that a family of proteins previously implicated in the regulation of the actin cytoskeleton also have powerful lipid bilayer-deforming properties via an N-terminal module (F-BAR) similar to the BAR domain. Several such proteins, like a subset of BAR domain proteins, bind to dynamin, a GTPase implicated in endocytosis and actin dynamics, via SH3 domains. The ability of BAR and F-BAR domain proteins to induce tubular invaginations of the plasma membrane is enhanced by disruption of the actin cytoskeleton and is antagonized by dynamin. These results suggest a close interplay between the mechanisms that control actin dynamics and those that mediate plasma membrane invagination and fission.     

A Coarse Grained Model for a Lipid Membrane with Physiological Composition and Leaflet Asymmetry

The resemblance of lipid membrane models to physiological membranes determines how well molecular dynamics (MD) simulations imitate the dynamic behavior of cell membranes and membrane proteins. Physiological lipid membranes are composed of multiple types of phospholipids, and the leaflet compositions are generally asymmetric. Here we describe an approach for self-assembly of a Coarse-Grained (CG) membrane model with physiological composition and leaflet asymmetry using the MARTINI force field. An initial set-up of two boxes with different types of lipids according to the leaflet asymmetry of mammalian cell membranes stacked with 0.5 nm overlap, reliably resulted in the self-assembly of bilayer membranes with leaflet asymmetry resembling that of physiological mammalian cell membranes. Self-assembly in the presence of a fragment of the plasma membrane protein syntaxin 1A led to spontaneous specific positioning of phosphatidylionositol(4,5)bisphosphate at a positively charged stretch of syntaxin consistent with experimental data. An analogous approach choosing an initial set-up with two concentric shells filled with different lipid types results in successful assembly of a spherical vesicle with asymmetric leaflet composition. Self-assembly of the vesicle in the presence of the synaptic vesicle protein synaptobrevin 2 revealed the correct position of the synaptobrevin transmembrane domain. This is the first CG MD method to form a membrane with physiological lipid composition as well as leaflet asymmetry by self-assembly and will enable unbiased studies of the incorporation and dynamics of membrane proteins in more realistic CG membrane models.     

Sequence-dependent backbone dynamics of a viral fusogen transmembrane helix

The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing as indicated by mutational studies and functional reconstitution of peptide mimics. Here, we demonstrate that mutations of a GxxxG motif or of Ile residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics as determined by deuterium/hydrogen-exchange kinetics. Thus, the backbone dynamics of these helices may be linked to their fusogenicity which is consistent with the known over-representation of Gly and Ile in viral fusogen transmembrane helices. The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing. Our present results demonstrate that mutations of certain residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics. Thus, the data suggest a relationship between sequence, backbone dynamics, and fusogenicity of transmembrane segments of viral fusogenic proteins.     

Biophysical properties of lipids and dynamic membranes

The lipid bilayer is a 3D assembly with a rich variety of physical features that modulate cell signaling and protein function. Lateral and transverse forces within the membrane are significant and change rapidly as the membrane is bent or stretched and as new constituents are added, removed or chemically modified. Recent studies have revealed how differences in structure between the two leaflets of the bilayer and between different areas of the bilayer can interact together with membrane deformation to alter the activities of transmembrane channels and peripheral membrane binding proteins. Here, we highlight some recent reports that the physical properties of the membrane can help control the function of transmembrane proteins and the motor-dependent elongation of internal organelles, such as the endoplasmic reticulum.     

The membrane environment modulates self-association of the human GpA TM domain—Implications for membrane protein folding and transmembrane signaling

The influence of lipid bilayer properties on a defined and sequence-specific transmembrane helix-helix interaction is not well characterized yet. To study the potential impact of changing bilayer properties on a sequence-specific transmembrane helix-helix interaction, we have traced the association of fluorescent-labeled glycophorin A transmembrane peptides by fluorescence spectroscopy in model membranes with varying lipid compositions. The observed changes of the glycophorin A dimerization propensities in different lipid bilayers suggest that the lipid bilayer thickness severely influences the monomer-dimer equilibrium of this transmembrane domain, and dimerization was most efficient under hydrophobic matching conditions. Moreover, cholesterol considerably promotes self-association of transmembrane helices in model membranes by affecting the lipid acyl chain ordering. In general, the order of the lipid acyl chains appears to be an important factor involved in determining the strength and stability of transmembrane helix-helix interactions. As discussed, the described influences of membrane properties on transmembrane helix-helix interactions are highly important for understanding the mechanism of transmembrane protein folding and functioning as well as for gaining a deeper insight into the regulation of signal transduction via membrane integral proteins by bilayer properties.     

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.