Asymmetry in the homodimeric ABC transporter MsbA recognized by a DARPin

ABC transporters harness the energy from ATP binding and hydrolysis to translocate substrates across the membrane. Binding of two ATP molecules at the nucleotide binding domains (NBDs) leads to the formation of an outward-facing state. The conformational changes required to reset the transporter to the inward-facing state are initiated by sequential hydrolysis of the bound nucleotides. In a homodimeric ABC exporter such as MsbA responsible for lipid A transport in Escherichia coli, sequential ATP hydrolysis implies the existence of an asymmetric conformation. Here we report the in vitro selection of a designed ankyrin repeat protein (DARPin) specifically binding to detergent-solubilized MsbA. Only one DARPin binds to the homodimeric transporter in the absence as well as in the presence of nucleotides, suggesting that it recognizes asymmetries in MsbA. DARPin binding increases the rate of ATP hydrolysis by a factor of two independent of the substrate-induced ATPase stimulation. Electron paramagnetic resonance (EPR) measurements are found to be in good agreement with the available crystal structures and reveal that DARPin binding does not affect the large nucleotide-driven conformational changes of MsbA. The binding epitope was mapped by cross-linking and EPR to the membrane-spanning part of the transmembrane domain (TMD). Using cross-linked DARPin-MsbA complexes, 8-azido-ATP was found to preferentially photolabel one chain of the homodimer, suggesting that the asymmetries captured by DARPin binding at the TMDs are propagated to the NBDs. This work demonstrates that in vitro selected binders are useful tools to study the mechanism of membrane proteins.     

Transmembrane gate movements in the type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.     

Conformational Cycle of the ABC Transporter MsbA in Liposomes: Detailed Analysis Using Double Electron-Electron Resonance Spectroscopy

Driven by the energy of ATP binding and hydrolysis, ATP-binding cassette transporters alternate between inward- and outward-facing conformations, allowing vectorial movement of substrates. Conflicting models have been proposed to describe the conformational motion underlying this switch in access of the transport pathway. One model, based on three crystal structures of the lipid flippase MsbA, envisions a large-amplitude motion that disengages the nucleotide-binding domains and repacks the transmembrane helices. To test this model and place the crystal structures in a mechanistic context, we use spin labeling and double electron-electron resonance spectroscopy to define the nature and amplitude of MsbA conformational change during ATP hydrolysis cycle. For this purpose, spin labels were introduced at sites selected to provide a distinctive pattern of distance changes unique to the crystallographic transformation. Distance changes in liposomes, induced by the transition from nucleotide-free MsbA to the highest energy intermediate, fit a simple pattern whereby residues on the cytoplasmic side undergo 20-30 Å closing motion while a 7- to 10-Å opening motion is observed on the extracellular side. The transmembrane helices undergo relative movement to create the outward opening consistent with that implied by the crystal structures. Double electron-electron resonance distance distributions reveal asymmetric backbone flexibility on the two sides of the transporter that correlates with asymmetric opening of the substrate-binding chamber. Together with extensive accessibility analysis, our results suggest that these structures capture features of the motion that couples ATP energy expenditure to work, providing a framework for the mechanism of substrate transport.     

EPR Spectroscopy of MolB2C2-A Reveals Mechanism of Transport for a Bacterial Type II Molybdate Importer

In bacteria, ATP-binding cassette (ABC) transporters are vital for the uptake of nutrients and cofactors. Based on differences in structure and activity, ABC importers are divided into two types. Type I transporters have been well studied and employ a tightly regulated alternating access mechanism. Less is known about Type II importers, but much of what we do know has been observed in studies of the vitamin B₁₂ importer BtuC₂D₂. MolB₂C₂ (formally known as HI1470/71) is also a Type II importer, but its substrate, molybdate, is ∼10-fold smaller than vitamin B₁₂. To understand mechanistic differences among Type II importers, we focused our studies on MolBC, for which alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed disulfide cross-linking and EPR spectroscopy. From these studies, we found that nucleotide binding is coupled to a conformational shift at the periplasmic gate. Unlike the larger conformational changes in BtuCD-F, this shift in MolBC-A is akin to unlocking a swinging door: allowing just enough space for molybdate to slip into the cell. The lower cytoplasmic gate, identified in BtuCD-F as “gate I,” remains open throughout the MolBC-A mechanism, and cytoplasmic gate II closes in the presence of nucleotide. Combining our results, we propose a peristaltic mechanism for MolBC-A, which gives new insight in the transport of small substrates by a Type II importer.     

On the Origin of Large Flexibility of P-glycoprotein in the Inward-facing State

P-glycoprotein (Pgp) is one of the most biomedically relevant transporters in the ATP binding cassette (ABC) superfamily due to its involvement in developing multidrug resistance in cancer cells. Employing molecular dynamics simulations and double electron-electron resonance spectroscopy, we have investigated the structural dynamics of membrane-bound Pgp in the inward-facing state and found that Pgp adopts an unexpectedly wide range of conformations, highlighted by the degree of separation between the two nucleotide-binding domains (NBDs). The distance between the two NBDs in the equilibrium simulations covers a range of at least 20 Å, including, both, more open and more closed NBD configurations than the crystal structure. The double electron-electron resonance measurements on spin-labeled Pgp mutants also show wide distributions covering both longer and shorter distances than those observed in the crystal structure. Based on structural and sequence analyses, we propose that the transmembrane domains of Pgp might be more flexible than other structurally known ABC exporters. The structural flexibility of Pgp demonstrated here is not only in close agreement with, but also helps rationalize, the reported high NBD fluctuations in several ABC exporters and possibly represents a fundamental difference in the transport mechanism between ABC exporters and ABC importers. In addition, during the simulations we have captured partial entrance of a lipid molecule from the bilayer into the lumen of Pgp, reaching the putative drug binding site. The location of the protruding lipid suggests a putative pathway for direct drug recruitment from the membrane.     

Conformational Transitions Underlying Pore Opening and Desensitization in Membrane-embedded Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Direct structural insight into the mechanisms underlying activation and desensitization remain unavailable for the pentameric ligand-gated channel family. Here, we report the structural rearrangements underlying gating transitions in membrane-embedded GLIC, a prokaryotic homologue, using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. We particularly probed the conformation of pore-lining second transmembrane segment (M2) under conditions that favor the closed and the ligand-bound desensitized states. The spin label mobility, intersubunit spin-spin proximity, and the solvent-accessibility parameters in the two states clearly delineate the underlying protein motions within M2. Our results show that during activation the extracellular hydrophobic region undergoes major changes involving an outward translational movement, away from the pore axis, leading to an increase in the pore diameter, whereas the lower end of M2 remains relatively immobile. Most notably, during desensitization, the intervening polar residues in the middle of M2 move closer to form a solvent-occluded barrier and thereby reveal the location of a distinct desensitization gate. In comparison with the crystal structure of GLIC, the structural dynamics of the channel in a membrane environment suggest a more loosely packed conformation with water-accessible intrasubunit vestibules penetrating from the extracellular end all the way to the middle of M2 in the closed state. These regions have been implicated to play a major role in alcohol and drug modulation. Overall, these findings represent a key step toward understanding the fundamentals of gating mechanisms in this class of channels.     

Uncoupling substrate transport from ATP hydrolysis in the Escherichia coli maltose transporter

Members of the ATP-binding cassette superfamily couple the energy from ATP hydrolysis to the active transport of substrates across the membrane. The maltose transporter, a well characterized model system, consists of a periplasmic maltose-binding protein (MBP) and a multisubunit membrane transporter, MalFGK(2). On the basis of the structure of the MBP-MalFGK(2) complex in an outward-facing conformation (Oldham, M. L., Khare, D., Quiocho, F. A., Davidson, A. L., and Chen, J. (2007) Nature 450, 515-521), we identified two mutants in transmembrane domains MalF and MalG that generated futile cycling; although interaction with MBP stimulated the ATPase activity of the transporter, maltose was not transported. Both mutants appeared to disrupt the normal transfer of maltose from MBP to MalFGK(2). In the first case, substitution of aspartate for glycine in the maltose-binding site of MalF likely generated a futile cycle by preventing maltose from binding to MalFGK(2) during the catalytic cycle. In the second case, a four-residue deletion of a periplasmic loop of MalG limited its reach into the maltose-binding pocket of MBP, allowing maltose to remain associated with MBP during the catalytic cycle. Retention of maltose in the MBP binding site in the deletion mutant, as well as insertion of this loop into the binding site in the wild type, was detected by EPR as a change in mobility of a nitroxide spin label positioned near the maltose-binding pocket of MBP.     

In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC₂; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC₂, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg²⁺. We were able to purify a full complex, RbsABC₂, in the presence of stable, transition state mimics (ATP, Mg²⁺, and VO₄); a RbsAC complex in the presence of ADP and Mg²⁺; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC₂ shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.     

Small Substrate Transport and Mechanism of a Molybdate ATP Binding Cassette Transporter in a Lipid Environment

Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria.     

Rigid core and flexible terminus: structure of solubilized light-harvesting chlorophyll a/b complex (LHCII) measured by EPR

The structure of the major light-harvesting chlorophyll a/b complex (LHCII) was analyzed by pulsed EPR measurements and compared with the crystal structure. Site-specific spin labeling of the recombinant protein allowed the measurement of distance distributions over several intra- and intermolecular distances in monomeric and trimeric LHCII, yielding information on the protein structure and its local flexibility. A spin label rotamer library based on a molecular dynamics simulation was used to take the local mobility of spin labels into account. The core of LHCII in solution adopts a structure very similar or identical to the one seen in crystallized LHCII trimers with little motional freedom as indicated by narrow distance distributions along and between α helices. However, distances comprising the lumenal loop domain show broader distance distributions, indicating some mobility of this loop structure. Positions in the hydrophilic N-terminal domain, upstream of the first trans-membrane α helix, exhibit more and more mobility the closer they are to the N terminus. The nine amino acids at the very N terminus that have not been resolved in any of the crystal structure analyses give rise to very broad and possibly bimodal distance distributions, which may represent two families of preferred conformations.     

Characterization of the Enzyme CbiH60 Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway

The anaerobic pathway for the biosynthesis of cobalamin (vitamin B₁₂) has remained poorly characterized because of the sensitivity of the pathway intermediates to oxygen and the low activity of enzymes. One of the major bottlenecks in the anaerobic pathway is the ring contraction step, which has not been observed previously with a purified enzyme system. The Gram-positive aerobic bacterium Bacillus megaterium has a complete anaerobic pathway that contains an unusual ring contraction enzyme, CbiH₆₀, that harbors a C-terminal extension with sequence similarity to the nitrite/sulfite reductase family. To improve solubility, the enzyme was homologously produced in the host B. megaterium DSM319. CbiH₆₀ was characterized by electron paramagnetic resonance and shown to contain a [4Fe-4S] center. Assays with purified recombinant CbiH₆₀ demonstrate that the enzyme converts both cobalt-precorrin-3 and cobalt factor III into the ring-contracted product cobalt-precorrin-4 in high yields, with the latter transformation dependent upon DTT and an intact Fe-S center. Furthermore, the ring contraction process was shown not to involve a change in the oxidation state of the central cobalt ion of the macrocycle.     

Desensitization mechanism in prokaryotic ligand-gated ion channel

Crystal structures of Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated prokaryotic homologue of pentameric ligand-gated ion channel (LGIC) from G. violaceus, have provided high-resolution models of the channel architecture and its role in selective ion conduction and drug binding. However, it is still unclear which functional states of the LGIC gating scheme these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. To elucidate the molecular events that constitute gating, we have carried out an extensive characterization of GLIC function and dynamics in reconstituted proteoliposomes by patch clamp measurements and EPR spectroscopy. We find that GLIC channels show rapid activation upon jumps to acidic pH followed by a time-dependent loss of conductance because of desensitization. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol. Many of these properties are parallel to functions observed in members of eukaryotic LGIC. Conformational changes in loop C, measured by site-directed spin labeling and EPR spectroscopy, reveal immobilization during desensitization analogous to changes in LGIC and acetylcholine binding protein. Together, our studies suggest conservation of mechanistic aspects of desensitization among LGICs of prokaryotic and eukaryotic origin.     

Membrane Region M2C2 in Subunit KtrB of the K+ Uptake System KtrAB from Vibrio alginolyticus Forms a Flexible Gate Controlling K+ Flux: AN ELECTRON PARAMAGNETIC RESONANCE STUDY*

Transmembrane stretch M_2C from the bacterial K⁺-translocating protein KtrB is unusually long. In its middle part, termed M_2C2, it contains several small and polar amino acids. This region is flanked by the two α-helices M_2C1 and M_2C3 and may form a flexible gate at the cytoplasmic side of the membrane controlling K⁺ translocation. In this study, we provide experimental evidence for this notion by using continuous wave and pulse EPR measurements of single and double spin-labeled cysteine variants of KtrB. Most of the spin-labeled residues in M_2C2 were shown to be immobile, pointing to a compact structure. However, the high polarity revealed for the microenvironment of residue positions 317, 318, and 327 indicated the existence of a water-accessible cavity. Upon the addition of K⁺ ions, M_2C2 residue Thr-318R1 (R1 indicates the bound spin label) moved with respect to M_2B residue Asp-222R1 and M_2C3 residue Val-331R1 but not with respect to M_2C1 residue Met-311R1. Based on distances determined between spin-labeled residues of double-labeled variants of KtrB in the presence and absence of K⁺ ions, structural models of the open and closed conformations were developed.     

Roles of Amphipathic Helices and the Bin/Amphiphysin/Rvs (BAR) Domain of Endophilin in Membrane Curvature Generation

Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63–86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63–75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic α-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.     

Determination of structural models of the complex between the cytoplasmic domain of erythrocyte band 3 and ankyrin-R repeats 13-24

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.     

Identification of Receptor Binding-induced Conformational Changes in Non-visual Arrestins

The non-visual arrestins, arrestin-2 and arrestin-3, belong to a small family of multifunctional cytosolic proteins. Non-visual arrestins interact with hundreds of G protein-coupled receptors (GPCRs) and regulate GPCR desensitization by binding active phosphorylated GPCRs and uncoupling them from heterotrimeric G proteins. Recently, non-visual arrestins have been shown to mediate G protein-independent signaling by serving as adaptors and scaffolds that assemble multiprotein complexes. By recruiting various partners, including trafficking and signaling proteins, directly to GPCRs, non-visual arrestins connect activated receptors to diverse signaling pathways. To investigate arrestin-mediated signaling, a structural understanding of arrestin activation and interaction with GPCRs is essential. Here we identified global and local conformational changes in the non-visual arrestins upon binding to the model GPCR rhodopsin. To detect conformational changes, pairs of spin labels were introduced into arrestin-2 and arrestin-3, and the interspin distances in the absence and presence of the receptor were measured by double electron electron resonance spectroscopy. Our data indicate that both non-visual arrestins undergo several conformational changes similar to arrestin-1, including the finger loop moving toward the predicted location of the receptor in the complex as well as the C-tail release upon receptor binding. The arrestin-2 results also suggest that there is no clam shell-like closure of the N- and C-domains and that the loop containing residue 136 (homolog of 139 in arrestin-1) has high flexibility in both free and receptor-bound states.     

Binding of the CYK-4 Subunit of the Centralspindlin Complex Induces a Large Scale Conformational Change in the Kinesin Subunit

Centralspindlin is a critical regulator of cytokinesis in animal cells. It is a tetramer consisting of ZEN-4/MKLP1, a kinesin-6 motor, and CYK-4/MgcRacGAP, a Rho GTPase-activating protein. At anaphase, centralspindlin localizes to a narrow region of antiparallel microtubule overlap and initiates central spindle assembly. Central spindle assembly requires complex formation between ZEN-4 and CYK-4. However, the structural consequences of CYK-4 binding to ZEN-4 are unclear as are the mechanisms of microtubule bundling. Here we investigate whether CYK-4 binding induces a conformational change in ZEN-4. Characterization of the structure and conformational dynamics of the minimal interacting regions between ZEN-4 and CYK-4 by continuous wave EPR and double electron-electron resonance (DEER) spectroscopy reveals that CYK-4 binding dramatically stabilizes the relative positions of the neck linker regions of ZEN-4. Additionally, our data indicate that each neck linker is similarly structured in the bound and unbound states. CYK-4 binding decreases the rate of ZEN-4-mediated microtubule gliding. These results constrain models for the molecular organization of centralspindlin.     

Generating Symmetry in the Asymmetric ATP-binding Cassette (ABC) Transporter Pdr5 from Saccharomyces cerevisiae

Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely “regenerated” Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters.     

Stabilization of G Domain Conformations in the tRNA-modifying MnmE-GidA Complex Observed with Double Electron Electron Resonance Spectroscopy

MnmE is a GTP-binding protein conserved between bacteria and eukarya. It is a dimeric three-domain protein where the two G domains have to approach each other for activation of the potassium-stimulated GTPase reaction. Together with GidA, in a heterotetrameric α₂β₂ complex, it is involved in the modification of the wobble uridine base U34 of the first anticodon position of particular tRNAs. Here we show, using various spin-labeled MnmE mutants and EPR spectroscopy, that GidA binding induces large conformational and dynamic changes in MnmE. It stimulates the GTPase reaction by stabilizing the GTP-bound conformation in a potassium-independent manner. Surprisingly, GidA binding influences not only the GTP- but also the GDP-bound conformation. Thus GidA is a new type of regulator for a G protein activated by dimerization.     

Conformational Changes in BAK,a Pore-forming Proapoptotic Bcl-2 Family Member,upon Membrane Insertion and Direct Evidence for the Existence of BH3-BH3 Contact Interface in BAK Homo-oligomers

During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein. Using a soluble form of BAK with a hexahistidine tag at the C terminus and a liposomal system containing the Ni²⁺-nitrilotriacetic acid lipid analog that can bind hexahistidine-tagged proteins, BAK oligomers were formed in the presence of the activator protein p7/p15Bid. In this system, we determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR to 13 different amino acid locations. Upon membrane insertion, the BH3 domains were reorganized, and the α5-α6 helical hairpin structure was partially exposed to the membrane environment. The monomer-monomer interface in the oligomeric structure was also mapped by measuring the distance-dependent spin-spin interactions for each residue location. Spin labels attached in the BH3 domain were juxtaposed within 5–10 Å distance in the oligomeric form in the membrane. These results are consistent with the current hypothesis that BAK or BAX forms homodimers, and these homodimers assemble into a higher order oligomeric pore. Detailed analyses of the data provide new insights into the structure of the BAX or BAK homodimer.