Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore.

In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the 'α3/α5' interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known 'α6:α6' interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH.

Organization of the mitochondrial apoptotic BAK pore: oligomerization of the BAK homodimers.

The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX "BH3-in-groove homodimer." Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a "worm hole."

Mitochondrial and Plasma Membrane Citrate Transporters: Discovery of Selective Inhibitors and Application to Structure/Function Analysis.

Cytoplasmic citrate is the prime carbon source for fatty acid, triacylglycerol, and cholesterol biosyntheses, and also regulates glucose metabolism via its allosteric inhibition of phosphofructokinase. It originates either via the efflux of citrate from the mitochondrial matrix on the inner membrane citrate transport protein (CTP) or via the influx of extracellular citrate on the plasma membrane citrate transporter (PMCT). Despite their common substrate, the two transport proteins share little sequence similarity and they transport citrate via fundamentally different mechanisms. We tested the ability of a set of previously identified CTP inhibitors, to inhibit the PMCT. We found that of the top 10 CTP inhibitors only one substantially inhibited the PMCT. Conversely, we identified two other inhibitors that inhibited the PMCT but had little effect on the CTP. All three identified PMCT inhibitors displayed a noncompetitive mechanism. Furthermore, models to explain inhibitor interactions with the CTP are proposed. As part of the present studies a PMCT homology model has been developed based on the crystal structure of the leucine transporter, and a possible citrate binding site has been identified and its composition compared with the two known citrate binding sites present within the CTP. The ability to selectively inhibit the PMCT may prove key to the pharmacologic amelioration of metabolic disorders resulting from the synthesis of excess lipid, cholesterol, and glucose, including human obesity, hyperlipidemia, hyper-cholesterolemia, and Type 2 diabetes.

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 Ni(2+)-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 alpha5-alpha6 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 A 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.

The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [(14)C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [(14)C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.

Inhibitors of the mitochondrial citrate transport protein: validation of the role of substrate binding residues and discovery of the first purely competitive inhibitor.

The mitochondrial citrate transport protein (CTP) is critical to energy metabolism in eukaryotic cells. We demonstrate that 1,2,3-benzenetricarboxylate (BTC), the classic and defining inhibitor of the mitochondrial CTP, is a mixed inhibitor of the reconstituted Cys-less CTP, with a strong competitive component [i.e., a competitive inhibition constant (K(ic)) of 0.12 +/- 0.02 mM and an uncompetitive inhibition constant (K(iu)) of 3.04 +/- 0.74 mM]. Based on docking calculations, a model for BTC binding has been developed. We then determined the K(ic) values for each of the eight substrate binding site cysteine substitution mutants and observed increases of 62- to 261-fold relative to the Cys-less control, thereby substantiating the importance of each of these residues in BTC binding. It is noteworthy that we observed parallel increases in the K(m) for citrate transport with each of these binding site mutants, thereby confirming that with these CTP variants, K(m) approximates the K(d) (for citrate) and is therefore a measure of substrate affinity. To further substantiate the importance of these binding site residues, in silico screening of a database of commercially available compounds has led to discovery of the first purely competitive inhibitor of the CTP. Docking calculations indicate that this inhibitor spans and binds to both substrate sites simultaneously. Finally, we propose a kinetic model for citrate transport in which the citrate molecule sequentially binds to the external and internal binding sites (per CTP monomer) before transport.