Hydrophobic Α-helices Research Articles

Structural biology of the Bcl-2 family of proteins

The proteins of the Bcl-2 family are important regulators of programmed cell death. Structural studies of Bcl-2 family members have provided many important insights into their molecular mechanism of action and how members of this family interact with one another. To date, structural studies have been performed on six Bcl-2 family members encompassing both anti- (Bcl-xL, Bcl-2, KSHV-Bcl-2, Bcl-w) and pro-apoptotic (Bax, Bid) members. They all show a remarkably similar fold despite an overall divergence in amino acid sequence and function (pro-apoptotic versus anti-apoptotic). The three-dimensional structures of Bcl-2 family members consist of two central, predominantly hydrophobic α-helices surrounded by six or seven amphipathic α-helices of varying lengths. A long, unstructured loop is present between the first two α-helices. The structures of the Bcl-2 proteins show a striking similarity to the overall fold of the pore-forming domains of bacterial toxins. This finding led to experiments which demonstrated that Bcl-xL, Bcl-2, and Bax all form pores in artificial membranes. A prominent hydrophobic groove is present on the surface of the anti-apoptotic proteins. This groove is the binding site for peptides that mimic the BH3 region of various pro-apoptotic proteins such as Bak and Bad. Structures of Bcl-xL in complex with these BH3 peptides showed that they bind as an amphipathic α-helix and make extensive hydrophobic contacts with the protein. These data have not only helped to elucidate the interactions important for hetero-dimerization of Bcl-2 family members but have also been used to guide the discovery of small molecules that block Bcl-xL and Bcl-2 function. In the recently determined structure of the anti-apoptotic Bcl-w protein, the protein was also found to have a hydrophobic groove on its surface capable of binding BH3-containing proteins and peptides. However, in the native protein an additional carboxy-terminal α-helix interacts with the hydrophobic groove. This is reminiscent of how the carboxy-terminal α-helix of the pro-apoptotic protein Bax binds into its hydrophobic groove. This interaction may play a regulatory role and for Bax may explain why it is found predominately in the cytoplasm prior to activation. The hydrophobic groove of the pro-apoptotic protein, Bid protein, is neither as long nor as deep as that found in Bcl-xL, Bcl-2, or Bax. In addition, Bid contains an extra α-helix, which is located between α1 and α2 with respect to Bcl-xL, Bcl-2, and Bax. Although there are still many unanswered questions regarding the exact mechanism by which the Bcl-2 family of proteins modulates apoptosis, structural studies of these proteins have deepened our understanding of apoptosis on the molecular level.

Cumulative effects of amino acid substitutions and hydrophobic mismatch upon the transmembrane stability and conformation of hydrophobic alpha-helices.

The effects of amino acid substitutions upon the behavior of poly(Leu)-rich alpha-helices inserted into model membrane vesicles were investigated. One or two consecutive Leu residues in the hydrophobic core of the helix were substituted with A, F, G, S, D, K, H, P, GG, SS, PG, PP, KK, or DD residues. A Trp placed at the center of the sequence allowed assessment of peptide behavior via fluorescence emission lambda(max) and dual quenching analysis of Trp depth [Caputo, G. A., and London, E. (2003) Biochemistry 42, 3265-3274]. In vesicles composed of dioleoylphosphatidylcholine (DOPC), all of the peptides with single substitutions adopted a transmembrane (TM) state. Experiments were also performed in thicker bilayers composed of dierucoylphosphatidylcholine (DEuPC). In DEuPC vesicles TM states were destabilized by mismatch between helix length and bilayer thickness. Nevertheless, in DEuPC vesicles TM states were still prevalent for peptides with single substitutions, although less so for peptides with P, K, H, or D substitutions. In contrast to single substitutions, certain consecutive double substitutions strongly interfered with formation of TM states. In both DOPC and DEuPC vesicles DD and KK substitutions abolished the normal TM state, but GG and SS substitutions had little effect. In even wider bilayers, a SS substitution reduced the formation of a TM state. A peptide with a PP substitution maintained the TM state in DOPC vesicles, but in DEuPC vesicles the level of formation of the TM state was significantly reduced. Upon disruption of normal TM insertion peptides moved close to the bilayer surface, with the exception of the KK-substituted peptide in DOPC vesicles, which formed a truncated TM segment. These studies begin to provide a detailed relationship between sequence and the stability of TM insertion and show that the influence of insertion-destabilizing residues upon hydrophobic helices can be strongly modulated by properties such as mismatch. For certain helix-forming hydrophobic sequences, sensitivity to lipid structure may be sufficient to induce large conformational changes in vivo.

TBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c

TNFR1/Fas engagement results in the cleavage of cytosolic BID to truncated tBID, which translocates to mitochondria. Immunodepletion and gene disruption indicate BID is required for cytochrome c release. Surprisingly, the three-dimensional structure of this BH3 domain-only molecule revealed two hydrophobic alpha-helices suggesting tBID itself might be a pore-forming protein. Instead, we demonstrate that tBID functions as a membrane-targeted death ligand in which an intact BH3 domain is required for cytochrome c release, but not for targeting. Bak-deficient mitochondria and blocking antibodies reveal tBID binds to its mitochondrial partner BAK to release cytochrome c, a process independent of permeability transition. Activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux, integrating the pathway from death receptors to cell demise.

The humanTDE gene homologue: Localization to 20q13.1-13.3 and variable expression in human tumor cell lines and tissue

Expression of the mouse testicular tumor differentially expressed (TDE) gene is increased in testicular tumors as well as in testicular tumor cell lines derived from transgenic mice carrying the polyomavirus large-T antigen under the control of the metallothionein-1 promoter. To determine whether the TDE gene has a role in the development of human cancers, we used the mouse TDE cDNA sequence to screen a human placental cDNA library. The human TDE cDNA homologue coded for a protein that was 78% homologous to the mouse TDE amino acid sequence. Like the mouse TDE protein, the human TDE protein had several predicted hydrophobic alpha-helices characteristic of transmembrane proteins. The human TDE gene was expressed in all cell lines and tissues examined. Four mRNA species were observed in placenta, where we identified an alternate splicing pattern and the use of two different polyadenylation sites. Using fluoresence in situ hybridization analysis, we localized the human TDE gene to the q13.1-13.3 region of chromosome 20, a region known to be amplified in several types of human cancers. We then observed, by northern blot analysis, that human TDE expression was increased in three of five lung tumors examined.

The human TDE gene homologue: Localization to 20q13.1–13.3 and variable expression in human tumor cell lines and tissue

Expression of the mouse testicular tumor differentially expressed (TDE) gene is increased in testicular tumors as well as in testicular tumor cell lines derived from transgenic mice carrying the polyomavirus large-T antigen under the control of the metallothionein-1 promoter. To determine whether the TDE gene has a role in the development of human cancers, we used the mouse TDE cDNA sequence to screen a human placental cDNA library. The human TDE cDNA homologue coded for a protein that was 78% homologous to the mouse TDE amino acid sequence. Like the mouse TDE protein, the human TDE protein had several predicted hydrophobic α-helices characteristic of transmembrane proteins. The human TDE gene was expressed in all cell lines and tissues examined. Four mRNA species were observed in placenta, where we identified an alternate splicing pattern and the use of two different polyadenylation sites. Using fluoresence in situ hybridization analysis, we localized the human TDE gene to the q13.1–13.3 region of chromosome 20, a region known to be amplified in several types of human cancers. We then observed, by northern blot analysis, that human TDE expression was increased in three of five lung tumors examined. Mol. Carcinog. 26:189–200, 1999. © 1999 Wiley-Liss, Inc.

Transmembrane Orientation of Hydrophobic α-Helices Is Regulated Both by the Relationship of Helix Length to Bilayer Thickness and by the Cholesterol Concentration

A fluorescence-based approach to evaluate the regulation of transmembrane orientation of alpha-helices has been developed to examine the behavior of a membrane-inserted alpha-helical peptide with a 19 residue hydrophobic sequence. The emission lambdamax of a Trp residue in the helix was used to determine its location in the bilayer. To calibrate this method, Trp lambdamax and depth (determined by parallax analysis of fluorescence quenching) were measured for transmembrane peptides with Trp at different positions. Transmembrane orientation of the alpha-helix was found to be destabilized by differences between the width of the bilayer and the length of the hydrophobic sequence (i.e., hydrophobic mismatch). When bilayer width exceeded the length of the hydrophobic segment, mismatch induced formation of a nontransmembraneous orientation close to the polar/hydrocarbon interface. By manipulation of bilayer width in situ, it was found that the transmembrane and nontransmembrane orientations could interconvert. Cholesterol altered the transmembrane/nontransmembrane equilibrium to a degree consistent with its tendency to increase bilayer thickness. Evaluation of the energetics of transmembrane vs nontransmembrane insertion showed increased mismatch of a helix with bilayer width by the equivalent of just one hydrophobic residue can destabilize transmembrane orientation by roughly 0.5 kcal/mol. Inclusion of 30 mol % cholesterol in a bilayer can alter transmembrane insertion stability by 3-5 kcal/mol. Thus, physiologically relevant variations in both the hydrophobic helix length/membrane thickness ratio and the cholesterol levels influence transmembrane insertion significantly.

Channel formation by antiapoptotic protein Bcl-2.

Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-XL, suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic alpha-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (Deltah5, 6) mutant did not. The most frequent conductance observed (18 +/- 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 +/- 2 pS and 90 +/- 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.

Increase in mRNA of multiple Eh pgp genes encoding P-glycoprotein homologues in emetine-resistant Entamoeba histolytica parasites

With the goal of understanding possible mechanisms of drug resistance by the protozoan parasite Entamoeba histolytica (Eh), two novel Eh P-glycoprotein (Pgp) genes ( Eh pgp5 and Eh pgp6) were sequenced, and the expression of four Eh pgp genes determined in wild-type (wt) clone A and emetine-resistant (Em R) clone C2 amebae. The Eh pgp5 gene encodes a 1301-amino acid (aa) protein that is similar to those of Eh pgpl (64% aa identity), Eh pgp2 (61%), Eh pgp6 (39%) and Homo sapiens MDR (multidrug-resistance-encoding)(Hs MDR1; 38%) genes. The 1282-aa Eh pgp6 open reading frame (ORF), which is 19-28 aa shorter than those encoded by other Eh pgp, is also similar to those of Eh pgp1 (46% aa identity), Eh pgp2 (38%), and Hs MDR1 (39%). Both Eh pgp5 and Eh pgp6 ORF predict two ATP-binding cassettes and twelve hydrophobic α-helices, which form the putative transmembrane channel. Em R clone C2 amebae, growing at all concentrations of drug, show increased amounts of Eh pgpl and Eh pgp6 mRNA when compared to wt clone A amebae. In contrast, only clone C2 amebae selected for growth at the highest concentrations of emetine (100-200 μg/ml) show increased Eh pgp5 mRNA, while mRNA of both clone C2 and clone A Eh amebae fail to bind an Eh pgp2-specific probe. It appears then that multiple Pgp may contribute to amebic Em resistance in vitro.

Cloning and characterization of a pharmacologically distinct A 1 adenosine receptor from guinea pig brain

Three full-length cDNA clones encoding the guinea pig A 1 adenosine receptor have been isolated by polymerase chain reaction (PCR) and low-stringency hybridization screening of a guinea pig brain cDNA library. These three cDNAs, though differing in their 5′ untranslated regions, contain the same open reading frame encoding a 326 amino acid residue protein with seven hydrophobic α-helices long enough to form the transmembrane domains, suggesting that it belongs to the G protein-coupled receptor superfamily. This protein is highly homologous to the A 1 adenosine receptors previously cloned from other species. Pharmacological characterization of this receptor transiently expressed in mammalian cells demonstrates that, despite its high homology to A 1 adenosine receptors of other species, the guinea pig A 1 adenosine receptor displays a unique pharmacological profile: high affinity for the A 1-selective antagonist CPX, yet very low affinity for some A 1-selective agonists such as CCPA, CHA and R-PIA. Northern blotting for different guinea pig tissues and in situ hybridization for guinea pig brain sections reveal an abundant and broad distribution of mRNA of this A 1 subtype receptor in the brain.

Integral membrane protein structure: transmembrane α-helices as autonomous folding domains: Current opinion in structural biology 1993, 3: 532–540

The transmembrane region of many integral membrane proteins is made up of a bundle of hydrophobic α-helices. Such a structure could result from a two-stage folding process, during which preformed transmembrane helices with independent stability pack without topological rearrangement. This view was originally prompted by experiments in which fragments of transmembrane regions were separately refolded into lipid bilayers and subsequently brought together to yield a functional protein. Other lines of evidence, including the existence of ‘one-helix’ miniproteins, gene-fusion experiments, helix-driven oligomerization of bitopic proteins, and sequence rearrangements in the course of evolution support this view. Although it forms a useful basis for structural predictions, the limitations of the two-stage folding hypothesis are not clearly defined, and the proportion of integral membrane proteins to which it applies remains uncertain. The papers discussed in the present review illustrate recent progress along these lines.

Localisation of methionine residues in bacteriorhodopsin by carbonyl 13C-NMR with sequence-specific assignments

High-resolution 13C-NMR experiments have been performed on bacteriorhodopsin biosynthetically labeled with carbonyl- 13C amino acids and solubilized in the detergent dodecylmaltoside. 13C-NMR spectra showing good resolution were obtained in the case of labeled amino acids moderately represented in the BR sequence. For BR labeled with [ 13C]carbonyl methionine, several sequence-specific assignment could be performed by co-labeling with 15N amino acids or proteolysis. These assignments were used to obtain structural data on BR. Water-exposure of methionine side chains in the protein was assessed by studying, using NMR, their oxidation by hydrogen peroxide. Local secondary structure at the level of methionine residues was monitored through the effect of 1H- 2H exchange on NMR spectra. It was concluded that Met 32, Met 68 and Met 163 are peripheral while all 6 other methionine residues are deeply embedded within hydrophobic α-helices. These results confirm the current model of the BR folding and secondary structure.

Glycine and β-branched residues support and modulate peptide helicity in membrane environments

Transmembrane (TM) segments of integral membrane proteins are putatively α-helical in conformation once inserted into the membrane, yet consist of primary sequences rich in residues known in soluble proteins as helix-breakers (Gly) and β-sheet promoters (Ile, Val, Thr). To examine the specific 2° structure propensities of such residues in membrane environments, we have designed and synthesized a series of 20-residue peptides with ‘guest’' hydrophobic segments — expected to provide three turns of incipient α-helix content — embedded in ‘host’ hydrophilic (Lys-Ser) matrices. Circular dichroism (CD) spectra of the model peptides in water showed that significant helical content was observed only for peptides with high Ala content; others behaved as ‘random coils’. However, in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, it was found that Gly can be accommodated as readily as Ala, and Ile or Val as readily as Leu, in hydrophobic α-helices. Further subtleties of structural preferences could be observed in electrically-neutral lyso-phosphatidylcholine (LPC) micelles, where helical propensity decreased in the order Ala-Leu-rich > Gly-Leu-rich > Gly-Ile(Val)-rich hydrophobic segments. The results conjure a role of environment-dependent helix-modulation for Gly, Ile, and Val residues — and suggest that these residues may provide, in part, the structural basis for conformational transitions within or adjacent to membrane domains, such as those accompanying membrane insertion and/or required for transport or signalling functions.

Errata

Several conflicting models have been proposed for the membrane arrangement of the major myelin proteolipid (PLP). We have compared features of the sequence of PLP with those of other eukaryotic integral membrane proteins, with the view of identifying the most likely transmembrane topology. A new, simple model is suggested, which features four hydrophobic α-helices spanning the whole thickness of the lipid bilayer. Its orientation may be such that both the N-and C-termini face the cytosol. None of the biochemical, biophysical or immunological experiments hitherto reported provides incontrovertible evidence against the model. The effect or absence thereof of various PLP mutations is discussed in the frame, of the proposed 4-helix topology.