Isolated Transmembrane Domain Research Articles

Thermodynamically reversible paths of the first fusion intermediate reveal an important role for membrane anchors of fusion proteins

Biological membrane fusion proceeds via an essential topological transition of the two membranes involved. Known players such as certain lipid species and fusion proteins are generally believed to alter the free energy and thus the rate of the fusion reaction. Quantifying these effects by theory poses a major challenge since the essential reaction intermediates are collective, diffusive and of a molecular length scale. We conducted molecular dynamics simulations in conjunction with a state-of-the-art string method to resolve the minimum free-energy path of the first fusion intermediate state, the so-called stalk. We demonstrate that the isolated transmembrane domains (TMDs) of fusion proteins such as SNARE molecules drastically lower the free energy of both the stalk barrier and metastable stalk, which is not trivially explained by molecular shape arguments. We relate this effect to the local thinning of the membrane (negative hydrophobic mismatch) imposed by the TMDs which favors the nearby presence of the highly bent stalk structure or prestalk dimple. The distance between the membranes is the most crucial determinant of the free energy of the stalk, whereas the free-energy barrier changes only slightly. Surprisingly, fusion enhancing lipids, i.e., lipids with a negative spontaneous curvature, such as PE lipids have little effect on the free energy of the stalk barrier, likely because of its single molecular nature. In contrast, the lipid shape plays a crucial role in overcoming the hydration repulsion between two membranes and thus rather lowers the total work required to form a stalk.

Death Receptor 5 Activation Is Energetically Coupled to Opening of the Transmembrane Domain Dimer

The precise mechanism by which binding of tumor necrosis factor ligands to the extracellular domain of their corresponding receptors transmits signals across the plasma membrane has remained elusive. Recent studies have proposed that activation of several tumor necrosis factor receptors, including Death Receptor 5, involves a scissorlike opening of the disulfide-linked transmembrane (TM) dimer. Using time-resolved fluorescence resonance energy transfer, we provide, to our knowledge, the first direct biophysical evidence that Death Receptor 5 TM-dimers open in response to ligand binding. Then, to probe the importance of the closed-to-open TM domain transition in the overall energetics of receptor activation, we designed point-mutants (alanine to phenylalanine) in the predicted, tightly packed TM domain dimer interface. We hypothesized that the bulky residues should destabilize the closed conformation and eliminate the ∼3 kcal/mol energy barrier to TM domain opening and the ∼2 kcal/mol energy difference between the closed and open states, thus oversensitizing the receptor. To test this, we used all-atom molecular dynamics simulations of the isolated TM domain in explicit lipid bilayers coupled to thermodynamic potential of mean force calculations. We showed that single point mutants at the interface altered the energy landscape as predicted, but were not enough to completely eliminate the barrier to opening. However, the computational model did predict that a double mutation at i, i+4 positions at the center of the TM domain dimer eliminates the barrier and stabilizes the open conformation relative to the closed. We tested these mutants in cells with time-resolved fluorescence resonance energy transfer and death assays, and show remarkable agreement with the calculations. The single mutants had a small effect on TM domain separation and cell death, whereas the double mutant significantly increased the TM domain separation and more than doubled the sensitivity of cells to ligand stimulation.

Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability.

Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes

The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation.

Structural Characterization of the Drug Translocation Path of MRP1/ABCC1

AbstractThe rapid clearance of drugs from human cells is carried out by MRP1 and other proteins of the ABC transporter superfamily. Selective mutations carried out by DeGorter indicated that replacement of Y324 by phenylalanine (but not by tryptophan or alanine) enhances the capacity of the protein to extrude various drugs. In this study we investigate the effect of mutation on the structure of the isolated transmembrane domain of MRP1 through molecular dynamics simulations of the protein embedded in a POPC membrane. The simulations reveal a persistent tendency of the translocation path to experience a partial constriction, losing ∼50 % of the water inside the conducting path. While the Wt, Y324W and Y324A transporters all experienced the same constriction, the Y324F transporter, the one having a higher clearance rate than the Wt, retains a fully open configuration. The structure of the Y324F mutant reveals an alternate set of stabilizing interactions that force a kink in transmembrane helix 6, which keeps the protein in a fully open outward‐facing configuration thus providing a molecular‐level account for the higher activity of the mutant. The ability of the simulations to corroborate the experimental observations implies that the homology model of MRP1 is a proper representation of the internal interactions between the residues in the protein, and can be used as a reliable model for studying the human multidrug resistance function of the MRP1 protein.

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L(535)X3G(539)X2A(542)X3V(546)X2L(549) rather than through the alternative glycine zipper motif A(536)X3G(540)X3G(544) (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr(588) and/or Tyr(594)) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.

Open and Closed Conformations of the Isolated Transmembrane Domain of Death Receptor 5 Support a New Model of Activation

It has long been presumed that activation of the apoptosis-initiating Death Receptor 5, as well as other structurally homologous members of the TNF-receptor superfamily, relies on ligand-stabilized trimerization of noninteracting receptor monomers. We and others have proposed an alternate model in which the TNF-receptor dimer—sitting at the vertices of a large supramolecular receptor network of ligand-bound receptor trimers—undergoes a closed-to-open transition, propagated through a scissorslike conformational change in a tightly bundled transmembrane (TM) domain dimer. Here we have combined electron paramagnetic resonance spectroscopy and potential-of-mean force calculations on the isolated TM domain of the long isoform of DR5. The experiments and calculations both independently validate that the opening transition is intrinsic to the physical character of the TM domain dimer, with a significant energy barrier separating the open and closed states.

FGFR3 Transmembrane Domain Interactions Persist in the Presence of Its Extracellular Domain

Isolated receptor tyrosine kinase transmembrane (TM) domains have been shown to form sequence-specific dimers in membranes. Yet, it is not clear whether studies of isolated TM domains yield knowledge that is relevant to full-length receptors or whether the large glycosylated extracellular domains alter the interactions between the TM helices. Here, we address this question by quantifying the effect of the pathogenic A391E TM domain mutation on the stability of the fibroblast growth factor receptor 3 dimer in the presence of the extracellular domain and comparing these results to the case of the isolated TM fibroblast growth factor receptor 3 domains. We perform the measurements in plasma membrane-derived vesicles using a Förster-resonance-energy-transfer-based method. The effect of the mutation on dimer stability in both cases is the same (∼−1.5 kcal/mol), suggesting that the interactions observed in simple TM-peptide model systems are relevant in a biological context.

Tryptophan at the transmembrane–cytosolic junction modulates thrombopoietin receptor dimerization and activation

Dimerization of single-pass membrane receptors is essential for activation. In the human thrombopoietin receptor (TpoR), a unique amphipathic RWQFP motif separates the transmembrane (TM) and intracellular domains. Using a combination of mutagenesis, spectroscopy, and biochemical assays, we show that W515 of this motif impairs dimerization of the upstream TpoR TM helix. TpoR is unusual in that a specific residue is required for this inhibitory function, which prevents receptor self-activation. Mutations as diverse as W515K and W515L cause oncogenic activation of TpoR and lead to human myeloproliferative neoplasms. Two lines of evidence support a general mechanism in which W515 at the intracellular juxtamembrane boundary inhibits dimerization of the TpoR TM helix by increasing the helix tilt angle relative to the membrane bilayer normal, which prevents the formation of stabilizing TM dimer contacts. First, measurements using polarized infrared spectroscopy show that the isolated TM domain of the active W515K mutant has a helix tilt angle closer to the bilayer normal than that of the wild-type receptor. Second, we identify second-site R514W and Q516W mutations that reverse dimerization and tilt angle changes induced by the W515K and W515L mutations. The second-site mutations prevent constitutive activation of TpoR W515K/L, while preserving ligand-induced signaling. The ability of tryptophan to influence the angle and dimerization of the TM helix in wild-type TpoR and in the second-site revertants is likely associated with its strong preference to be buried in the headgroup region of membrane bilayers.

The A391E mutation enhances FGFR3 activation in the absence of ligand

The A391E mutation in the transmembrane domain of fibroblast growth factor receptor 3 leads to aberrant development of the cranium. It has been hypothesized that the mutant glutamic acid stabilizes the dimeric receptor due to hydrogen bonding and enhances its ligand-independent activation. We previously tested this hypothesis in lipid bilayers and showed that the mutation stabilizes the isolated transmembrane domain dimer by − 1.3 ° kcal/mol. Here we further test the hypothesis, by investigating the effect of the A391E mutation on the activation of full-length fibroblast growth factor receptor 3 in Human Embryonic Kidney 293T cells in the absence of ligand. We find that the mutation enhances the ligand-independent activation propensity of the receptor by − 1.7 ° kcal/mol. This value is consistent with the observed strength of hydrogen bonds in membranes, and supports the above hypothesis.

Interactions of mouse glycophorin A with the dRTA-related mutant G719D of the mouse Cl–/HCO3–exchanger Ae1This paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

The AE1 mutation G701D, associated with recessive distal renal tubular acidosis (dRTA), produces only minimal erythroid phenotype, reflecting erythroid-specific expression of stimulatory AE1 subunit glycophorin A (GPA). GPA transgene expression could theoretically treat recessive dRTA in patients and in mice expressing cognate Ae1 mutation G719D. However, human (h) GPA and mouse (m) Gpa amino acid sequences are widely divergent, and mGpa function in vitro has not been investigated. We therefore studied in Xenopus oocytes the effects of coexpressed mGpa and hGPA on anion transport by erythroid (e) and kidney (k) isoforms of wild-type mAe1 (meAe1, mkAe1) and of mAe1 mutant G719D. Coexpression of hGPA or mGpa enhanced the function of meAe1 and mkAe1 and rescued the nonfunctional meAe1 and mkAe1 G719D mutants through increased surface expression. Progressive N-terminal truncation studies revealed a role for meAe1 amino acids 22-28 in GPA-responsiveness of meAe1 G719D. MouseN-cyto/humanTMD and humanN-cyto/mouseTMD kAE1 chimeras were active and GPA-responsive. In contrast, whereas chimera mkAe1N-cyto/hkAE1 G701DTMD was GPA-responsive, chimera hkAE1N-cyto/mkAe1 G719DTMD was GPA-insensitive. Moreover, whereas the isolated transmembrane domain (TMD) of hAE1 G701D was GPA-responsive, that of mAe1 G719D was GPA-insensitive. Thus, mGpa increases surface expression and activity of meAe1 and mkAe1. However, the G719D mutation renders certain mAe1 mutant constructs GPA-unresponsive and highlights a role for erythroid-specific meAe1 amino acids 22-28 in GPA-responsiveness.

NMR Study of General Anesthetic Interactions with the Neuronal N-Acetylcholine Receptor Transmembrane Domain

General anesthetics have been shown to inhibit neuronal nicotinic acetylcholine receptors (nAChRs) channel function but the mechanisms of this action are not very clear. Here we present the study of the interactions of the anesthetics halothane, isoflurane, and ketamine with the transmembrane domains of the human nAChRs (α4)2(β2)3 and (α4)3(β2)2 using high-resolution solution NMR. The isolated transmembrane domains of α4 and β2 subunits, with the extracellular and intracellular domains removed by mutagenesis, were expressed in E. coli, purified into detergent micelles and mixed in the indicated ratio. Residues affected by anesthetics were determined on the basis of their chemical shift changes in 15N-1H HSQC spectra upon titration by anesthetics. In addition to intrasubunit binding sites identified recently, anesthetic interaction sites at the subunit interfaces were also detected. The main site was located near the extracellular interface involving the beginning of TM1 of one of the subunits and the loop between TM2 and TM3 (or residues near the loop) of the other subunit. Moreover, two leucines in the middle of TM2 (one of which facing the pore is the putative hydrophobic gate of the channel) were also significantly affected by anesthetics. Both interaction sites were common for halothane and isoflurane. The intravenous anesthetic ketamine affected a number of residues in TM4, suggesting that ketamine could bind at the protein-detergent interface. The ketamine binding site at the extracellular interface appeared to be similar to the halothane or isoflurane one. Anesthetics also affected NMR peak intensity and line width of specific residues and in some cases resulted in peak splitting due to conformational exchange, indicating modulations of protein dynamics on the microsecond-to-millisecond timescale, which directly related to protein function. This work was supported by NIH grants: R01GM066358, R01GM056257, and R37GM049202.

Anesthetic Binding Sites in the Neuronal N-Acetylcholine Receptor Transmembrane Domain

Neuronal nicotinic acetylcholine receptors (nAChRs) are sensitive to general anesthetics at physiologically relevant concentrations, but the locations and mechanisms of relevant anesthetic interactions are unknown. The interactions of the anesthetics halothane, isoflurane, and ketamine with the transmembrane domains of the human nAChR α4 and β2 subunits were studied using fluorescence spectroscopy and high-resolution solution NMR. The isolated transmembrane domains, with the extracellular and intracellular domains removed by mutagenesis, were expressed in E. coli and purified into detergent micelles. Multi-angle light scattering experiments on a mixture of α4 and β2 subunits demonstrated their interaction in detergent micelles. Anesthetics quenched the intrinsic tryptophan fluorescence of these proteins in a manner consistent with specific binding. Direct interaction with anesthetic halothane, isoflurane, and ketamine was confirmed by NMR saturation transfer difference spectroscopy. The anesthetics induced chemical shift changes for specific resonances in the NMR spectra of the α4 and β2 transmembrane domains. Two specific interaction sites for the volatile anesthetics halothane and isoflurane have been identified at the extracellular and intracellular interfaces near the ends of the transmembrane domain in the α4 subunit. Differing from halothane and isoflurane, the intravenous anesthetic ketamine affected a set of residues at a more interior location near the extracellular interface. Changes in NMR peak intensity and line width indicated modulations of protein motion by all three anesthetics. This study demonstrated that general anesthetics interact at specific sites within the isolated transmembrane domain of nAChR and affect protein dynamics on a time scale consistent with protein function. This work was supported by NIH grants: R01GM066358, R01GM056257, R37GM049202, and R01GM069766.

Quantitative Measurements of Receptor Interactions in Mammalian Cells: Implications for Human Pathologies

Receptor Tyrosine kinases (RTKs) are family of single-pass cell membrane receptors with extracellular ligand-binding domains and intracellular kinase domains, which conduct biochemical signals via lateral dimerization in the plasma membrane. Mutations in the transmembrane (TM) domains of these receptors are known to promote unregulated signaling. An example is the Ala391Glu mutation in the TM domain of FGFR3, which leads to pathologies such as Crouzon syndrome and bladder cancer. This mutation has been shown to stabilize the isolated TM domain dimers in lipid membranes, but it is not known if it stabilizes the full length FGFR3 receptor dimers in the plasma membrane of mammalian cells. To address the effect of the mutation in mammalian cells, we have determined free energies of dimerization for the wild type and mutant FGFR3 in mammalian (HEK293T and CHO) plasma membranes using the QI Förster resonance energy transfer (FRET) technique [Li et al.,2008]. The measured change in the dimerization free energy due to the Ala391Glu mutation is −1.2 kcal/mol, consistent with previous reports of hydrogen bond strength in proteins, as well as results for the isolated FGFR3 TM domains. Thus, we have shown that the mutation stabilizes the full length FGFR3 dimers in mammalian cells. We propose that this dimer stabilization is the major cause for FGFR3 overactivation and human pathologies. Li E,Placone J,Merzlyakov M, Hristova K (2008) Quantitative measurements of protein interactions in a crowded cellular environment.Anal Chem 80:5976-5985.

Ligand Binding in the Conserved Interhelical Loop of CorA, a Magnesium Transporter from Mycobacterium tuberculosis

CorA is a constitutively expressed magnesium transporter in many bacteria. The crystal structures of Thermotoga maritima CorA provide an excellent structural framework for continuing studies. Here, the ligand binding properties of the conserved interhelical loop, the only portion of the protein exposed to the periplasmic space, are characterized by solution nuclear magnetic resonance spectroscopy. Through titration experiments performed on the isolated transmembrane domain of Mycobacterium tuberculosis CorA, it was found that two CorA substrates (Mg2+ and Co2+) and the CorA-specific inhibitor (Co(III) hexamine chloride) bind in the loop at the same binding site. This site includes the glutamic acid residue from the conserved "MPEL" motif. The relatively large dissociation constants indicate that such interactions are weak but not atypical for channels. The present data support the hypothesis that the negatively charged loop could act as an electrostatic ring, increasing local substrate concentrations before transport across the membrane.

NMR Structural Studies of Interactions of a Small, Nonpeptidyl Tpo Mimic with the Thrombopoietin Receptor Extracellular Juxtamembrane and Transmembrane Domains

Thrombopoietin (Tpo) is a glycoprotein growth factor that supports hematopoietic stem cell survival and expansion and is the principal regulator of megakaryocyte growth and differentiation. Several small, nonpeptidyl molecules have been identified as selective human Tpo receptor (hTpoR) agonists. To understand how the small molecule Tpo mimic SB394725 interacts and activates hTpoR, we performed receptor domain swap and mutagenesis studies. The results suggest that SB394725 interacts specifically with the extracellular juxtamembrane region (JMR) and the transmembrane (TM) domain of hTpoR. Solution and solid-state NMR structural studies using a peptide containing the JMR-TM sequences showed that this region of hTpoR, unexpectedly, consists of two alpha-helices separated by a few nonhelical residues. SB394725 interacts specifically with His-499 in the TM domain and a few distinct residues in the JMR-TM region and affects several specific C-terminal TM domain residues. The unique structural information provided by these studies both sheds light on the distinctive mechanism of action of SB394725 and provides valuable insight into the mechanism of ligand-induced cytokine receptor activation.

Probing the Molecular Dynamics of the ABC Multidrug Transporter LmrA by Deuterium Solid-State Nuclear Magnetic Resonance

The molecular dynamics of the 64 kDa ABC multidrug efflux pump LmrA from Lactococcus lactis within lipid membranes has been investigated by deuterium solid-state NMR. Deuteriomethyl-labeled alanine has been used to probe global protein backbone dynamics. A comparison of static deuterium NMR spectra of full-length LmrA in the resting state and its isolated transmembrane domain revealed a high mobility for the nucleotide binding domains. Their motional freedom is restricted upon ATP binding as seen for LmrA in complex with AMP-PNP, a nonhydrolyzable ATP analogue. LmrA returns to full motional flexibility in the posthydrolysis, vanadate-trapped state. These experiments provide insight into the molecular dynamics of a full-length ABC transporter during the catalytic cycle. Data are discussed in the context of known biochemical data and structural models of ABC transporters.

Role of calnexin in the glycan-independent quality control of proteolipid protein.

The endoplasmic (ER) quality control apparatus ensures that misfolded or unassembled proteins are not deployed within the cell, but are retained in the ER and degraded. A glycoprotein-specific system involving the ER lectins calnexin and calreticulin is well documented, but very little is known about mechanisms that may operate for non-glycosylated proteins. We have used a folding mutant of a non- glycosylated membrane protein, proteolipid protein (PLP), to examine the quality control of this class of polypeptide. We find that calnexin associates with newly synthesized PLP molecules, binding stably to misfolded PLP. Calnexin also binds stably to an isolated transmembrane domain of PLP, suggesting that this chaperone is able to monitor the folding and assembly of domains within the ER membrane. Notably, this glycan-independent interaction with calnexin significantly retards the degradation of misfolded PLP. We propose that calnexin contributes to the quality control of non-glycosylated polytopic membrane proteins by binding to misfolded or unassembled transmembrane domains, and discuss our findings in relation to the role of calnexin in the degradation of misfolded proteins.

Synthetic null-cysteine phospholamban analogue and the corresponding transmembrane domain inhibit the Ca-ATPase.

Chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) have been used to analyze the structure and function of phospholamban (PLB), a 52-residue integral membrane protein that regulates the calcium pump (Ca-ATPase) in cardiac sarcoplasmic reticulum (SR). PLB exists in equilibrium between monomeric and pentameric forms, as observed by SDS-PAGE, EPR, and fluorescence. It has been proposed that inhibition of the pump is due primarily to the monomeric form, with both pentameric stability and inhibition dependent primarily on the transmembrane (TM) domain. To test these hypotheses, we have studied the physical and functional properties of a synthetic null-cysteine PLB analogue that is entirely monomeric on SDS-PAGE, and compared it with the synthetic null-cysteine TM domain (residues 26-52). The TM domain was found to be primarily oligomeric on SDS-PAGE, and boundary lipid spin label analysis in lipid bilayers verified that the isolated TM domain is more oligomeric than the full-length parent molecule. These results indicate that the stability of the PLB pentamer is due primarily to attractive interactions between hydrophobic TM domains, overcoming the repulsive electrostatic interactions between the cationic cytoplasmic domains (residues 1-25). When reconstituted into liposomes containing the Ca-ATPase, the null-cysteine TM domain had the same inhibitory function as that of the full-length parent molecule. We conclude that the TM domain of PLB is sufficient for inhibitory function, the oligomeric stability of PLB does not determine its inhibitory activity, and the three Cys residues in the TM domain are not required for inhibitory function.

Interaction of the isolated transmembrane domain of diphtheria toxin with membranes.

Insertion of diphtheria toxin's T (transmembrane) domain into the endosomal membrane under acidic conditions is known to promote translocation of its catalytic domain across the membrane and into the cytosol. The T domain, a cysteine-free bundle of alpha-helices, was expressed as a discrete protein in Escherichia coli and purified. The isolated domain was stable and largely monomeric at pH 8.0. Like the holotoxin it bound the hydrophobic fluorophore, 2-p-toluidinylnaphthalene 6-sulfonate, upon acidification, but the transition pH was higher than with the holotoxin (pH 5.6 vs 5.1) and broader, reflecting the absence of interdomain interactions. The domain also permeabilized large unilamellar vesicles under acidic conditions, as demonstrated by release of entrapped solutes. Mutant forms of T domain, each with a single residue replaced by cysteine, were derivatized with a thiol-reactive nitroxide-containing spin label and analyzed by electron paramagnetic resonance (EPR). EPR spectra and solvent accessibilities of the labels at pH 8.0 were consistent with the environments predicted from the toxin's crystallographic structure. Acidification in the presence of large unilamellar vesicles caused a nitroxide label at position 332 on helix TH8 to move from a buried site in the water soluble state to a lipid-exposed surface site at a depth of approximately 15 A within the bilayer. This is consistent with the concept that the TH8-TH9 helix pair inserts into the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)