Theoretical study of the packing of α-helices into possible transmembrane bundles. Sequences including alanines,leucines and serines

Energy optimizations are carried out on packages of two and five α-helices containing leucines on their faces of contact and made otherwise of alanines. The effect of these bulky side-chains on the optimal arrangements is analysed and compared to the results previously obtained for pure poly(l-alanine) packages; the essential pairing properties are conserved (near antiparallelism, preponderous role of the non-bonded interactions, possibility of existence of parallel pairs); five α-helices made of 8 alanines and 6 leucines (three on each interface) can pack in different stable P5L bundles including various holes, according to the tilt and relative sliding of the helices. Substitution of serines to the alanines lying on the inner wall affects very little the interhelix packing. The seryl side chains adapt their conformation at best to their surroundings. The P5L packages can be used to represent individual subunits arranged in ‘superbundles’ around a central pit in a channel-forming protein.     

Mutations in the cytoplasmic domain of a paramyxovirus fusion glycoprotein rescue syncytium formation and eliminate the hemagglutinin-neuraminidase protein requirement for membrane fusion

SER virus is closely related to the paramyxovirus simian virus 5 (SV5) but is defective in syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT) domain that has been shown to inhibit membrane fusion, and this inhibitory effect could be eliminated by truncation of the C-terminal sequence (S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). To study the sequence requirements for regulation of fusion, codons for SER virus F protein residues spanning amino acids 535 to 542 and 548 were mutated singly to alanines, and the two leucine residues at positions 539 and 548 were mutated doubly to alanines. We found that leu-539 and leu-548 in the CT domain played a critical role in the inhibition of fusion, as mutation of the two leucines singly to alanines partially rescued fusion, and the double mutation L539, 548A completely rescued syncytium formation. Mutation of charged residues to alanines had little effect on the suppression of fusion activity, whereas the mutation of serine residues to alanines enhanced fusion activity significantly. The L539, 548A mutant also showed extensive syncytium formation when expressed without the SER virus HN protein. By constructing a chimeric SV5-SER virus F CT protein, we also found that the inhibitory effect of the long CT of the SER virus F protein could be partially transferred to the SV5 F protein. These results demonstrate that an elongated CT of a paramyxovirus F protein interferes with membrane fusion in a sequence-dependent manner.     

Different functional recognition of basolateral signals in Caco-2 and MDCK cells

Using the basolateral mutant PS of the normally apical neurotrophin receptor p75 (p75NTR) we have identified two cytoplasmic determinants responsible for this reversed localization in the human intestinal cell line, Caco2. These signals are based on two consecutive leucines (322-323) and a tyrosine (Y308). Truncation of the cytoplasmic tail removing the two leucines or their replacement by alanines led to a nonpolarized expression of the resulting mutants in Caco2 cells. To our surprise, the same mutations had no effect on the basolateral localization of the mutant PS in MDCK cells. In MDCK cells, the basolateral localization was entirely dependent on a cytoplasmic tyrosine Y308, while in Caco-2 cells this tyrosine signal was functional as a basolateral signal only when the cytoplasmic domain of PS was truncated shortly after it. These data indicate for the first time that there is a differential recognition of basolateral signals between MDCK and Caco-2 cells.     

A leucine repeat motif in AbiA is required for resistance of Lactococcus lactis to phages representing three species.

The abiA gene encodes an abortive bacteriophage infection mechanism that can protect Lactococcus species from infection by a variety of bacteriophages including three unrelated phage species. Five heptad leucine repeats suggestive of a leucine zipper motif were identified between residues 232 and 266 in the predicted amino acid sequence of the AbiA protein. The biological role of residues in the repeats was investigated by incorporating amino acid substitutions via site-directed mutagenesis. Each mutant was tested for phage resistance against three phages, φ31, sk1, and c2, belonging to species P335, 936, and c2, respectively. The five residues that comprise the heptad repeats were designated L234, L242, A249, L256, and L263. Three single conservative mutations of leucine to valine in positions L235, L242, and L263 and a double mutation of two leucines (L235 and L242) to valines did not affect AbiA activity on any phages tested. Non-conservative single substitutions of charged amino acids for three of the leucines (L235, L242, and L256) virtually eliminated AbiA activity on all phages tested. Substitution of the alanine residue in the third repeat (A249) with a charged residue did not affect AbiA activity. Replacement of L242 with an alanine elimination phage resistance against φ31, but partial resistance to sk1 and c2 remained. Two single proline substitutions for leucines L242 and L263 virtually eliminated AbiA activity against all phages, indicating that the predicted alpha-helical structure of this region is important. Mutations in an adjacent region of basic amino acids had various effects on phage resistance, suggesting that these basic residues are also important for AbiA activity. This directed mutagenesis analysis of AbiA indicated that the leucine repeat structure is essential for conferring phage resistance against three species of lactococcal bacteriophages.     

Single proline substitutions in predicted alpha-helices of murine granulocyte-macrophage colony-stimulating factor result in a loss in bioactivity and altered glycosylation

Contributions of alpha-helices to biological activity in murine granulocyte-macrophage colony-stimulating factor were analyzed using site-directed mutagenesis and protein expression in COS-1 cells. A series of single proline substitutions were made for residues within the four predicted alpha-helices as a means of disrupting local helical secondary structure. Mutations in three of the four helices resulted in marked reductions in bioactivity. Five mutants E21P, L56P, E60P, L63P, and L107P showed 10(2)-10(4)-fold reduction in bioactivity as well as hyperglycosylation. The same Pro substitutions made on non-N-glycosylated molecules had a similar loss in bioactivity implying that a Pro-induced structural change and not hyperglycosylation was responsible for the major decrease in bioactivity. Additional amino acid substitutions at these residues which conserved charge or hydrophobicity, or replaced the original residue with an Ala, verified that conformational changes in the protein structure were specifically due to steric constraints imposed by the Pro residue rather than loss of important side chain functions.     

The effect of peptide/lipid hydrophobic mismatch on the phase behavior of model membranes mimicking the lipid composition in Escherichia coli membranes

The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by (31)P NMR spectroscopy. The peptides (WALPn peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at approximately 60 degrees C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by approximately 15 degrees C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H(II)) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes.     

Solid-state NMR investigations of interaction contributions that determine the alignment of helical polypeptides in biological membranes

Helical peptides reconstituted into oriented phospholipid bilayers were studied by proton-decoupled 15N solid-state NMR spectroscopy. Whereas hydrophobic channel peptides, such as the N-terminal region of Vpu of HIV-1, adopt transmembrane orientations, amphipathic peptide antibiotics are oriented parallel to the bilayer surface. The interaction contributions that determine the alignment of helical peptides in lipid membranes were analysed using model sequences, and peptides that change their topology in a pH-dependent manner have been designed. The energy contributions of histidines, lysines, leucines and alanines as well as the alignment of peptides and phospholipids under conditions of hydrophobic mismatch have been investigated in considerable detail.     

A combined approach of mass spectrometry, molecular modeling, and site-directed mutagenesis highlights key structural features responsible for the thermostability of Sulfolobus solfataricus carboxypeptidase

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme, consisting of four identical subunits with a M(r) of 43,000. In a previous paper (Occhipinti et al., Biophys J 2003; 85:1165-1175), we developed a structure of the enzyme by molecular modeling and validated it by site-directed mutagenesis and small angle X-ray scattering. Here, we report investigations aimed at further validating the model, as well as at identifying molecular determinants responsible for thermostability. To this end, we took advantage of mass spectrometry techniques, notably LC-MS/MS. The structure was confirmed by such approaches, in that they lead to the identification of a disulfide bridge formed by Cys286 and Cys293, whose location in the model is well suited for giving rise to the crosslink. More notably, we also identified a protease-resistant core consisting of the N- and C-terminal antiparallel alpha-helices, which in the model are predicted to interact with each other via hydrophobic quadrants. On the basis of the model, we also tentatively identified the most tightly interacting residues as Leu7, Ala380, and Leu376. Although the replacement of Ala380 by serine did not detectably impair protein stability, a dramatic drop in thermostability was observed when the two leucines were replaced by either aspartate (L7D; L376D) or asparagine (L7N; L376N). We then investigated the kinetic thermal stability of the wild type and the mutants by determining the thermodynamic activation parameters, DeltaG++, DeltaH++, and DeltaS++. Besides highlighting the key role of the hydrophobic core in thermostability, these results suggest clearly different mechanisms of destabilization by the single mutations, depending on whether the leucines are replaced by asparagines or aspartates.     

The amino terminus of Epstein-Barr virus glycoprotein gH is important for fusion with epithelial and B cells

Epstein-Barr virus (EBV) infects B lymphocytes and epithelial cells. While the glycoproteins required for entry into these two cell types differ, the gH/gL glycoprotein complex is essential for entry into both epithelial and B cells. Analysis of gH protein sequences from three gammaherpesviruses (EBV, marmoset, and rhesus) revealed a potential coiled-coil domain in the N terminus. Four leucines located in this region in EBV gH were replaced by alanines by site-directed mutagenesis and analyzed for cell-cell membrane fusion with B cells and epithelial cells. Reduction in fusion activity was observed for mutants containing L65A and/or L69A mutations, while substitutions in L55 and L74 enhanced the fusion activity of the mutant gH/gL complexes with both cell types. All of the mutants displayed levels of cell surface expression similar to those of wild-type gH and interacted with gL and gp42. The observation that a conservative mutation of leucine to alanine in the N terminus of EBV gH results in fusion-defective mutant gH/gL complexes is striking and points to an important role for this region in EBV-mediated membrane fusion with B lymphocytes and epithelial cells.     

Structural locus of the pH gate in the Kir1.1 inward rectifier channel

The closed-state crystal structure of prokaryotic inward rectifier, KirBac1.1, has implicated four inner helical phenylalanines near the cytoplasmic side as a possible locus of the channel gate. In the present study, we investigate whether this structural feature corresponds to the physiological pH gate of the renal inward rectifier, Kir1.1 (ROMK, KCNJ1). Kir1.1 is endogenous to the mammalian renal collecting duct and the thick ascending limb of Henle and is strongly gated by internal pH in the physiological range. It has four leucines (L160-Kir1.1b), homologous to the phenylalanines of KirBac1.1, which could function as steric gates near the convergence of the inner (M2) helices. Replacing these Leu-160 residues of Kir1.1b by smaller glycines abolished pH gating; however, replacement with alanines, whose side chains are intermediate in size between leucine and glycine, did not eliminate normal pH gating. Furthermore, a double mutant, constructed by adding the I163M-Kir1.1b mutation to the L160G mutation, also lacked normal pH gating, although the I163M mutation by itself enhanced the pH sensitivity of the channel. In addition to size, side-chain hydrophobicity at 160-Kir1.1b was also important for normal pH gating. Mutants with polar side chains (L160S, L160T) did not gate normally and were as insensitive to internal pH as the L160G mutant. Hence, either small or highly polar side chains at 160-Kir1.1b stabilize the open state of the channel. A homology model of the Kir1.1 closed state, based on the crystal structure of KirBac1.1, was consistent with our electrophysiological data and implies that closure of the Kir1.1 pH gate results from steric occlusion of the permeation path by the convergence of four leucines at the cytoplasmic apex of the inner transmembrane helices. In the open state, K crosses the pH gate together with its hydration shell.     

Influence of proline-14 substitution on the secondary structure in a synthetic analogue of alamethicin

Due to the bend introduced by proline 14 in the conformation of alamethicin (AcUPUAUQUVUGLU P VUUEQFol), the role of this residue was assumed essential in the barrel-stave model for voltage-gated ion channels. Taking advantage of a previous synthetic alamethicin analogue (L2) in which all eight α-aminoisobutyric(U) Were replaced by leucines (Ac L P L A L AQ L V L GL L PV LL EQFol), another analogue (L5) was synthesized in order to test the effects ofproline-14 substitution by an alanine (Ac L P L A L AQ L V L G LL PV LL EQFol). Previous conductance experiments showed that both high voltage dependence and multistate behavior were conserved. In order to complement these functional results, a conformational study of L5 has been undertaken and compared to L2 using CD. high field nmr, and molecular dynamics. Results show that L5 presents a better ordered structure than L2 particularly in the region of the substitution and in the C-terminal part. These results are discussed as regards the previous hypothesis of the nonessential character of helix bending for the gating of voltage-dependent ion channels. © 1995 John Wiley & Sons, Inc.     

The C-terminal half of the colicin A pore-forming domain is active in vivo and in vitro

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of Escherichia coli from the periplasmic side and forms a functional channel. The soluble structure of pfColA consists of a ten-helix bundle containing a hydrophobic helical hairpin. Here, we generated a series of mutants in which an increasing number of sp-pfColA alpha-helices was deleted. These peptides were tested for their ability to form ion channels in vivo and in vitro. We found that the shortest sp-pfColA mutant protein that killed Escherichia coli was composed of the five last alpha-helices of sp-pfColA, whereas the shortest peptide that formed a channel in planar lipid bilayer membranes similar to that of intact pfColA was the protein composed of the last six alpha-helices. The peptide composed of the last five alpha-helices of pfColA generated a voltage-independent conductance in planar lipid bilayer with properties very different from that of intact pfColA. Thus, helices 1 to 4 are unnecessary for channel formation, while helix 5, or some part of it, is important but not absolutely necessary. Voltage-dependence of colicin is evidently controlled by the first four alpha-helices of pfColA.     

Defining the SNARE complex binding surface of alpha-SNAP: implications for SNARE complex disassembly

N-Ethylmaleimide-sensitive factor (NSF) and its adaptor protein alpha-soluble NSF attachment protein (alpha-SNAP) sustain membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE) complexes that form during membrane fusion. To better understand the role of alpha-SNAP in this process, we used site-directed mutagenesis to identify residues in alpha-SNAP that interact with SNARE complexes. We find that mutations in charged residues distributed over a concave surface formed by the N-terminal nine alpha-helices of alpha-SNAP affect its ability to bind synaptic SNARE complex and promote its disassembly by NSF. Replacing basic residues on this surface with alanines reduced SNARE complex binding and disassembly, whereas replacing acidic residues with alanines enhanced alpha-SNAP efficacy in both assays. These findings show that the ability of NSF to take apart SNARE complexes depends upon electrostatic interactions between alpha-SNAP and the acidic surface of the SNARE complex and provide insight into how NSF and alpha-SNAP work together to drive disassembly.     

Distal leucines are key functional determinants of Alix-binding simian immunodeficiency virus SIV(smE543) and SIV(mac239) type 3 L domains

In addition to PTAP L domains, primate lentiviruses carry Alix-binding motifs that include the recently described type 3 SREKPYKEVTEDLLHLNSLF sequence. We examined the requirements for the type 3 sequence motif in simian immunodeficiency virus SIV(smE543) and identified the (499)LNSLF(503) sequence as a key functional determinant. Mutation of distal leucines (499)L and (502)L (LL mutant) caused an inhibitory effect on Alix-dependent SIV(smE543) release that was quantitatively similar to that observed following disruption of the type 3 L domain or RNA interference (RNAi) depletion of Alix. Similar results were obtained with the SIV(mac239) LL mutant. Thus, distal leucines are key determinants of SIV(smE543) and SIV(mac239) type 3 L domains.     

Redox-dependent oligomerization through a leucine zipper motif is essential for MG53-mediated cell membrane repair

We recently discovered that MG53, a muscle-specific tripartite motif (TRIM) family protein, functions as a sensor of oxidation to nucleate the assembly of cell membrane repair machinery. Our data showed that disulfide bond formation mediated by Cys242 is critical for MG53-mediated translocation of intracellular vesicles toward the injury sites. Here we test the hypothesis that leucine zipper motifs in the coiled-coil domain of MG53 constitute an additional mechanism that facilitates oligomerization of MG53 during cell membrane repair. Two leucine zipper motifs in the coiled-coil domain of MG53 (LZ1 - L176/L183/L190/V197 and LZ2 - L205/L212/L219/L226) are highly conserved across the different animal species. Chemical cross-linking studies show that LZ1 is critical for MG53 homodimerization, whereas LZ2 is not. Mutations of the conserved leucines into alanines in LZ1, not in LZ2, diminish the redox-dependent oligomerization of MG53. Live cell imaging studies demonstrate that the movement of green fluorescent protein (GFP)-tagged MG53 mutants (GFP-LA1 and GFP-LA2) is partially compromised in response to mechanical damage of the cell membrane, and the GFP-LA1/2 double mutant is completely ineffective in translocation toward the injury sites. In addition to the leucine zipper-mediated intermolecular interaction, redox-dependent cross talk between MG53 appears to be an obligatory step for cell membrane repair, since in vivo modification of cysteine residues with alkylating reagents can prevent the movement of MG53 toward the injury sites. Our data show that oxidation of the thiol group of Cys242 and leucine zipper-mediated interaction among the MG53 molecules both contribute to the nucleation process for MG53-mediated cell membrane repair.     

Structure of a packaging-defective mutant of minute virus of mice indicates that the genome is packaged via a pore at a 5-fold axis

The parvovirus minute virus of mice (MVM) packages a single copy of its linear single-stranded DNA genome into preformed capsids, in a process that is probably driven by a virus-encoded helicase. Parvoviruses have a roughly cylindrically shaped pore that surrounds each of the 12 5-fold vertices. The pore, which penetrates the virion shell, is created by the juxtaposition of 10 antiparallel β-strands, two from each of the 5-fold-related capsid proteins. There is a bottleneck in the channel formed by the symmetry-related side chains of the leucines at position 172. We report here the X-ray crystal structure of the particles produced by a leucine-to-tryptophan mutation at position 172 and the analysis of its biochemical properties. The mutant capsid had its 5-fold channel blocked, and the particles were unable to package DNA, strongly suggesting that the 5-fold pore is the packaging portal for genome entry.     

Functional roles of conserved transmembrane prolines in the human VPAC(1) receptor

The importance of three conserved transmembrane prolines of the human vasoactive intestinal polypeptide (VPAC)(1) receptor was examined by single alanine substitution. P266A, P300A and P348A reduced the expression level, but maintained the binding to VIP. P266A showed decreased ability to stimulate cAMP, while P300A and P348A displayed an increased potency in cAMP production combined with a high sensitivity towards GTP compared to the wild type receptor. In addition, substitutions of two conserved leucines located in position -2 and +1 from P348 were investigated. L346A and L349A reduced the receptor expression, influenced the G protein coupling and decreased the receptor activity. These observations, which are the first on conserved transmembrane prolines within this family of receptors, indicate that these residues are important for receptor expression, G protein coupling and receptor activity.     

Investigations into the mechanisms used by the C-terminal anchors of Escherichia coli penicillin-binding proteins 4, 5, 6 and 6b for membrane interaction.

Escherichia coli low molecular mass penicillin-binding proteins (PBPs) include PBP4, PBP5, PBP6 and PBP6b. Evidence suggests that these proteins interact with the inner membrane via C-terminal amphiphilic alpha-helices. Nonetheless, the membrane interactive mechanisms utilized by the C-terminal anchors of PBP4 and PBP6b show differences to those utilized by PBP5 and PBP6. Here, hydrophobic moment-based analyses have predicted that, in contrast to the PBP4 and PBP6b C-termini, those of PBP5 and PBP6 are candidates to form oblique orientated alpha-helices. Consistent with these predictions, Fourier transform infrared spectroscopy (FTIR) has shown that peptide homologs of the PBP4 and PBP5 C-terminal regions, P4 and P5, respectively, both possessed the ability to adopt alpha-helical structure in the presence of lipid. However, whereas P4 appeared to show a preference for interaction with the surface regions of dimyristoylglycerophosphoethanolamine and dimyristoylglycerophosphoglycerol membranes, P5 appeared to show deep penetration of both these latter membranes and dimyristoylglycerophosphocholine membranes. Based on these results, we have suggested that in contrast to the membrane anchoring of the PBP4 and PBP6b C-terminal alpha-helices, the PBP5 and PBP6 C-terminal alpha-helices may possess hydrophobicity gradients and penetrate membranes in an oblique orientation.     

A nuclear transport signal in mammalian target of rapamycin is critical for its cytoplasmic signaling to S6 kinase 1

The mammalian target of rapamycin (mTOR) regulates nutrient-dependent cell growth and proliferation through cytoplasmic targets, such as S6 kinase 1 (S6K1). Consistent with its main function in the cytoplasm, mTOR is predominantly cytoplasmic. However, previously we have found that mTOR shuttles between the nucleus and cytoplasm, and we have proposed that the nucleocytoplasmic shuttling of mTOR is required for the maximal activation of S6K1. The intrinsic signals directing mTOR nuclear transport and the underlying mechanisms are unknown. In this study we initially set out to identify nuclear export signals in mTOR. A systematic scan of the mTOR sequence revealed 16 peptides conforming to the canonical leucine-rich nuclear export signal, of which 3 were found by reporter assays to contain leptomycin B-sensitive and leucine-dependent nuclear export activity. Unexpectedly, mTOR proteins with those conserved leucines mutated to alanines were unable to enter the nucleus. Further investigation revealed that the L982A/L984A and L1287A/L1289A mutations likely induced a global structural change in mTOR, whereas the L545A/L547A mutation directly impaired the nuclear import of the protein, potentially regulated by a nucleocytoplasmic shuttling signal. The loss of nuclear import was accompanied by the significantly reduced ability of the L545A/L547A mutant to activate S6K1 in cells. Most importantly, when nuclear import was restored in the L545A/L547A mutant by the addition of an exogenous nuclear import signal, signaling to S6K1 was rescued. Taken together, our observations suggest the existence of a nuclear shuttling signal in mTOR and provide definitive evidence for the requirement of mTOR nuclear import in its cytoplasmic signaling to S6K1.