Amphipathic Motif Research Articles

Membrane Anchoring of the AgrD N-terminal Amphipathic Region Is Required for Its Processing to Produce a Quorum-sensing Pheromone in Staphylococcus aureus

Quorum-sensing pheromones are signal molecules that are secreted from Gram-positive bacteria and utilized by these bacteria to communicate among individual cells to regulate their activities as a group through a cell density-sensing mechanism. Typically, these pheromones are processed from precursor polypeptides. The mechanisms of trafficking, processing, and modification of the precursor to generate a mature pheromone are unclear. In Staphylococcus aureus, AgrD is the propeptide for an autoinducing peptide (AIP) pheromone that triggers the Agr cell density-sensing system upon reaching a threshold and subsequently regulates expression of virulence factor genes. The transmembrane protein AgrB, encoded in the agr locus, is necessary for the processing of AgrD to produce mature AIP; however, it is not clear how AgrD interacts with AgrB and how this interaction results in the generation of mature AIP. In this study, we found that the AgrD propeptide was integrated into the cytoplasmic membrane by a conserved alpha-helical amphipathic motif in its N-terminal region. We demonstrated that membrane targeting of AgrD by this motif was required for the stabilization of AgrD and the production of mature AIP, although this region was not specifically involved in the interaction with AgrB. An artificial amphipathic peptide replacing the N-terminal amphipathic motif of AgrD directed the protein to the cytoplasmic membrane and enabled the production of AIP. Analysis of Bacillus ComX precursor protein sequences suggested that the amphipathic membrane-targeting motif might also exist in pheromone precursors of other Gram-positive bacteria.

Unfolding of class A amphipathic peptides on a lipid surface.

The folding of polypeptides associated with biomembranes is a ubiquitous phenomenon, yet the thermodynamics underlying the process are poorly understood. In the present work we examine the unfolding of a series of alpha-helical amphipathic membrane-associated peptides using guanidine hydrochloride as a denaturant. The peptides are based on the class A amphipathic helix motif, and each contains a single tryptophan at sequence position 2, 3, 7, 12, or 14. The isothermal unfolding process was monitored by circular dichroism ellipticity at 222 nm to monitor changes in the helical structure of the peptide. Tryptophan fluorescence was used to probe the local changes in the environment about the indole fluorophore. The unfolding curves generated from the two experimental techniques for each peptide-lipid complex were non-coincidental, suggesting the presence of stable intermediate(s) in the unfolding. A three-state model could adequately account for the data and yielded parameters which were consistent with the presence of a partially folded intermediate structure which (i) is closer in Gibb's free energy to the folded state than the unfolded state and (ii) retains much of the interfacial and amphipathic character of the folded state. Denaturant-induced peptide dissociation from the peptide-lipid complexes was found to be negligible as confirmed by size exclusion chromatography. The results are compared with related thermodynamic data and discussed in terms of current models of peptide folding at membrane interfaces.

The N-terminal 1000 residues of apolipoprotein B associate with microsomal triglyceride transfer protein to create a lipid transfer pocket required for lipoprotein assembly.

Apolipoprotein (apo) B, the major protein component of the atherogenic low-density lipoprotein (LDL), has a pentapartite structure, NH2-betaalpha1-beta1-alpha2-beta2-alpha3-COOH, the beta domains containing multiple amphipathic beta strands and the alpha domains containing multiple amphipathic alpha helixes. We recently reported that the first 1000 residues of human apoB-100 have sequence and amphipathic motif homologies to the lipid-pocket of lamprey lipovitellin (LV) [Segrest, J. P., Jones, M. K., and Dashti, N. (1999) J. Lipid Res. 40, 1401-1416]. The lipid-pocket of LV is a small triangular space lined by three antiparallel amphipathic beta sheets, betaA, betaB, and betaD. The betaA and betaB sheets are joined together by an antiparallel alpha helical bundle, alpha domain. We proposed [Segrest, J. P., Jones, M. K., and Dashti, N. (1999) J. Lipid Res. 40, 1401-1416] that formation of a LV-like lipid-pocket is necessary for lipid-transfer to apoB-containing lipoprotein particles and that this pocket is formed by association of the region of the betaalpha1 domain homologous to the betaA and betaB sheets of LV with a betaD-like amphipathic beta sheet from microsomal triglyceride transfer protein (MTP). To test this hypothesis, we generated four truncated cDNA constructs terminating at or near the juncture of the betaalpha1 and beta1 domains: Residues 1-800 (apoB:800), 1-931 (apoB:931), 1-1000 (apoB:1000), and 1-1200 (apoB:1200). Characterization of particles secreted by stable transformants of the McA-RH7777 cell line demonstrated that (i) ApoB:800, missing the betaB domain, was secreted as a lipid-poor aggregate. (ii) ApoB:931, containing most, but not all, of the betaB domain, was secreted as lipid-poor particles unassociated with MTP. (iii) ApoB:1000, containing the entire betaB domain, was secreted as a relatively lipid-rich particle associated hydrophobically with MTP. (iv) ApoB:1200, containing the betaalpha1 domain plus 200 residues of the beta1 domain, was secreted predominantly as a lipid-poor particle but also as a minor relatively lipid-rich, MTP-associated particle. We thus have captured an intermediate in apoB-containing particle assembly, a lipid transfer competent pocket formed by association of the complete betaalpha1 domain of apoB with MTP.

Trialysin, a Novel Pore-forming Protein from Saliva of Hematophagous Insects Activated by Limited Proteolysis

We have characterized a pore-forming lytic protein from the saliva of the hematophagous insect Triatoma infestans, a vector of Chagas disease. This protein, named trialysin, has 22 kDa and is present in the saliva at about 200 microg/ml. Purified trialysin forms voltage-dependent channels in planar lipid bilayers with conductance of 880 +/- 40 pS. It lyses protozoan parasites and bacteria indicating that it has a role in the control of microorganism growth in the salivary glands. At higher concentrations, but below those found in saliva, trialysin can also permeabilize and lyse mammalian cells, suggesting that it might also facilitate insect blood feeding by interfering with the cell response of the host. The translated cDNA sequence of trialysin shows a basic, lysine-rich protein in which the N-terminal region is predicted to form an amphipathic alpha-helical structure with positive charges on one side and hydrophobic amino acids on the opposite side. A synthetic peptide corresponding to this cationic amphipathic alpha-helix induces protozoan lysis and mammalian cell permeabilization, showing that this region is involved in lytic activity. However, the lytic peptide G6V32 is 10-fold less efficient than trialysin in lysing parasites and 100-fold less efficient in permeabilizing mammalian cells. Trialysin activity is about 10-fold reduced in salivary gland homogenates prepared in the presence of an irreversible serine-protease inhibitor. Since trialysin precursor contains an anionic pro-sequence of 33 amino acids contiguous to the cationic amphipathic putative alpha-helix, we propose that removal of the acidic pro-sequence by limited proteolysis activates trialysin by exposing this lytic basic amphipathic motif.

Removal of amphipathic epitopes from genetically engineered antibodies: production of modified immunoglobulins with reduced immunogenicity.

Several approaches have been developed to reduce the human immune response to nonhuman antibodies. However, chimeric antibodies and humanized antibodies often have decreased binding affinity. We described a new approach for reducing the immunogenicity of chimeric antibodies while maintaining the affinity. This approach seeks to prevent the recognition of murine immunogenic peptides from the antibody variable region by human lymphocytes. Putative immunogenic epitopes in the variable region are identified and subjected to site directed mutagenesis to make them human and/or to break the amphipathic motifs. The R3 antibody, which blocks the epidermal growth factor (EGF) receptor, was used as a model system to test this approach. Four segments containing possible amphipathic epitopes were found in the heavy variable domain using the program AMPHI. Six amino acids within two of these segments were substituted by the corresponding residues from a homologous human sequence. No mutations were made in the murine light variable domain. Experiments in monkeys suggested that the "detope" R3 antibody was less immunogenic than its chimeric analogue. A search for possible amphipathic epitopes in the Kabat database revealed the presence of conserved patterns in the different families of variable region sequences, suggesting that the proposed method may be of general applicability.

N-terminal domain of apolipoprotein B has structural homology to lipovitellin and microsomal triglyceride transfer protein: a “lipid pocket” model for self-assembly of apoB-containing lipoprotein particles

The process of assembly of apolipoprotein (apo) B-containing lipoprotein particles occurs co-translationally after disulfide-dependent folding of the N-terminal domain of apoB but the mechanism is not understood. During a recent database search for protein sequences that contained similar amphipathic β strands to apoB-100, four vitellogenins, the precursor form of lipovitellin, an egg yolk lipoprotein, from chicken, frog, lamprey, and C. elegans appeared on the list of candidate proteins. The X-ray crystal structure of lamprey lipovitellin is known to contain a “lipid pocket” lined by antiparallel amphipathic β sheets. Here we report that the first 1000 residues of human apoB-100 (the α1 domain plus the first 200 residues of the β1 domain) have sequence and amphipathic motif homologies to the lipid-binding pocket of lamprey lipovitellin. We also show that most of the α1 domain of human apoB-100 has sequence and amphipathic motif homologies to human microsomal triglyceride transfer protein (MTP), a protein required for assembly of apoB-containing lipoproteins.▪Based upon these results, we suggest that an LV-like “proteolipid” intermediate containing a “lipid pocket” is formed by the N-terminal portion of apoB alone or, more likely, as a complex with MTP. This intermediate produces a lipid nidus required for assembly of apoB-containing lipoprotein particles; pocket expansion through the addition of amphipathic β strands from the β1 domain of apoB results in the formation of a progressively larger high density lipoprotein (HDL)-like, then very low density lipoprotein (VLDL)-like, spheroidal lipoprotein particle.—Segrest, J. P., M. K. Jones, and N. Dashti. N-terminal domain of apolipoprotein B has structural homology to lipovitellin and microsomal triglyceride transfer protein: a “lipid pocket” model for self-assembly of apoB-containing lipoprotein particles. J. Lipid Res. 1999. 40: 1401–1416.

Combinatorial synthesis and modification of functional porphyrin libraries: identification of new, amphipathic motifs for biomolecule binding.

ADVERTISEMENT RETURN TO ISSUEPREVReportNEXTCombinatorial Synthesis and Modification of Functional Porphyrin Libraries: Identification of New, Amphipathic Motifs for Biomolecule BindingCharles Michael Drain, Xianchang Gong, Vanessa Ruta, Clifford E. Soll, and Philippe F. ChicoineauView Author Information Department of Chemistry, Hunter College of the City University of New York, 695 Park Avenue, New York, New York 10021 Cite this: J. Comb. Chem. 1999, 1, 4, 286–290Publication Date (Web):June 8, 1999Publication History Received18 March 1999Published online8 June 1999Published inissue 1 July 1999https://doi.org/10.1021/cc990011sCopyright © 1999 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views597Altmetric-Citations25LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (71 KB) Supporting Info (1)»Supporting Information Supporting Information SUBJECTS:Alkyls,Functional groups,Genetics,Hydrophobicity,Pyrroles Get e-Alerts

Conformational studies of an amphipathic peptide corresponding to human apolipoprotein A-II residues 18-30 with a C-terminal lipid binding motif EWLNS.

A peptide was designed and synthesized to enhance the lipid binding properties of a 13-residue fragment of apolipoprotein A-II. The peptide, VTDYGKDLMEKVKEWLNS [apoA-II(18-30)+], contains a five-residue amphipathic motif, EWLNS, at the C-terminus of apolipoprotein A-II residues 18-30. The lipid binding properties of apoA-II(18-30)+ were assessed using optical spectroscopy in the presence of sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), tetradecyltrimethyl ammonium chloride (TMA) and dimyristoylphosphatidylcholine (DMPC). The fluorescence emission spectra and the circular dichroism data suggested that apoA-II(18-30)+ interacted most strongly with SDS and most weakly with DMPC. An ensemble of structures for apoA-II(18-30)+ in aqueous solution containing SDS was calculated using distance geometry/simulated annealing methods from 308 NOE-based distance restraints. The backbone (N-C-C = O) RMSD from the average structure of an ensemble of 15 out of 20 calculated structures was 0.54 +/- 0.16 A. Apart from some dynamic fraying at both termini, the distance geometry and simulated annealing calculations showed that apoA-II(18-30)+ adopted a well defined amphipathic helix with distinct hydrophobic and hydrophilic faces.