RESUMO
The interaction of the receptor-binding domain (R domain) of diphtheria toxin with a pure lipid membrane has been characterized by several approaches. Using a photoactivatable lipid, the R domain has been shown to deeply insert in the lipid membrane. Three regions of the R domain (residues 380-421, 422-441, and 442 to about 483) are protected by their interaction with the membrane from externally added proteases. At least one of these regions is deeply interacting with the lipid membrane, as evidenced by the location of Cys 461 and 471 determined by fluorescence experiments. Binding of the R domain to the lipid membrane is characterized by the appearance of an alpha-helical component whose orientation is compatible with a transmembrane orientation.
Assuntos
Toxina Diftérica/química , Lipídeos de Membrana/química , Fragmentos de Peptídeos/química , Receptores de Superfície Celular/química , Cromatografia Líquida de Alta Pressão , Toxina Diftérica/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intercelular , Lipossomos/química , Fragmentos de Peptídeos/isolamento & purificação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteolipídeos/química , Receptores de Superfície Celular/isolamento & purificação , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , TriptofanoRESUMO
The diphtheria toxin (DT) membrane topology was investigated by proteolysis experiments. Diphtheria toxin was incubated with asolectin liposomes at pH5 in order to promote its membrane insertion, and the protein domains located outside the lipid vesicles were digested with proteinase K (which is a non-specific protease). The protected peptides were separated by electrophoresis and identified by microsequence analysis. Their orientation with respect to the lipid bilayer and their accessibility to the aqueous phase were determined by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). These data, combined with those provided by proteolytic cleavage with a specific protease (endoproteinase Glu-C), led us to propose a topological model of the N-terminal part of the diphtheria toxin B fragment inserted into the lipid membrane. In this model, two alpha-helices adopt a transmembrane orientation, with their axes parallel to the lipid acyl chains, while a third alpha-helix could adopt a transmembrane topology only in a small proportion of DT molecules.
Assuntos
1,2-Dipalmitoilfosfatidilcolina , Toxina Diftérica/química , Bicamadas Lipídicas , Fosfolipídeos , Sequência de Aminoácidos , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Cinética , Dados de Sequência Molecular , Fosfatidilcolinas , Análise de SequênciaRESUMO
Diphtheria toxin (DT) is a bacterial protein that crosses the membrane of endosomes of target cells in response to the low endosomal pH. In this paper, we have inserted diphtheria toxin in asolectin vesicles at pH 5.0 and treated the reconstituted system with pronase. The peptides that were protected from digestion were separated by gel electrophoresis, transferred to a membrane and their N-terminal sequences were determined. All peptides belong to the B fragment of DT and cover residues 194-223, 265-375 and 429-528. The secondary structures of the peptides inserted in the membrane, determined by Fourier-transformed infrared spectroscopy, were shown to be mostly alpha-helices and beta-sheets (44% and 53%, respectively). On the basis of these data and the recently published X-ray structure of DT, we are proposing a topology for the DTB fragment in the membrane.
