title: Crucial role of perfringolysin O D1 domain in orchestrating structural transitions leading to membrane-perforating pores: a hydrogen-deuterium exchange study.
abstract: Perfringolysin O (PFO) is a toxic protein that binds to cholesterol-containing membranes, oligomerizes, and forms a β-barrel transmembrane pore, leading to cell lysis. Previous studies have uncovered the sequence of events in this multistage structural transition to a considerable detail, but the underlying molecular mechanisms are not yet fully understood. By measuring hydrogen-deuterium exchange patterns of peptide bond amide protons monitored by mass spectrometry (MS), we have mapped structural changes in PFO and its variant bearing a point mutation during incorporation to the lipid environment. We have defined all regions that undergo structural changes caused by the interaction with the lipid environment both in wild-type PFO, thus providing new experimental constraints for molecular modeling of the pore formation process, and in a point mutant, W165T, for which the pore formation process is known to be inefficient. We have demonstrated that point mutation W165T causes destabilization of protein solution structure, strongest for domain D1, which interrupts the pathway of structural transitions in other domains necessary for proper oligomerization in the membrane. In PFO, the strongest changes accompanying binding to the membrane focus in D1; the C-terminal part of D4; and strands β1, β4, and β5 of D3. These changes were much weaker for PFO(W165T) lipo where substantial stabilization was observed only in D4 domain. In this study, the application of hydrogen-deuterium exchange analysis monitored by MS provided new insight into conformational changes of PFO associated with the membrane binding, oligomerization, and lytic pore formation.
authors.affiliation: From the Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawinskiego St., 02-106 Warsaw, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Kacprzyk-Stokowiec
authors.affiliation: From the Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawinskiego St., 02-106 Warsaw, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Kulma
authors.affiliation: Department of Cell Biology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland, and.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Traczyk
authors.affiliation: Department of Cell Biology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland, and.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kwiatkowska
authors.affiliation: Department of Cell Biology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland, and.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Sobota
authors.affiliation: From the Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawinskiego St., 02-106 Warsaw, Poland, Department of Biology, Institute of Genetics and Biotechnology, Warsaw University, 1 Miecznikowa Street, 02-185 Warsaw, Poland michald@ibb.waw.pl.
authors.forename: Michał
authors.initials: M
authors.lastname: Dadlez
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry; D003016:Clostridium perfringens / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013109:Sphingomyelins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D053835:Unilamellar Liposomes / Q000737:chemistry*
chemical_list: D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D013109:Sphingomyelins; D053835:Unilamellar Liposomes; C020452:Clostridium perfringens theta-toxin; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine
keywords: Cholesterol; Hydrogen-Deuterium Exchange; Lipid-binding Protein; Protein Conformation; Toxin