title: Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis.
abstract: The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain.
authors.affiliation: Department of Chemistry, Centre for Molecular Informatics, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Alexandra
authors.initials: A
authors.lastname: East
authors.affiliation: Unit of Structural Microbiology, Institut Pasteur, CNRS UMR3528 and University Paris Diderot, Sorbonne, 25 rue du Docteur Roux, 75724 Paris, France.
authors.forename: Ariel E
authors.initials: AE
authors.lastname: Mechaly
authors.affiliation: Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, CNRS ERL3526, 25 rue du Docteur Roux, 75724 Paris, France.
authors.forename: Gerard H M
authors.initials: GHM
authors.lastname: Huysmans
authors.affiliation: Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, CNRS ERL3526, 25 rue du Docteur Roux, 75724 Paris, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Bernarde
authors.affiliation: Unit of Structural Microbiology, Institut Pasteur, CNRS UMR3528 and University Paris Diderot, Sorbonne, 25 rue du Docteur Roux, 75724 Paris, France.
authors.forename: Diana
authors.initials: D
authors.lastname: Tello-Manigne
authors.affiliation: Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, CNRS ERL3526, 25 rue du Docteur Roux, 75724 Paris, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Nadeau
authors.affiliation: Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, CNRS ERL3526, 25 rue du Docteur Roux, 75724 Paris, France.
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Pugsley
authors.affiliation: Unit of Structural Microbiology, Institut Pasteur, CNRS UMR3528 and University Paris Diderot, Sorbonne, 25 rue du Docteur Roux, 75724 Paris, France.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Buschiazzo
authors.affiliation: Unit of Structural Microbiology, Institut Pasteur, CNRS UMR3528 and University Paris Diderot, Sorbonne, 25 rue du Docteur Roux, 75724 Paris, France.
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Alzari
authors.affiliation: Department of Chemistry, Centre for Molecular Informatics, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK; Bioinformatics Institute (A(∗)STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Singapore; Department of Biological Sciences, Faculty of Science, National University of Singapore, 5A Engineering Drive 1, Singapore 117411, Singapore. Electronic address: peterjb@bii.a-star.edu.sg.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Bond
authors.affiliation: Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, CNRS ERL3526, 25 rue du Docteur Roux, 75724 Paris, France. Electronic address: ofrancet@pasteur.fr.
authors.forename: Olivera
authors.initials: O
authors.lastname: Francetic
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005089:Exocytosis; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D007709:Klebsiella / Q000201:enzymology; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008074:Lipoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008074:Lipoproteins; D008565:Membrane Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase
keywords: POPE; X-ray crystallography; lipoprotein sorting; liposomes; molecular dynamics; peripheral membrane proteins; pullulanase; secretion signal; type II secretion