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Structure. 2016 Jan 5;24(1):92-104. doi: 10.1016/j.str.2015.10.023. Epub 2015 Dec 10.

Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis.

Author information

1
Department of Chemistry, Centre for Molecular Informatics, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
2
Unit of Structural Microbiology, Institut Pasteur, CNRS UMR3528 and University Paris Diderot, Sorbonne, 25 rue du Docteur Roux, 75724 Paris, France.
3
Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, CNRS ERL3526, 25 rue du Docteur Roux, 75724 Paris, France.
4
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.
5
Laboratory of Macromolecular Systems and Signalling, Institut Pasteur, CNRS ERL3526, 25 rue du Docteur Roux, 75724 Paris, France. Electronic address: ofrancet@pasteur.fr.

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.

KEYWORDS:

POPE; X-ray crystallography; lipoprotein sorting; liposomes; molecular dynamics; peripheral membrane proteins; pullulanase; secretion signal; type II secretion

Comment in

PMID:
26688215
DOI:
10.1016/j.str.2015.10.023
[Indexed for MEDLINE]
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