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

Alexandra East (1) , Ariel E Mechaly (2) , Gerard H M Huysmans (3) , Cédric Bernarde (3) , Diana Tello-Manigne (2) , Nathalie Nadeau (3) , Anthony P Pugsley (3) , Alejandro Buschiazzo (2) , Pedro M Alzari (2) , Peter J Bond (1, 4) , Olivera Francetic (3)

1 Department of Chemistry [Cambridge, UK]
2 Microb. Struc. (UMR_3528 / U-Pasteur_5) - Microbiologie structurale - Structural Microbiology
3 Systèmes macromoléculaires et Signalisation
4 Bioinformatics Institute [Singapore]

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.

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

Domaines: Sciences du Vivant [q-bio]

Alexandra East, Ariel E Mechaly, Gerard H M Huysmans, Cédric Bernarde, Diana Tello-Manigne, et al.. Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis.. Structure, 2016, 24 (1), pp.92-104. ⟨10.1016/j.str.2015.10.023⟩. ⟨pasteur-01498343⟩