Structure of Outward-Facing PglK and Molecular Dynamics of Lipid-Linked Oligosaccharide Recognition and Translocation

Camilo Perez; Ahmad Reza Mehdipour; Gerhard Hummer; Kaspar P Locher

doi:10.1016/j.str.2019.01.013

Structure of Outward-Facing PglK and Molecular Dynamics of Lipid-Linked Oligosaccharide Recognition and Translocation

Structure. 2019 Apr 2;27(4):669-678.e5. doi: 10.1016/j.str.2019.01.013. Epub 2019 Feb 21.

Authors

Camilo Perez¹, Ahmad Reza Mehdipour², Gerhard Hummer³, Kaspar P Locher⁴

Affiliations

¹ Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland; Biozentrum, University of Basel, 4056 Basel, Switzerland.
² Department of Theoretical Biophysics, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany.
³ Department of Theoretical Biophysics, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany; Institute of Biophysics, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany. Electronic address: gerhard.hummer@biophys.mpg.de.
⁴ Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland. Electronic address: locher@mol.biol.ethz.ch.

PMID: 30799077
DOI: 10.1016/j.str.2019.01.013

Abstract

PglK is a lipid-linked oligosaccharide (LLO) flippase essential for asparagine-linked protein glycosylation in Campylobacter jejuni. Previously we have proposed a non-alternating-access LLO translocation mechanism, where postulated outward-facing states play a primary role. To investigate this unusual mechanistic proposal, we have determined a high-resolution structure of PglK that displays an outward semi-occluded state with the two nucleotide binding domains forming an asymmetric closed dimer with two bound ATPγS molecules. Based on this structure, we performed extensive molecular dynamics simulations to investigate LLO recognition and flipping. Our results suggest that PglK may employ a "substrate-hunting" mechanism to locally increase the LLO concentration and facilitate its jump into the translocation pathway, for which sugars from the LLO head group are essential. We further conclude that the release of LLO to the outside occurs before ATP hydrolysis and is followed by the closing of the periplasmic cavity of PglK.

Keywords: ABC transporter; X-ray crystallography; computational simulation; flipping; mechanism; membrane protein; molecular dynamics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenosine Triphosphate / analogs & derivatives*
Adenosine Triphosphate / chemistry
Adenosine Triphosphate / metabolism
Bacterial Proteins / chemistry*
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Binding Sites
Biological Transport
Campylobacter jejuni / chemistry*
Campylobacter jejuni / enzymology
Campylobacter jejuni / genetics
Cloning, Molecular
Crystallography, X-Ray
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Glycosyltransferases / chemistry*
Glycosyltransferases / genetics
Glycosyltransferases / metabolism
Hydrolysis
Kinetics
Lipopolysaccharides / chemistry*
Lipopolysaccharides / metabolism
Molecular Dynamics Simulation
Mutation
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Interaction Domains and Motifs
Proteolipids / chemistry
Proteolipids / metabolism
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Substrate Specificity
Thermodynamics

Substances

Bacterial Proteins
Lipopolysaccharides
Proteolipids
Recombinant Proteins
lipid-linked oligosaccharides
proteoliposomes
adenosine 5'-O-(3-thiotriphosphate)
Adenosine Triphosphate
Glycosyltransferases