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Structural and mechanistic studies of components of bacterial protein N-glycosylation pathway and of vitamin B12 transport

English title Structural and mechanistic studies of components of bacterial protein N-glycosylation pathway and of vitamin B12 transport
Applicant Locher Kaspar
Number 166672
Funding scheme Project funding
Research institution Institut für Molekularbiologie und Biophysik Deptartement für Biologie ETH Zurich
Institution of higher education ETH Zurich - ETHZ
Main discipline Biophysics
Start/End 01.04.2016 - 30.09.2019
Approved amount 918'000.00
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All Disciplines (2)

Discipline
Biophysics
Biochemistry

Keywords (8)

X-ray crystallography; Lipid flippase; Protein N-glycosylation; Transcobalamin receptor; Vitamin B12 transport; Lipid-linked oligosaccharide; Crystal structure; ABC transporter

Lay Summary (German)

Lead
Membranständige Enyzme katalysieren wichtige Prozesse in der N-Glykosylierung von Akzeptorproteinen sowie in der Aufnahme von essentiellen Vitaminen, inklusive Vitamin B12. Die strukturellen und mechanistischen Grundlagen dazu sind nur ansatzweise bekannt. Das Projekt zielt darauf ab, die entsprechenden Reaktionen auf molekularer Ebene zu beleuchten.
Lay summary

Das Verständnis von Reaktionsmechanismen von membranständigen Enyzmen hängt davon ab, die dreidimensionale Struktur dieser Proteine zu bestimmen. Im vorliegenden Projekt sollen drei Proteine untersucht werden, die im pathogenen Bakterium Campylobacter jejuni an der N-Glykosylierung von Proteinen, also an der Koppelung von Zucker an Eiweisse, beteiligt sind. Diese Reaktionen sind von grosser Bedeutung für das Ueberleben des Organismus. Zudem wird durch die Studie das Verständnis der entsprechenden Reaktionsmechanismen in Eukaryoten ermöglicht. In Bakterien  gibt es auch eine potentielle Anwendung , indem der Stoffwechselweg gezielt dahingehend modifiziert werden kann, Proteine mit neuartigen Zuckern zu beladen.

Der zweite Teil des Projektes befasst sich mit der Aufnahme des essentiellen Vitamins B12 (Cobalamin) in Bakterien sowie in menschlichen Zellen. Die entsprechenden Proteinsysteme haben Gemeinsamkeiten, aber auch Unterschiede. Auch hier soll mittels Strukturaufklärung (Röntgenkristallographie) sowie durch funktionelle Untersuchung der Reaktionsmechanismus im Detail beschrieben werden, um daraus Einblick in mögliche Anwendungen zu erhalten. Mögliche Anwendungen sind im bakteriellen System eine Inhibierung, die allenfalls das Wachstum verlangsamen könnte (antibiotische Anwendung), während im menschlichen System das Verständnis von B12-defizienten Krankheiten im Vordergrund steht.

 

Direct link to Lay Summary Last update: 31.03.2016

Responsible applicant and co-applicants

Employees

Publications

Publication
Structure of Outward-Facing PglK and Molecular Dynamics of Lipid-Linked Oligosaccharide Recognition and Translocation
Perez Camilo, Mehdipour Ahmad Reza, Hummer Gerhard, Locher Kaspar P. (2019), Structure of Outward-Facing PglK and Molecular Dynamics of Lipid-Linked Oligosaccharide Recognition and Translocation, in Structure, 27(4), 669-678.e5.
Structure of bacterial oligosaccharyltransferase PglB bound to a reactive LLO and an inhibitory peptide
(2018), Structure of bacterial oligosaccharyltransferase PglB bound to a reactive LLO and an inhibitory peptide, in Sci Rep, 8(1), 16297.
Binding Specificities of Nanobody•Membrane Protein Complexes Obtained from Chemical Cross-Linking and High-Mass MALDI Mass Spectrometry
Köhler Martin, Neff Christoph, Perez Camilo, Brunner Cyrill, Pardon Els, Steyaert Jan, Schneider Gisbert, Locher Kaspar P., Zenobi Renato (2018), Binding Specificities of Nanobody•Membrane Protein Complexes Obtained from Chemical Cross-Linking and High-Mass MALDI Mass Spectrometry, in Analytical Chemistry, 90(8), 5306-5313.
Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD
Mireku S. A., Sauer M. M., Glockshuber R., Locher K. P. (2017), Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD, in Scientific Reports, 7(1), 14296-14296.
Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase
Napiórkowska Maja, Boilevin Jérémy, Sovdat Tina, Darbre Tamis, Reymond Jean-Louis, Aebi Markus, Locher Kaspar P (2017), Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase, in Nature Structural & Molecular Biology, 24(12), 1100-1106.
Structure of the human transcobalamin beta domain in four distinct states
Bloch Joël S., Ruetz Markus, Kräutler Bernhard, Locher Kaspar P. (2017), Structure of the human transcobalamin beta domain in four distinct states, in PLOS ONE, 12(9), e0184932-e0184932.
Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody
Perez Camilo, Köhler Martin, Janser Daniel, Pardon Els, Steyaert Jan, Zenobi Renato, Locher Kaspar P. (2017), Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody, in Scientific Reports, 7(1), 46641-46641.
Conformational Change of a Tryptophan Residue in BtuF Facilitates Binding and Transport of Cobinamide by the Vitamin B12 Transporter BtuCD-F
Mireku S. A., Ruetz M., Zhou T., Korkhov V. M., Kräutler B., Locher K. P. (2017), Conformational Change of a Tryptophan Residue in BtuF Facilitates Binding and Transport of Cobinamide by the Vitamin B12 Transporter BtuCD-F, in Scientific Reports, 7(1), 41575-41575.
Structural basis of transcobalamin recognition by human CD320 receptor
Alam Amer, Woo Jae-Sung, Schmitz Jennifer, Prinz Bernadette, Root Katharina, Chen Fan, Bloch Joël S., Zenobi Renato, Locher Kaspar P. (2016), Structural basis of transcobalamin recognition by human CD320 receptor, in Nature Communications, 7(1), 12100-12100.
Mechanistic diversity in ATP-binding cassette (ABC) transporters
Locher Kaspar P (2016), Mechanistic diversity in ATP-binding cassette (ABC) transporters, in Nature Structural & Molecular Biology, 23(6), 487-493.

Collaboration

Group / person Country
Types of collaboration
Beat Meier / ETH Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Jan Steyaert / Free University of Brussels Belgium (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Renato Zenobi / ETH Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Markus Aebi / ETH Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Gunnar Jeschke / ETH Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Jean-Louis Reymond / University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Bernhard Kraeutler / University of Innsbruck Austria (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Eurocarb 2019 Talk given at a conference Structural insight into eukaryotic oligosaccharyltransferase 01.07.2019 Leiden, Netherlands Locher Kaspar;
GRC Glycobiology Talk given at a conference Structural insight into eukaryotic oligosaccharyltransferase 11.03.2019 Il Ciocco, Italy Locher Kaspar;
University of Guelph MCB Seminar Speaker Series Individual talk Structural and functional studies of bacterial and eukaryotic membrane-bound glycosyltransferases facilitating protein N-glycosylation 31.10.2018 Guelph, Canada Locher Kaspar;
COMPPÅ Symposium “Membrane protein production and analysis” Talk given at a conference Structural insight into bacterial and eukaryotic glycosyltransferases facilitating protein N-glycosylation 17.06.2017 New York, United States of America Locher Kaspar;
GRC Conference on Gycobiology Talk given at a conference Structural Studies of Bacterial Oligosaccharyltransferase PglB 19.03.2017 Ventura, CA, United States of America Napiorkowska Maja;
Caltech Biochemistry Seminar Individual talk Structural and mechanistic insight into bacterial and eukaryotic N-glycosylation reactions 01.03.2017 Pasadena, United States of America Locher Kaspar;
John Innes Centre Afternoon Friday Seminar Series Individual talk Understanding the molecular mechanisms with which bacteria glycosylate proteins 03.02.2017 Norwich, Great Britain and Northern Ireland Locher Kaspar;
Society of Glycobiology Annual Meeting Talk given at a conference Structural and mechanistic studies of LLO flippase and oligosaccharyltransferase 20.11.2016 New Orleans, United States of America Locher Kaspar;
Cold Spring Harbor Asia Conference Membrane Proteins: Structure & Function Talk given at a conference Structural and mechanistic studies of ABC transporters facilitating translocation or flipping reactions 16.05.2016 Suzhou, China Locher Kaspar;


Associated projects

Number Title Start Funding scheme
146191 Reaction mechanism of bacterial ABC transporters and oligosaccharyltransferase 01.04.2013 Project funding
146191 Reaction mechanism of bacterial ABC transporters and oligosaccharyltransferase 01.04.2013 Project funding
189111 Structural and mechanistic studies of liver ABC transporters 01.10.2019 Project funding
170808 Acquisition of a Talos Arctica transmission electron microscope for single particle analysis and cryo-tomography 01.05.2017 R'EQUIP
147632 Transglyco: Chemistry, Enzymology and Physiology of Oligosaccharyltransferase 01.11.2013 Sinergia
173709 GlycoSTART: Structure and function of eukaryotic oligosaccharyltransferase 01.06.2017 Sinergia

Abstract

This project has two topics: First, essential enzymes of the protein glycosylation machinery of the pathogenic bacterium Campylobacter jejuni (pgl locus) will be studied. The proteins include the ATP-driven, lipid-linked oligosaccharide flippase PglK, the oligosaccharyltransferase PglB, and the processive glycosyltransferase PglH. All three proteins catalyze reactions of great importance in glycobiology, and can serve as model systems of many other processes (in bacteria and eukaryotes). The flippase PglK and the OST PglB are integral membrane proteins that mediate particularly challenging reactions. The goal of this project is to unravel the detailed reaction mechanisms by a combination of structural biology and biochemical techniques.The second subproject deals with vitamin B12 transport processes in bacteria and in humans. Bacteria that are dependent on B12 uptake catalyze ATP-powered B12 transport across the plasma membrane. The E. coli BtuCD-F system is an example and a useful model system of such a transport machinery. BtuCD is an ABC transporter and BtuF is the cognate, periplasmic B12-binding protein. The specificity of the system for cobalamin and cobinamide will be studied using structural and functional techniques. In humans, the structural basis of cellular B12 uptake depends on the cell-surface receptor (CD320) that recognizes and binds B12-bound transcobalamin (TC) from blood plasma. We plan to reveal the structural basis of TC binding by CD320 and also of the release of B12 from TC at low pH.For both sub-projects, the techniques applied include the expression and purification of membrane proteins, their crystallization from detergent solution, and X-ray crystallography for high-resolution structure determination. In addition, functional glycosylation and transport assays will be used to study variants of the proteins. Also, biophysical techniques including substrate binding studies, electron paramagnetic resonance and nuclear magnetic resonance spectroscopy will be performed (in collaboration) to assess dynamic and substrate binding characteristics.The planned research will help define the reaction mechanisms of the studied proteins in sufficient detail to serve as models for related systems. In addition, the techniques and assays developed will be useful to study homologous, eukaryotic proteins that are biochemically even more challenging in the future.
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