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Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach

Kaur, Hundeep and Hartmann, Jean-Baptiste and Jakob, Roman P. and Zahn, Michael and Zimmermann, Iwan and Maier, Timm and Seeger, Markus A. and Hiller, Sebastian. (2019) Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach. Journal of Biomolecular NMR, 73. pp. 375-384.

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Official URL: https://edoc.unibas.ch/73642/

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Abstract

The insertase BamA is an essential protein of the bacterial outer membrane. Its 16-stranded transmembrane β-barrel contains a lateral gate as a key functional element. This gate is formed by the C-terminal half of the last β-strand. The BamA barrel was previously found to sample different conformations in aqueous solution, as well as different gate-open, gate-closed, and collapsed conformations in X-ray crystallography and cryo-electron microscopy structures. Here, we report the successful identification of conformation-selective nanobodies that stabilize BamA in specific conformations. While the initial candidate generation and selection protocol was based on established alpaca immunization and phage display selection procedures, the final selection of nanobodies was enhanced by a solution NMR-based screening step to shortlist the targets for crystallization. In this way, three crystal structures of BamA-nanobody complexes were efficiently obtained, showing two types of nanobodies that indeed stabilized BamA in two different conformations, i.e., with open and closed lateral gate, respectively. Then, by correlating the structural data with high resolution NMR spectra, we could for the first time assign the BamA conformational solution ensemble to defined structural states. The new nanobodies will be valuable tools towards understanding the client insertion mechanism of BamA and towards developing improved antibiotics.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Hiller)
01 Faculty of Theology > Ehemalige Organisationseinheiten Theologie > Praktische Theologie (Grözinger)
05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Maier)
UniBasel Contributors:Hiller Odermatt, Sebastian and Maier, Timm
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Springer
ISSN:0925-2738
e-ISSN:1573-5001
Note:Publication type according to Uni Basel Research Database: Journal article
Identification Number:
Last Modified:27 Apr 2020 14:25
Deposited On:10 Feb 2020 16:34

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