Journal of Biomolecular NMR

, Volume 73, Issue 6–7, pp 375–384 | Cite as

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

  • Hundeep Kaur
  • Jean-Baptiste Hartmann
  • Roman P. Jakob
  • Michael Zahn
  • Iwan Zimmermann
  • Timm Maier
  • Markus A. Seeger
  • Sebastian HillerEmail author


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.


Solution NMR spectroscopy Outer membrane protein X-ray crystallography Protein structure Protein folding Conformational selection Specific binders Nanobodies Lipid gate 



We thank Tim Sharpe for scientific support and discussions and sciCORE at University of Basel for support with high performance computing. S. Štefanić from the Nanobody Service Facility of the University of Zurich is acknowledged for alpaca immunizations. X-ray diffraction data were collected at beamline PXI of the Paul Scherrer Institute, Villigen, Switzerland; we acknowledge excellent support by the facility team. Funding from the Swiss National Science Foundation via the NFP 72 (407240_167125) to S.H., by an SNSF Professorship of the Swiss National Science Foundation (PP00P3_144823) to M.A.S. and by a BioEntrepreneur-Fellowship of the University of Zurich (BIOEF-17-002) to I.Z. is gratefully acknowledged.

Supplementary material

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.BiozentrumUniversity of BaselBaselSwitzerland
  2. 2.Institute of Medical MicrobiologyUniversity of ZurichZurichSwitzerland

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