Structural Basis of Nanobody-Mediated Blocking of BtuF, the Cognate Substrate-Binding Protein of the Escherichia Coli Vitamin B12 Transporter BtuCD

Sci Rep. 2017 Oct 30;7(1):14296. doi: 10.1038/s41598-017-14512-8.

Abstract

Bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing co-factors. Recently, an IgG antibody targeting the external binding protein of the Staphylococcus aureus Mn(II) ABC importer was reported to inhibit transport activity and reduce bacterial cell growth. We here explored the possibility of using alpaca-derived nanobodies to inhibit the vitamin B12 transporter of Escherichia coli, BtuCD-F, as a model system by generating nanobodies against the periplasmic binding protein BtuF. We isolated six nanobodies that competed with B12 for binding to BtuF, with inhibition constants between 10-6 and 10-9 M. Kinetic characterization of the nanobody-BtuF interactions revealed dissociation half-lives between 1.6 and 6 minutes and fast association rates between 104 and 106 M-1s-1. For the tightest-binding nanobody, we observed a reduction of in vitro transport activity of BtuCD-F when an excess of nanobody over B12 was used. The structure of BtuF in complex with the most effective nanobody Nb9 revealed the molecular basis of its inhibitory function. The CDR3 loop of Nb9 reached into the substrate-binding pocket of BtuF, preventing both B12 binding and BtuCD-F complex formation. Our results suggest that nanobodies can mediate ABC importer inhibition, providing an opportunity for novel antibiotic strategies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / antagonists & inhibitors*
  • ATP-Binding Cassette Transporters / immunology
  • ATP-Binding Cassette Transporters / metabolism
  • Animals
  • Biological Transport / drug effects*
  • Camelids, New World / immunology
  • Crystallography, X-Ray
  • Escherichia coli / growth & development*
  • Escherichia coli / immunology
  • Escherichia coli Proteins / antagonists & inhibitors*
  • Escherichia coli Proteins / immunology
  • Escherichia coli Proteins / metabolism*
  • Models, Molecular
  • Periplasmic Binding Proteins / antagonists & inhibitors*
  • Periplasmic Binding Proteins / immunology
  • Periplasmic Binding Proteins / metabolism*
  • Protein Binding / physiology
  • Protein Conformation
  • Single-Domain Antibodies / immunology*
  • Vitamin B 12 / metabolism*

Substances

  • ATP-Binding Cassette Transporters
  • Escherichia coli Proteins
  • Periplasmic Binding Proteins
  • Single-Domain Antibodies
  • btuF protein, E coli
  • Vitamin B 12