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Cellular and Molecular Life Sciences

, Volume 72, Issue 7, pp 1417–1432 | Cite as

Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5

  • Jonas Protze
  • Miriam Eichner
  • Anna Piontek
  • Stefan Dinter
  • Jan Rossa
  • Kinga Grażyna Blecharz
  • Peter Vajkoczy
  • Joerg Piontek
  • Gerd Krause
Research Article

Abstract

Clostridium perfringens enterotoxin (CPE) binds to distinct claudins (Clds), which regulate paracellular barrier functions in endo- and epithelia. The C-terminal domain (cCPE) has the potential for selective claudin modulation, since it only binds to a subset of claudins, e.g., Cld3 and Cld4 (cCPE receptors). Cld5 (non-CPE receptor) is a main constituent in tight junctions (TJ) of the blood-brain barrier. We aimed to reveal claudin recognition mechanisms of cCPE and to create a basis for a Cld5-binder. By utilizing structure-based interaction models, mutagenesis and assays of cCPE-binding to the TJ-free cell line HEK293, transfected with human Cld1 and murine Cld5, we showed how cCPE-binding to Cld1 and Cld5 is prevented by two residues in extracellular loop 2 of Cld1 (Asn150 and Thr153) and Cld5 (Asp149 and Thr151). Binding to Cld5 is especially attenuated by the lack of a bulky hydrophobic residue like leucine at position 151. By downsizing the binding pocket and compensating for the lack of this leucine residue, we created a novel cCPE-variant; cCPEY306W/S313H binds Cld5 with nanomolar affinity (K d 33 ± 10 nM). Finally, the effective binding to endogenously Cld5-expressing blood-brain barrier model cells (murine microvascular endothelial cEND cell line) suggests cCPEY306W/S313H as basis for Cld5-specific modulation to improve paracellular drug delivery, or to target claudin overexpressing tumors.

Keywords

Tight junctions Protein–protein interactions Membrane proteins Drug delivery system Structure–function study Mutagenesis Molecular modeling 

Abbreviations

TJ

Tight junctions

CPE

Clostridium perfringens Enterotoxin

cCPE

C-terminal domain of Clostridium perfringens enterotoxin

Cld

Claudin

ECL

Extracellular loop

PDB

Protein data bank

RMSD

Root mean square deviation

Notes

Acknowledgments

This work was supported by Deutsche Forschungsgemeinschaft (DFG) grants KR 1273/3-2, PI 837/2-1 and by the Sonnenfeld Stiftung (PhD-scholarship for Miriam Eichner).

Conflict of interest

The authors declare no conflict of interests.

Supplementary material

18_2014_1761_MOESM1_ESM.pdf (2.6 mb)
Supplementary material 1 (PDF 2632 kb)

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Copyright information

© Springer Basel 2014

Authors and Affiliations

  • Jonas Protze
    • 1
  • Miriam Eichner
    • 1
  • Anna Piontek
    • 1
  • Stefan Dinter
    • 1
  • Jan Rossa
    • 1
  • Kinga Grażyna Blecharz
    • 2
  • Peter Vajkoczy
    • 2
  • Joerg Piontek
    • 3
  • Gerd Krause
    • 1
  1. 1.Leibniz-Institut für Molekulare PharmakologieBerlinGermany
  2. 2.Department of NeurosurgeryCharité-Universitätsmedizin BerlinBerlinGermany
  3. 3.Institute of Clinical PhysiologyCharité-Universitätsmedizin BerlinBerlinGermany

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