caa a22 4500 60617544X CHVBK 20210128100743.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00018-014-1761-6 doi (NATIONALLICENCE)springer-10.1007/s00018-014-1761-6 Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5 [Elektronische Daten] [Jonas Protze, Miriam Eichner, Anna Piontek, Stefan Dinter, Jan Rossa, Kinga Blecharz, Peter Vajkoczy, Joerg Piontek, Gerd Krause] 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±10nM). 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. Springer Basel, 2014 Tight junctions nationallicence Protein-protein interactions nationallicence Membrane proteins nationallicence Drug delivery system nationallicence Structure-function study nationallicence Mutagenesis nationallicence Molecular modeling nationallicence TJ : Tight junctions nationallicence CPE : Clostridium perfringens Enterotoxin nationallicence cCPE : C-terminal domain of Clostridium perfringens enterotoxin nationallicence Cld : Claudin nationallicence ECL : Extracellular loop nationallicence PDB : Protein data bank nationallicence RMSD : Root mean square deviation nationallicence Protze Jonas Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut Eichner Miriam Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut Piontek Anna Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut Dinter Stefan Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut Rossa Jan Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut Blecharz Kinga Department of Neurosurgery, Charité-Universitätsmedizin Berlin, 13353, Berlin, Germany aut Vajkoczy Peter Department of Neurosurgery, Charité-Universitätsmedizin Berlin, 13353, Berlin, Germany aut Piontek Joerg Institute of Clinical Physiology, Charité-Universitätsmedizin Berlin, 12203, Berlin, Germany aut Krause Gerd Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut Cellular and Molecular Life Sciences Springer Basel 72/7(2015-04-01), 1417-1432 1420-682X 72:7<1417 2015 72 18 https://doi.org/10.1007/s00018-014-1761-6 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00018-014-1761-6 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Protze Jonas Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut NATIONALLICENCE 700 1- Eichner Miriam Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut NATIONALLICENCE 700 1- Piontek Anna Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut NATIONALLICENCE 700 1- Dinter Stefan Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut NATIONALLICENCE 700 1- Rossa Jan Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut NATIONALLICENCE 700 1- Blecharz Kinga Department of Neurosurgery, Charité-Universitätsmedizin Berlin, 13353, Berlin, Germany aut NATIONALLICENCE 700 1- Vajkoczy Peter Department of Neurosurgery, Charité-Universitätsmedizin Berlin, 13353, Berlin, Germany aut NATIONALLICENCE 700 1- Piontek Joerg Institute of Clinical Physiology, Charité-Universitätsmedizin Berlin, 12203, Berlin, Germany aut NATIONALLICENCE 700 1- Krause Gerd Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany aut NATIONALLICENCE 773 0- Cellular and Molecular Life Sciences Springer Basel 72/7(2015-04-01), 1417-1432 1420-682X 72:7<1417 2015 72 18