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Sci Rep. 2018 Mar 26;8(1):5171. doi: 10.1038/s41598-018-23554-5.

Identification of an Arg-Leu-Arg tripeptide that contributes to the binding interface between the cytokine MIF and the chemokine receptor CXCR4.

Author information

1
Department of Vascular Biology, Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig-Maximilians-University of Munich, Feodor-Lynen-Str. 17, D-81377, Munich, Germany.
2
Division of Peptide Biochemistry, Technische Universität München, D-85354, Freising-Weihenstephan, Germany.
3
Department of Anaesthesiology, RWTH Aachen University Hospital, D-52074, Aachen, Germany.
4
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, USA.
5
Institute for Cardiovascular Prevention, Klinikum der Universität München, Ludwig-Maximilians-University of Munich, Pettenkofer Str. 8, D-80336, Munich, Germany.
6
Munich Heart Alliance, D-80802, Munich, Germany.
7
Cardiovascular Research Institute Maastricht, Maastricht University, 6229, Maastricht, The Netherlands.
8
Department of Vascular Biology, Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig-Maximilians-University of Munich, Feodor-Lynen-Str. 17, D-81377, Munich, Germany. juergen.bernhagen@med.uni-muenchen.de.
9
Munich Heart Alliance, D-80802, Munich, Germany. juergen.bernhagen@med.uni-muenchen.de.
10
Munich Cluster for Systems Neurology, D-81377, Munich, Germany. juergen.bernhagen@med.uni-muenchen.de.
11
Division of Peptide Biochemistry, Technische Universität München, D-85354, Freising-Weihenstephan, Germany. akapurniotu@wzw.tum.de.

Abstract

MIF is a chemokine-like cytokine that plays a role in the pathogenesis of inflammatory and cardiovascular disorders. It binds to the chemokine-receptors CXCR2/CXCR4 to trigger atherogenic leukocyte migration albeit lacking canonical chemokine structures. We recently characterized an N-like-loop and the Pro-2-residue of MIF as critical molecular determinants of the CXCR4/MIF binding-site and identified allosteric agonism as a mechanism that distinguishes CXCR4-binding to MIF from that to the cognate ligand CXCL12. By using peptide spot-array technology, site-directed mutagenesis, structure-activity-relationships, and molecular docking, we identified the Arg-Leu-Arg (RLR) sequence-region 87-89 that - in three-dimensional space - 'extends' the N-like-loop to control site-1-binding to CXCR4. Contrary to wildtype MIF, mutant R87A-L88A-R89A-MIF fails to bind to the N-terminal of CXCR4 and the contribution of RLR to the MIF/CXCR4-interaction is underpinned by an ablation of MIF/CXCR4-specific signaling and reduction in CXCR4-dependent chemotactic leukocyte migration of the RLR-mutant of MIF. Alanine-scanning, functional competition by RLR-containing peptides, and molecular docking indicate that the RLR residues directly participate in contacts between MIF and CXCR4 and highlight the importance of charge-interactions at this interface. Identification of the RLR region adds important structural information to the MIF/CXCR4 binding-site that distinguishes this interface from CXCR4/CXCL12 and will help to design MIF-specific drug-targeting approaches.

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