Analysis of the interaction of Plexin-B1 and Plexin-B2 with Rnd family proteins

PLoS One. 2017 Oct 17;12(10):e0185899. doi: 10.1371/journal.pone.0185899. eCollection 2017.


The Rnd family of proteins, Rnd1, Rnd2 and Rnd3, are atypical Rho family GTPases, which bind to but do not hydrolyse GTP. They interact with plexins, which are receptors for semaphorins, and are hypothesised to regulate plexin signalling. We recently showed that each Rnd protein has a distinct profile of interaction with three plexins, Plexin-B1, Plexin-B2 and Plexin-B3, in mammalian cells, although it is unclear which region(s) of these plexins contribute to this specificity. Here we characterise the binary interactions of the Rnd proteins with the Rho-binding domain (RBD) of Plexin-B1 and Plexin-B2 using biophysical approaches. Isothermal titration calorimetry (ITC) experiments for each of the Rnd proteins with Plexin-B1-RBD and Plexin-B2-RBD showed similar association constants for all six interactions, although Rnd1 displayed a small preference for Plexin-B1-RBD and Rnd3 for Plexin-B2-RBD. Furthermore, mutagenic analysis of Rnd3 suggested similarities in its interaction with both Plexin-B1-RBD and Plexin-B2-RBD. These results suggest that Rnd proteins do not have a clear-cut specificity for different Plexin-B-RBDs, possibly implying the contribution of additional regions of Plexin-B proteins in conferring functional substrate selection.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • COS Cells
  • Calorimetry / methods
  • Chlorocebus aethiops
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Kinetics
  • Mutation
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Neural Cell Adhesion Molecules / genetics
  • Neural Cell Adhesion Molecules / metabolism*
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Thermodynamics
  • rho GTP-Binding Proteins / genetics
  • rho GTP-Binding Proteins / metabolism*


  • Nerve Tissue Proteins
  • Neural Cell Adhesion Molecules
  • PLXNB1 protein, human
  • PLXNB2 protein, human
  • PLXNB3 protein, human
  • RND1 protein, human
  • Receptors, Cell Surface
  • Recombinant Proteins
  • RND2 protein, human
  • RND3 protein, human
  • rho GTP-Binding Proteins

Grant support

This project was supported by a BBSRC PhD studentship (TW) and Cancer Research UK grant C6620/A15961 (AJR). We are grateful to the Wellcome Trust for funding the King’s College London Centre for Biomolecular Spectroscopy (MRC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.