CARD domain of rat RIP2 kinase: Refolding, solution structure, pH-dependent behavior and protein-protein interactions

PLoS One. 2018 Oct 23;13(10):e0206244. doi: 10.1371/journal.pone.0206244. eCollection 2018.


RIP2, one of the RIP kinases, interacts with p75 neurotrophin receptor, regulating the neuron survival, and with NOD1 and NOD2 proteins, causing the innate immune response against gram-negative and gram-positive bacteria via its caspase recruitment domain (CARD). This makes RIP2 a prospective target for novel therapies, aimed to modulate the inflammatory diseases and neurogenesis/neurodegeneration. Several studies report the problems with the stability of human RIP2 CARD and its production in bacterial hosts, which is a prerequisite for the structural investigation with solution NMR spectroscopy. In the present work, we report the high yield production and refolding protocols and resolve the structure of rat RIP2 CARD. The structure reveals the important differences to the previously published conformation of the homologous human protein. Using solution NMR, we characterized the intramolecular mobility and pH-dependent behavior of RIP2 CARD, and found the propensity of the protein to form high-order oligomers at physiological pH while being monomeric under acidic conditions. The oligomerization of protein may be explained, based on the electrostatic properties of its surface. Analysis of the structure and sequences of homologous proteins reveals the residues which are significant for the unusual fold of RIP2 CARD domains from different species. The high-throughput protein production/refolding protocols and proposed explanation for the protein oligomerization, provide an opportunity to design the stabilized variants of RIP2 CARD, which could be used to study the structural details of RIP2/NOD1/NOD2 interaction and perform the rational drug design.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Caspase Activation and Recruitment Domain*
  • Hydrogen-Ion Concentration
  • Magnetic Resonance Spectroscopy / methods
  • Models, Molecular
  • Nod1 Signaling Adaptor Protein / chemistry
  • Nod1 Signaling Adaptor Protein / genetics
  • Nod1 Signaling Adaptor Protein / metabolism
  • Nod2 Signaling Adaptor Protein / chemistry
  • Nod2 Signaling Adaptor Protein / genetics
  • Nod2 Signaling Adaptor Protein / metabolism
  • Protein Binding
  • Protein Multimerization*
  • Protein Refolding*
  • Receptor-Interacting Protein Serine-Threonine Kinase 2 / chemistry*
  • Receptor-Interacting Protein Serine-Threonine Kinase 2 / genetics
  • Receptor-Interacting Protein Serine-Threonine Kinase 2 / metabolism
  • Sequence Homology, Amino Acid
  • Solutions
  • Static Electricity


  • NOD2 protein, rat
  • Nod1 Signaling Adaptor Protein
  • Nod2 Signaling Adaptor Protein
  • Solutions
  • Receptor-Interacting Protein Serine-Threonine Kinase 2

Grant support

The work is supported by the Russian Science Foundation, grant #14-14-00573. Experiments were partially carried out using the equipment provided by the IBCH core facility (CKP IBCH, supported by Russian Ministry of Education and Science, grant RFMEFI62117X0018). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.