posters 5th Asia-Pacific NMR Symposium 2013

19F NMR: a valuable tool in probing ligand interactions of the SPRY-domain containing SOCS box 2 (SPSB2) protein (#167)

Eleanor W. W. Leung 1 , Hiromasa Yagi 1 , Raymond S Norton 1
  1. Monash University, Parkville, VIC, Australia

Inducible nitric-oxide synthase (iNOS) plays a pivotal role in combatting intracellular pathogens such as Leishmaniasis and tuberculosis through nitric oxide (NO) production. iNOS is negatively regulated by ubiquitination and proteasomal degradation. iNOS contains a highly conserved pentapeptide motif (DINNN) in its N-terminal region that strongly interacts with the SPRY-domain containing SOCS box proteins (SPSBs). Upon binding, SPSBs recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, targeting it for proteasomal degradation. By blocking this interaction between SPSB2 and iNOS, the intracellular lifetime of iNOS can be prolonged, and which in turn enhances the killing of infectious pathogens such as bacteria and Leishmania major parasites.1-3

We have explored 19F NMR as a means of probing the environment of Trp residues in SPSB2 and gaining insight into the DINNN-SPSB2 interaction. Trp was chosen based on a previous study showing that the W207A mutation abrogates DINNN-SPSB2 interaction.4 In agreement with the previously solved crystal structure,5 W207 is in fact, situated within close-proximity to the DINNN binding site. We have successfully replaced 6 Trp residues in SPSB2 with 5-fluorotryptophan by utilizing a Trp auxotroph E. coli strain.6 19F NMR results clearly shows that one of the 5-fluorotryptophan resonances is perturbed significantly upon binding with DINNN. Therefore, 19F NMR will be a valuable approach in identifying potential inhibitors that bind to the DINNN binding site. Inhibitors that target this site are potentially novel anti-infective agents.

  1. Kuang et al. (2010) J Cell Biol 190, 129-141.
  2. Kuang et al. (2009) J Mol Biol 386, 662-674.
  3. Lewis et al. (2011) J Immunol 187, 3798-3805.
  4. Masters et al. (2006) Nat Struc Mol Biol 13, 77-84
  5. Filippakopoulos et al. (2010) J Mol Biol 401, 389-402
  6. Anderluh et al. (2005) J Mol Biol 347, 27-39