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19 F NMR as a Probe of Ligand Interactions with the iNOS Binding site of SPRY Domain‐Containing SOCS Box Protein 2
Author(s) -
Leung Eleanor W. W.,
Yagi Hiromasa,
Harjani Jitendra R.,
Mulcair Mark D.,
Scanlon Martin J.,
Baell Jonathan B.,
Norton Raymond S.
Publication year - 2014
Publication title -
chemical biology and drug design
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.59
H-Index - 77
eISSN - 1747-0285
pISSN - 1747-0277
DOI - 10.1111/cbdd.12355
Subject(s) - peptide , binding site , mutagenesis , chemistry , ligand (biochemistry) , escherichia coli , biochemistry , stereochemistry , nuclear magnetic resonance spectroscopy , binding domain , receptor , mutation , gene
SPRY domain‐containing SOCS box protein 2 (SPSB2) regulates inducible nitric oxide synthase ( iNOS ) by targeting it for proteasomal degradation. Inhibiting this interaction prolongs the intracellular lifetime of iNOS , leading in turn to enhanced killing of infectious pathogens such as bacteria and parasites. SPSB2 recognizes a linear motif (DINNN) in the disordered N‐terminus of iNOS , and ligands that target the DINNN binding site on SPSB2 are potentially novel anti‐infective agents. We have explored 19 F NMR as a means of probing ligand binding to SPSB2. All six Trp residues in SPSB2 were replaced with 5‐fluorotryptophan (5‐F‐Trp) by utilizing a Trp auxotroph strain of Escherichia coli . The labeled protein was well folded and bound a DINNN‐containing peptide with similar affinity to native SPSB2. Six well‐resolved 5‐F‐Trp resonances were observed in the 19 F NMR spectrum and were assigned using site‐directed mutagenesis. The 19 F resonance of W207 was significantly perturbed upon binding to DINNN‐containing peptides. Other resonances were perturbed to a lesser extent although in a way that was sensitive to the composition of the peptide. Analogues of compounds identified in a fragment screen also perturbed the W207 resonance, confirming their binding to the iNOS peptide‐binding site on SPSB2. 19 F NMR promises to be a valuable approach in developing inhibitors that bind to the DINNN binding site.