Molecular Insights into the Interaction Between the SPRY Domain-Containing SOCS Box Protein SPSB2 and Peptides Based on the Binding Motif from iNOS
Eleanor W. W. Leung A E , Mark D. Mulcair A E , Beow Keat Yap A B , Sandra E. Nicholson C D , Martin J. Scanlon A F and Raymond S. Norton A FA Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic. 3052, Australia.
B Current address: School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Gelugor, Penang, Malaysia.
C The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic. 3052, Australia.
D The Department of Medical Biology, University of Melbourne, Parkville, Vic. 3052, Australia.
E These authors contributed equally to this work.
F Corresponding authors. Email: martin.scalon@monash.edu; ray.norton@monash.edu
Australian Journal of Chemistry 70(2) 191-200 https://doi.org/10.1071/CH16510
Submitted: 14 September 2016 Accepted: 31 October 2016 Published: 25 November 2016
Abstract
SPRY domain-containing SOCS box proteins SPSB1, 2, and 4 mediate the proteasomal degradation of inducible nitric oxide synthase (iNOS) and thereby modulate the amount of NO available for combating infectious organisms. A highly conserved Asp-Ile-Asn-Asn-Asn (DINNN) motif found at the N-terminus of iNOS binds to SPSB2 with nanomolar affinity. The design of specific and potent inhibitors of iNOS–SPSB interactions will be aided by a better understanding of the interactions of this DINNN sequence with SPSB2. Although crystal structures of SPSB complexes with DINNN peptides are available, aspects of the interaction between peptide and protein are still not fully understood. Here, our results from surface plasmon resonance and NMR spectroscopy indicate that residues flanking the DINNN motif, which make no direct contact with SPSB2 in the available crystal structures, nonetheless play an important role in enhancing the binding affinity to SPSB2, by up to 80-fold. Mutational analysis of the DINNN sequence showed that mutation of the Asp or the first Asn residue to Ala reduced the binding affinity by 200- or 600-fold respectively, whereas mutation of the third Asn made binding undetectable. Ala substitution of the second Asn residue caused a 30-fold drop in binding affinity. Substitution of the Ile had very little effect on the binding affinity and substitutions with bulky residues were tolerated. This provides an opportunity for further modification for therapeutic applications. These results highlight the complex interplay of peptide sequence and protein binding and inform efforts to design peptide therapeutics to disrupt the iNOS–SPSB interaction.
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