Abstract
Neuronal nitric oxide synthase is widely regarded as an important contributor to a number of disorders of excitable tissues. Recently the adaptor protein NOS1AP has emerged as a contributor to several nNOS-linked conditions. As a consequence, the unexpectedly complex mechanisms of interaction between nNOS and its effector NOS1AP have become a particularly interesting topic from the point of view of both basic research and the potential for therapeutic applications. Here we demonstrate that the concerted action of two previously described motif regions contributing to the interaction of nNOS with NOS1AP, the ExF region and the PDZ ligand motif, efficiently excludes an alternate ligand from the nNOS-PDZ ligand-binding pocket. Moreover, we identify an additional element with a denaturable structure that contributes to interaction of NOS1AP with nNOS. Denaturation does not affect the functions of the individual motifs and results in a relatively mild drop, ∼3-fold, of overall binding affinity of the C-terminal region of NOS1AP for nNOS. However, denaturation selectively prevents the concerted action of the two motifs that normally results in efficient occlusion of the PDZ ligand-binding pocket, and results in 30-fold reduction of competition between NOS1AP and an alternate PDZ ligand.
Highlights
NMDA receptor mediated signaling through nNOS is increasingly recognized as a contributor to a number of neurological conditions including stroke, depression and neuropathic pain (Florio et al, 2009; Hill et al, 2012; Doucet et al, 2013; Lee et al, 2015)
This was consistent with a ∼5-fold lowering of the off-rate of interaction of the nNOSNOS1AP complex when the PDZ ligand motif was present that we observed using an independent assay system
The increased affinity of the essentially non-binding ExF point mutants lacking of PDZ ligand is an expected consequence of denaturation, but these proteins still have very low affinity for nNOS and this effect is unlikely to influence the specific binding of NOS1AP fragments with functional ExF motifs
Summary
NMDA receptor mediated signaling through nNOS is increasingly recognized as a contributor to a number of neurological conditions including stroke, depression and neuropathic pain (Florio et al, 2009; Hill et al, 2012; Doucet et al, 2013; Lee et al, 2015). Conflicting with this, is that the last 9–20 residues of NOS1AP are not sufficient for interaction to be detected by either pulldown or immunoprecipitation methods (Li et al, 2015; Candemir et al, 2016) The explanation for this finding is that the C-terminal PDZ ligand motif alone has exceedingly low affinity for nNOS (Li et al, 2015) but it contributes stabilization to a primary interaction with an internal NOS1AP sequence containing an ExF motif. This results in ∼5-fold increased affinity and a ∼5fold slower off-rate and, in the context of multiple wash steps of a conventional pulldown assay this amounts to the difference between strong binding and no binding (Li et al, 2015)
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