Efficient Binding of the NOS1AP C-Terminus to the nNOS PDZ Pocket Requires the Concerted Action of the PDZ Ligand Motif, the Internal ExF Site and Structural Integrity of an Independent Element

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.

ZLc002, a putative small-molecule inhibitor of nNOS interaction with NOS1AP, suppresses inflammatory nociception and chemotherapy-induced neuropathic pain and synergizes with paclitaxel to reduce tumor cell viability

“…To determine whether disruption of nNOS–NOS1AP interactions induced by ZLc002 resulted from a direct mechanism, we used a cell-free AlphaScreen biochemical binding assay employing purified nNOS and NOS1AP fragments containing the interacting interface. 15 , 16 , 18 Intriguingly, we failed to detect the disruption by ZLc002 of His-nNOS 1-299 -GST-NOS1AP 400-506 binding with the fragments that are critical for nNOS–NOS1AP interactions 15 in this reductionist assay. By contrast, the peptide nNOS–NOS1AP disruptor TAT-GESV disrupted these interactions in the same AlphaScreen assay, whereas a putative inactive peptide TAT-GESVΔ1, lacking the terminal valine residue, failed to do so, consistent with the known critical role of the peptide ligand terminal valine in target recognition.…”

“…We documented that, in a cell-free binding assay, ZLc002 did not directly disrupt binding between purified nNOS and NOS1AP containing the known interacting sites. 15,16 We also established that, in intact HEK293T cells transfected with the full-length tagged proteins, ZLc002 disrupted the co-immunoprecipitation of nNOS with NOS1AP. We demonstrated that ZLc002 suppressed formalin-evoked pain behavior and neuronal activation in lumbar spinal dorsal horn.…”

“…Although both of these interactions involve the extended PDZ domain at the first 130 N-terminal amino acids of nNOS, the stable NOS1AP interaction requires the docking of a PDZ ligand motif into the nNOS-PDZ pocket, 15 whereas PSD95-PDZ2 interacts with a distinct β-finger extension of the core nNOS-PDZ domain. 36 The two sites of interaction are, therefore, spatially very close to one another but are distinct, 15,16,18 making this comparison a good evaluation of selectivity. Importantly, because endogenous nNOS, PSD95, or NMDAR subunits can be expected to be absent in HEK293T cells and ZLc002 disrupts the constitutive nNOS–NOS1AP interaction in this system, ZLc002 is unlikely to reduce co-immunoprecipitation of nNOS and NOS1AP in cultured neurons by acting through NMDA-evoked mechanisms involving extraneous targets that are present in neurons but not measured herein.…”

Disruption of nNOS–NOS1AP protein–protein interactions suppresses neuropathic pain in mice

“…In addition to PSD95, nNOS interacts with nitric oxide synthase 1 adaptor protein (NOS1AP), also known as Carboxy-terminal PDZ ligand of nNOS (CAPON) [24]. Binding of nNOS to NOS1AP occurs through a class III PDZ-PDZ interaction between the canonical PDZ (postsynaptic density 95, P SD95; discs large, D lg; zonula occludens-1, Z O-1) of nNOS (amino acid (a.a.) 11–98) and both the stabilizing C-terminal tail and indispensable internal ExF motif (a.a. 429–431) of NOS1AP [24; 28; 30; 48]. Interactions between nNOS and NOS1AP are implicated in neuropathological conditions including stroke, anxiety and schizophrenia, and disrupting these interactions is neuroprotective [6; 9; 29; 49; 57].…”