Our biochemical and NMR data of the NIPP1 PP1-binding domain demonstrates that NIPP1 is an intrinsically disordered protein. NIPP1 undergoes a folding-upon-binding transition upon complex formation with PP1 to form the functional NIPP1:PP1 holoenzyme. As anticipated, NIPP1 binds in the RVxF-binding groove of PP1. However, additional interaction sites, including a hydrophobic interaction with the ΦΦ motif binding grove formed by PP1 Tyr78 and β-strand 14 are necessary for its nanomolar interaction with PP1. Notably, this Tyr78 is conserved in PP1 in nearly all isoforms and species, including all human isoforms and in organisms as diverse as Schistosoma mansoni and Trichoplax adhaerens (the only organism identified without a tyrosine at this position is Lepeophtheirus salmonis, where it is replaced by a cysteine). In contrast, in PP2A and PP3 (PP3, calcineurin), the structurally equivalent residue is replaced by an isoleucine or valine, respectively. Thus, the specificity of PP1 for these regulators is likely mediated in part by the presence of a tyrosine residue at this binding pocket in PP1.
Interestingly, NIPP1 residues 160–175, which only show a ~20% populated α-helix in unbound NIPP1, become fully populated when bound to PP1. In contrast with the RVxF and the ΦΦ hydrophobic pocket interactions, which are mainly stabilized by hydrophobic contacts, the NIPP1helix
interaction is stabilized predominately by electrostatic interactions. Importantly, the ΦΦ hydrophobic pocket interaction was also observed in the spinophilin:PP1 complex and is thus the first structurally confirmed interaction site, outside of the RVxF binding groove, that has been detected in multiple PP1 holoenzymes (Peti et al., 2012
; Ragusa et al., 2010
). Therefore, there is now optimism that the analysis of additional PP1 holoenzymes will lead to a common understanding of how PP1 regulatory proteins bind and direct the activity of PP1.
Direct comparison with the structure of the spinophilin:PP1 holoenzyme shows that while both spinophilin and NIPP1 are IDPs, spinophilin folds completely upon binding to PP1 and becomes rigid, while NIPP1 retains some flexibility as electron density for NIPP1 residues 185–198 was not observed. This shows that different IDP PP1 regulators adopt different levels of structure and rigidity when bound to PP1. We previously showed that spinophilin inhibits the dephosphorylation of the canonical PP1 substrate glycogen phosphorylase a
by binding to the C-terminal substrate binding groove and blocking its access to PP1 residue D71, a residue previously shown to be critical for glycogen phosphorylase a
binding. Here, we show that a stretch of dynamic polybasic amino acids, N-terminal to the RVxF-motif of NIPP1, is necessary for the inhibition of the dephosphorylation of a subset of PP1 substrates, including glycogen phosphorylase a
. These results suggest that the increased, localized positive charge on NIPP1 influences glycogen phosphorylase a
substrate binding and/or positioning and thus likely inhibits its PP1-mediated dephosphorylation via a mechanism similar to that proposed for substrate selection by the MYPT1:PP1 holoenzyme, i.e. altered electrostatics (Terrak et al., 2004
Thus far, two distinct modes of substrate selection by PP1 holoenzymes have been reported. Spinophilin:PP1 selects substrates by sterically occluding specific substrate binding sites (Ragusa et al., 2010
). This mechanism for altering the substrate specificity of PP1 is different to that reported for the MYPT1:PP1 holoenzyme, which was proposed to be determined by altered electrostatics, as well as by potentially extending substrate binding grooves (Terrak et al., 2004
). The NIPP1:PP1 holoenzyme appears to function in a manner more similar to that of MYPT1:PP1. Specifically, the presence of NIPP1 significantly changes the electrostatic charge distribution of the surface of the NIPP1:PP1 holoenzyme when directly compared with PP1. We have also shown that the NIPP1 FHA domain plays a role in substrate recruitment. Recent peptide:NIPP1 FHA domain NMR interaction studies showed that this interaction is weak, but strong enough to achieve further selectivity for specific phosphorylated substrates (Kumeta et al., 2008
These observations begin to explain the striking diversity of PP1 holoenzymes, each of which may form a truly unique enzyme with distinctive properties. This is made even more intriguing by the fact that the number of identified PP1 targeting proteins (~200) is still increasing (Bollen et al., 2010
; Hendrickx et al., 2009
; Peti et al., 2012
). If the diversity of interactions observed for PP1 is conserved across ser/thr phosphatases, it would allow the ~40 ser/thr protein phosphatases to form hundreds of unique holoenzymes, ensuring that they are as specific as the 428 known ser/thr protein kinases.