Spinophilin, MYPT1 and I-2 all modulate the activity of PP1. They do so by either inhibiting the catalytic site (I-2) or by modifying the PP1 surface so specific substrates can be efficiently dephosphorylated (spinophilin and MYPT1). Despite their related functions and common binding partner, a comparison of the unbound structures of these three IDPs shows that they have different structural and dynamic characteristics in both the free and PP1-bound states.
First, spinophilin is highly dynamic in its unbound form; e.g. nearly no positive 15
H}-NOEs are observed [19
]. In contrast, I-2 possess a nearly 100% populated α
-helix, in which many strong (i
+ 1) HN
NOEs from residues in this helix are readily measured in a three-dimensional 15
H NOESY spectrum [38
]. In addition, residues near this α
-helix also have reduced backbone flexibility. Finally, at the other end of the IDP spectrum, only the 40 N-terminal residues are unstructured in MYPT1 and even these residues have restricted short-timescale backbone dynamics [35
Secondly, all three IDPs have pre-formed secondary-structural elements that are used to bind PP1. However, the number of pre-formed structures varies significantly between the three IDPs. The most dynamic protein, spinophilin, has the least amount of pre-formed secondary structure. Conversely, I-2 has three pre-formed α-helices. However, whereas some of these pre-formed secondary-structure elements in I-2 have a significant role in PP1 binding and biological function (e.g. the 100% populated α-helix in the free state binds PP1 and blocks its catalytic site, explaining the inhibitory mechanism of I-2), some pre-formed secondary-structural elements in I-2 are not used for interacting with PP1. Lastly, MYPT1 also has a pre-formed α-helix that is important for PP1 binding.
Thirdly, and as might be expected, spinophilin, I-2 and MYPT1 have different structural ensembles in their unbound forms [19
]. However, they also adopt significantly different conformations in their bound forms, i.e. when they are bound to PP1 to form PP1 holoenzymes (spinophilin–PP1, I-2–PP1 and MYPT1–PP1) (). Indeed, these structures reveal that, beyond the RVXF binding motif, there are no common PP1-binding motifs among the IDPs. Instead, these structures have revealed that PP1 is a protein interaction hub and that many PP1 surfaces are potential protein–protein interaction sites. In fact, using the average SASA (solvent-accessible surface area) of the RVXF motif and more recently identified PP1-binding pockets as a measure of the SASA needed for a single PP1-binding site, and comparing it with the SASA of the entire PP1 protein, we predict that PP1 may have up to 30 non-overlapping regulatory protein-binding sites [5
]. IDPs, because of their increased flexibility and extended structures, have a significant advantage compared with folded proteins when binding PP1 because they can easily interact with a single or multiple PP1-binding pockets using a minimal number of residues. In contrast, if PP1 holoenzymes were formed by folded regulatory proteins, these proteins would need to be much larger (~4-fold) to engage the equivalent PP1-binding surfaces. The diversity of binding sites available on PP1, and the inherent flexibility of IDPs, explains why, for this system, the conformations of IDPs bound to PP1 are distinct.
Fourthly, spinophilin, I-2 and MYPT1 use different mechanisms to bind PP1. Two major processes can lead to the selection of a single folded conformation when IDPs bind to a folded protein: conformational selection and/or induced fit. In the first case, the IDP has an intrinsic preference for its binding conformer and therefore the interacting protein is only a scaffold, i.e. it does not direct the formation of the bound conformation. This conformational selection model of binding requires that a limited population of the IDP adopts the bound-state conformation in its free state [39
]. This behaviour is observed in spinophilin and MYPT1. Both unbound states have pre-formed structures that are similar to the structures they adopt in their bound forms. This is not the case for I-2, which probably follows an ‘induced-fit’ process. Here, I-2 conformations in the unbound form are different from the PP1-bound conformer. In this case, the bound conformation is energetically accessible only in the presence of its binding partner [40
], PP1, and is either not detected in the unbound state or is so minimally populated that its presence is undetectable using the techniques that we have used in our studies. Clearly, the highly populated α
-helix in I-2 might play a significant role in initiating the induced-fit events that define the interaction of I-2 with PP1.
Fifthly, there are significant differences in the residual flexibility of these three IDPs in their PP1-bound states. Spinophilin, the most dynamic IDP in its unbound form, becomes completely rigid when bound to PP1. It does so by forming both a β
-sheet, which extends a β
-sheet in PP1, and an α
-helix, which was minimally populated in the unbound state. All residues have excellent electron density in the spinophilin–PP1 holoenzyme crystal structure. This behaviour is similar to that observed for the MYPT1–PP1 complex [36
], where MYPT1 becomes entirely structured when bound to PP1. This is interesting, as the most flexible (spinophilin) and most rigid (MYPT1) IDPs in their unbound states adopt fully rigid structures when bound to PP1. In contrast, I-2 is well-structured and more rigid than spinophilin in its unbound form. However, only ~25% of the I-2 residues adopt a rigid structure in the I-2–PP1 crystal structure [37
]; 75% of all I-2 residues stay flexible. Using NMR spectroscopy and SAXS, we were able to determine the ensemble structure of the I-2–PP1 holoenzyme in solution, which showed that the I-2 residues that stay flexible in the I-2–PP1 complex form two extended loop structures [19
]. Interestingly, using chemical shift data, we identified a ~60% populated α
-helix in unbound I-2, which is located in one of these extended loops. It is possible that this α
-helix forms an additional protein–protein interaction site, enabling this IDP to interact with other proteins while bound to PP1 [41