Effect of Phosphorylation on Supertertiary Structure and Ligand Binding Affinity on Scaffold Proteins

PSD-95 and PSD-93 are multidomain scaffold proteins with a similar tertiary structure and a common set of interaction partners. Despite the high sequence homology in their protein binding domains these isoforms play opposing roles in learning and memory. The origin of their functional differences remains a mystery. The most apparent physical difference between them is the length and composition of their interdomain linkers as well as the number and position of the phosphorylation sites. In PSD-95, the binding domains partition into two structurally-independently supramodules separated by an intrinsically disordered linker. PSD-93 has a similar intrinsically disordered region but the supertertiary structure has yet to be determined. We used single molecule fluorescence resonance energy transfer (FRET) to compare the supertertiary structures and found PSD-93 has a greater separation between supramodules. This could arise from the increased linker length but also a more even distribution of positive and negative charges in PSD-93. The number and spatial organization of charges determines the extension of intrinsically-disordered peptides. Interestingly, in vitro phosphorylation by Src kinase decreased the separation in PSD-93 but increased the separation in PSD-95 resulting a similar separation between supramodules upon phosphorylation. As with structural studies, binding between PSD-95 and its ligands has been extensively studied while the affinity of PSD-93 for their shared ligands is has not been measured. Using smFRET we found that the two scaffolds have similar binding affinity, before and after phosphorylation, for their common ligands. Furthermore, we found that phosphorylation selectively increased the affinity for some ligands while leaving others unchanged. Our results show that phosphorylation can remodel the supertertiary structure of multidomain scaffold proteins. Additionally, phosphorylation can change the specificity by selectively altering binding affinity for specific ligands.