Mapping Phosphorylation-Specific Pin1–CRMP2 Interactions Using an Integrated Mass Spectrometry Approach

Abnormal protein phosphorylation is a fundamental trigger in the pathogenesis of Alzheimer’s Disease, leading to the formation of neurofibrillary tangles. Thus, molecular determination of the critical factors in controlling phosphorylation is desirable. Pin1, a cis–trans prolyl isomerase has recently been implicated in Alzheimer’s Disease progression. Moreover, Pin1 specifically targets phosphoproteins, regulating their function. Here, we reveal a novel interaction interface between Pin1 and the Collapsin Response Mediator Protein-2 (CRMP2), a protein found hyperphosphorylated alongside Tau within neurofibrillary tangles. Using native mass spectrometry, we show that Pin1 binds to the disordered C-terminus of CRMP2 in a phosphorylation-dependent manner with residues Thr509 and Thr514 on CRMP2 important for enhanced binding affinity. Hydrogen–deuterium exchange mass spectrometry experiments further localized this binding site to the WW domain of Pin1. Together, these findings provide novel insight into a putative regulatory role of Pin1 in modulating hyperphosphorylation of CRMP2.