The tau protein plays essential roles in neurons. In healthy individuals, tau is critical to early neuronal development and helps sustain memory as we age. In neurodegenerative disease, including Alzheimer’s and several types of frontotemporal dementias, aberrant tau functioning may often be implicated.
Tau is an intrinsically disordered protein. The fact that it has no known folded structure has hindered its study. We propose that tau does adopt specific conformations in some crucial scenarios; namely, when it is in complex with protein interaction partners, which are critical to many of its functions. Here we aimed to determine one such tau conformation. Specifically, we employed crosslinking technologies to help determine the conformation of tau when in complex with the post-synaptic density protein 95 (PSD95), with which it interacts to regulate post-synaptic signals.
Firstly, we used crosslinking mass spectrometry (XL-MS) with the cleavable crosslinker DSSO to screen tau-PSD95 interaction interfaces. Utilising stepped HCD on an Orbitrap Exploris 480 and mass-difference triggered MS3 on an Orbitrap Tribrid Eclipse, we identified two domains with interaction interfaces on tau and one on PSD95.
Guided by these results, we then sought to pinpoint specific interacting residues using protein interface catalyzed capture (PICC), a novel covalent crosslinking strategy using on-protein chemistry. This involved the design and synthesis of tau peptides associated with interacting regions and modified with endogenous crosslinking residues. Both unlabeled (light) and heavy isotope labeled (heavy) endogenous crosslinking peptides were synthesised. Light and heavy crosslinked samples were mixed, digested, and subjected to LC-MS/MS, which provided capacity to confirm crosslinking through identification of co-eluting heavy and light peptide pairs using in-house built software (MethylQuant).
Together these experiments helped characterise tau-PSD95 interaction sites within the microtubule-binding domain and disordered regions of tau, and provided unprecendented insight into the conformations adopted by tau within a protein complex.