Targeting the interface of the pathological complex of α-synuclein and TPPP/p25

•TPPP/p25 and α-synuclein (SYN) display physiological and pathological functions.•Mutants/fragments of SYN were produced to identify the interface of TPPP/p25-SYN complex.•121–140 aa segment of SYN is crucial for TPPP/p25 binding.•Targeting interface of proteins with multiple functions is an innovative strategy for drug discovery.

The pathological interaction of intrinsically disordered proteins, such as α-synuclein (SYN) and Tubulin Polymerization Promoting Protein (TPPP/p25), is often associated with neurodegenerative disorders. These hallmark proteins are co-enriched and co-localized in brain inclusions of Parkinson's disease and other synucleinopathies; yet, their successful targeting does not provide adequate effect due to their multiple functions. Here we characterized the interactions of the human recombinant wild type SYN, its truncated forms (SYN1–120, SYN95–140), a synthetized peptide (SYN126–140) and a proteolytic fragment (SYN103–140) with TPPP/p25 to identify the SYN segment involved in the interaction. The binding of SYN103–140 to TPPP/p25 detected by ELISA suggested the involvement of a segment within the C-terminal of SYN. The studies performed with ELISA, Microscale Thermophoresis and affinity chromatography proved that SYN95–140 and SYN126–140 – in contrast to SYN1–120 – displayed significant binding to TPPP/p25. Fluorescence assay with ANS, a molten globule indicator, showed that SYN, but not SYN1–120 abolished the zinc-induced local folding of both the full length as well as the N- and C-terminal-free (core) TPPP/p25; SYN95–140 and SYN126–140 were effective as well. The aggregation-prone properties of the SYN species with full length or core TPPP/p25 visualized by immunofluorescent microscopy demonstrated that SYN95–140 and SYN126–140, but not SYN1–120, induced co-enrichment and massive intracellular aggregation after their premixing and uptake from the medium. These data with their innovative impact could contribute to the development of anti-Parkinson drugs with unique specificity by targeting the interface of the pathological TPPP/p25-SYN complex.