3D structure, ligand binding, autoinhibition, aggregation, and cellular distribution of wild-type, mutant, and labeled alpha-synuclein

CARLOS W. BERTONCINI; DMITRY CHERNY; CLAUDIO O. FERNANDEZ; ELIZABETH GARFINKEL; CHRISTIAN GRIESINGER; WOLFGANG HOYER; ELIZABETH A JARES-ERIJMAN; THOMAS M. JOVIN; DEREK MARSH; RODOLFO M. RASIA; MARÍA JULIA ROBERTI; VINOD SUBRAMANIAM; MARKUS ZWECKSTETTER

Long Beach, CA

Congreso; 49th Biopysical Society Meeting; 2005

The misfolding/dysfunction of alpha-synuclein (AS) is a feature of neurodegenerative disorders. The monomeric 140-aa protein has been presumed to be unstructured but adopts a beta-stranded conformation in the aggregated fibrils characteristic of the Lewy bodies in Parkinson´s disease (PD). Biogenic polyamines and Cu(II) promote aggregation of AS in vitro and may modulate the pathogenesis of PD. We have studied the binding of these agents to AS by biochemical, spectroscopic, and high-resolution microscopy (scanning probe, e.m.) techniques. The polyamine binding site was assigned by NMR to the C-terminal aa109-140, with distal secondary changes in aa22-93. These and other biochemical (aggregation kinetics, CD) and NMR [residual dipolar couplings (RDC) and paramagnetic relaxation enhancement (PRE) by nitroxide spin labels] measurements of wt and mutant AS labels reveal the existence of an aggregation-autoinhibited structure of native AS that is destabilized by ligands or by introduction of known (A30P and A53T) disease-related mutations. NMR, EPR, and absorption spectroscopy also reveal well-defined hierarchical binding sites for Cu(II), reinforcing the notion that AS adopts a restricted set of non-random conformations in solution. Thus, binding of natural or synthetic ligands may act as conformational switches and function as potential therapeutic or disease preventive agents. Two other approaches have been directed at defining the mechanism, intermediates and products of aggregation in vitro and in vivo: (1) temperature controlled in situ tapping mode AFM in solution to monitor the self-assembly of AS into nanostructures of various morphologies, including elongated sheets and spheroidal structures similar to those reported for several amyloidogenic proteins and linked to neurotoxicity; and (2) construction, characterization, and introduction into cells of fluorescent (cysteine-conjugates, inserted bisarsenical binding peptide motifs) derivatives of AS.