Structural Role of Compensatory Amino Acid Replacements in the α-Synuclein Protein Valeria Losasso, †,‡ Adriana Pietropaolo, § Claudio Zannoni, ∥ Stefano Gustincich,* ,† and Paolo Carloni* ,‡ † International School for Advanced Studies (SISSA), 34136 Trieste, Italy ‡ German Research School for Simulation Sciences, FZ-Jülich and RWTH Aachen, Wilhelm-Johnen-Strasse, 52428 Jülich, Germany § Universita ̀ di Catanzaro, Dipartimento di Scienze Farmacobiologiche, 88100 Catanzaro, Italy ∥ Universita ̀ di Bologna, Dipartimento di Chimica Fisica ed Inorganica, 40136 Bologna, Italy * S Supporting Information ABSTRACT: A subset of familial Parkinson’s disease (PD) cases is associated with the presence of disease-causing point mutations in human α-synuclein [huAS(wt)], including A53T. Surprisingly, the human neurotoxic amino acid 53T is present in non-primate, wild-type sequences of α-synucleins, including that expressed by mice [mAS(wt)]. Because huAS(A53T) causes neurodegeneration when expressed in rodents, the amino acid changes between the wild-type human protein [huAS(wt)] and mAS(wt) might act as intramolecular suppressors of A53T toxicity in the mouse protein, restoring its physiological structure and function. The lack of structural information for mAS(wt) in aqueous solution has prompted us to conduct a comparative molecular dynamics study of huAS(wt), huAS(A53T), and mAS(wt) in water at 300 K. The calculations are based on an ensemble of nuclear magnetic resonance-derived huAS(wt) structures. huAS(A53T) turns out to be more flexible and less compact than huAS(wt). Its central (NAC) region, involved in fibril formation by the protein, is more solvent-exposed than that of the wild-type protein, in agreement with nuclear magnetic resonance data. The compactness of mAS(wt) is similar to that of the human protein. In addition, its NAC region is less solvent-exposed and more rigid than that of huAS(A53T). All of these features may be caused by an increase in the level of intramolecular interactions on passing from huAS(A53T) to mAS(wt). We conclude that the presence of “compensatory replacements” in the mouse protein causes a significant change in the protein relative to huAS(A53T), restoring features not too dissimilar to those of the human protein. P arkinson’s disease (PD) is a neurodegenerative disease affecting ∼5 million people worldwide. 1 In post-mortem brains of sporadic PD patients, proteinaceous fibrillar aggregates, called Lewy bodies, represent the neuropathological hallmark of the disease. The major components of Lewy bodies are fibrils of human α-synuclein [huAS(wt)] 2 whose function has not been fully elucidated. 3 huAS(wt) exists both in cytosol and bound to the membrane. 4 While it assumes a partially helical conformation in micelles, 5 NMR, 6−8 electron and fluorescence microscopy, 9 and circular dichroism (CD) 10−12 showed that huAS(wt) is a naturally unfolded protein in aqueous solution (Figure 1b) where it may establish long-range interactions. In vitro and in vivo, huAS(wt) fibrillates, forming a heterogeneous set of amyloid-like filaments and oligomers that are intimately involved in pathogenesis. 10,13 huAS(wt) features three domains (Figure 1a). The N- terminal (amino acids 1−60) amphipathic domain contains four 11-residue imperfect repeats. The hydrophobic domain of residues 61−95 contains two additional repeats and the amyloidogenic NAC (non-amyloid component) region. Hydro- phobic residues 71−82 of this region are involved in the conversion into fibrillar species. 14,15 Indeed, fluorimetry, electron microscopy, immunoelectron microscopy, and circular dichroism spectroscopy 15−18 have shown that these residues are crucial for fibril formation. In addition, deletion of residues 76 and 77 strongly impairs aggregation. 18 The SNCA gene encoding AS 19 has been found mutated in rare familial cases of PD. Together with pathological duplications, three point mutations, A30P, 20 E46K, 21 and A53T, 22 have been identified. Interestingly, the latter two fibrillate in vitro faster than huAS(wt), 23,24 while huAS(A30P) fibrillates slower. 23 In the resulting fibrils, the C-terminal domain of huAS(A53T) is more compact than that of huAS(wt), as revealed by solid-state NMR spectroscopy as well as electron and atomic force microscopy. 23,25,26 A30P and E46K mutations involve conserved amino acids across all species as expected for a disease-provoking mutation (Figure S1 of the Supporting Information). Instead, A53 is not evolutionarily conserved. 22 Intriguingly, in non-primate mam- mals, A is replaced by T, which is the familial mutation in PD. 53T is accompanied by as many as six other substitutions in mouse and rat, and seven in horse. Because huAS(A53T) causes neurodegeneration when expressed in rodents, 19,27−32 Received: May 16, 2011 Revised: July 5, 2011 Published: July 8, 2011 Article pubs.acs.org/biochemistry © 2011 American Chemical Society 6994 dx.doi.org/10.1021/bi2007564 | Biochemistry 2011, 50, 6994−7001