Objective/Rationale: The alpha-synuclein is implicated in the pathogenesis of Parkinson’s disease. An aggregated form of synuclein containing many molecules has been proposed to interact destructively with nerve membranes. In contrast, our studies showed that one or a few molecules of synuclein form specific protein channels in membranes. These channels could have a positive or dissipative function in the metabolism of the central nervous system. Our identification of a specific synuclein function, channel formation in membranes, suggests that it may be possible to obtain a detailed (atomic level) structure of synuclein, which could allow identification of potential sites for drug action in the synuclein molecule.
For purposes of crystallization, genetically defined alpha-synuclein in a single molecule form will be expressed, purified, and characterized by its differential absorption of polarized light and ability to make channels in membranes. Using three different methods of protein crystallization, it is proposed to obtain protein crystals of alpha-synuclein that would ultimately allow determination of its atomic structure. The crystals would be formed in an environment resembling a biological membrane. Formation of threedimensional crystals will be tested in an environment consisting mainly of (i) detergent and/or (ii) of lipid. The structure of these crystals will be analyzed through the pattern of transmitted x-rays. Similarly, two dimensional crystals will be sought from which a structure of synuclein in a membrane environment can be obtained by analysis of the electron beam that passes though the crystal, measured with an electron microscope.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
There is substantial precedent for believing that the determination of the atomic structure of a medically relevant protein, a determinant of tissue or organ function, or of a disease, can identify potential drug targets that can, respectively, modify function or treat the disease. One example of a protein embedded in a membrane for which important information on drug binding sites has recently been obtained from determination of its crystal structure is the β-adrenergic receptor, a receptor for beta-blockers. This receptor is an integral membrane protein for which determination of the atomic structure has led to an understanding of the sites of action of cardiac, hypertension, and asthma drugs.
It is anticipated that crystals of alpha-synuclein will be obtained. If the structure obtained from these crystals describes an ion channel associated with function of the synuclein, then a likely site of drug action would be in the lumen of the channel, thereby blocking channel function. It is noted that, from our experience in studies on membrane proteins, it must be anticipated that the first crystals we obtain will not be good enough to resolve the structure of synuclein at atomic resolution. Even if they are imperfect, these crystals should provide substantial information about the nature of crystallization conditions that can lead to this goal.
3-D crystals and alpha-synuclein: Small crystals have been obtained of alpha synuclein in a model of biological membranes (bicelles). Preliminary analysis indicates that the crystals originate from the protein and not the bicelle lipid-detergent environment.
Structure of the alpha-synuclein channel: Experiments with alpha-synuclein have yielded well defined and positive results, and the possibility of mapping the topology and structure of the voltage-gated alpha-synuclein channel in the membrane. Through the use of a specific residue trapping strategy, these experiments emphatically suggest a physiological role for alpha-synuclein in synaptic transfer, as proposed previously (Zakharov et al., Helical α-Synuclein Forms Highly Conductive Ion Channels,” Biochemistry, 46: 14369-14379, 2007).