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Structure of membrane-bound alpha-synuclein from site-directed spin labeling and computational refinement.

ABSTRACT: alpha-Synuclein is known to play a causative role in Parkinson disease. Although its physiological functions are not fully understood, alpha-synuclein has been shown to interact with synaptic vesicles and modulate neurotransmitter release. However, the structure of its physiologically relevant membrane-bound state remains unknown. Here we developed a site-directed spin labeling and EPR-based approach for determining the structure of alpha-synuclein bound to a lipid bilayer. Continuous-wave EPR was used to assign local secondary structure and to determine the membrane immersion depth of lipid-exposed residues, whereas pulsed EPR was used to map long-range distances. The structure of alpha-synuclein was built and refined by using simulated annealing molecular dynamics restrained by the immersion depths and distances. We found that alpha-synuclein forms an extended, curved alpha-helical structure that is over 90 aa in length. The monomeric helix has a superhelical twist similar to that of right-handed coiled-coils which, like alpha-synuclein, contain 11-aa repeats, but which are soluble, oligomeric proteins (rmsd = 0.82 A). The alpha-synuclein helix extends parallel to the curved membrane in a manner that allows conserved Lys and Glu residues to interact with the zwitterionic headgroups, while uncharged residues penetrate into the acyl chain region. This structural arrangement is significantly different from that of alpha-synuclein in the presence of the commonly used membrane-mimetic detergent SDS, which induces the formation of two antiparallel helices. Our structural analysis emphasizes the importance of studying membrane protein structure in a bilayer environment.

SUBMITTER: Jao CC

PROVIDER: S-EPMC2605001 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Structure of membrane-bound alpha-synuclein from site-directed spin labeling and computational refinement.

Jao Christine C CC Hegde Balachandra G BG Chen Jeannie J Haworth Ian S IS Langen Ralf R

Proceedings of the National Academy of Sciences of the United States of America 20081209 50

alpha-Synuclein is known to play a causative role in Parkinson disease. Although its physiological functions are not fully understood, alpha-synuclein has been shown to interact with synaptic vesicles and modulate neurotransmitter release. However, the structure of its physiologically relevant membrane-bound state remains unknown. Here we developed a site-directed spin labeling and EPR-based approach for determining the structure of alpha-synuclein bound to a lipid bilayer. Continuous-wave EPR w ...[more]

PMID: 19066219

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Project description:Protein structures are not static but sample different conformations over a range of amplitudes and time scales. These fluctuations may involve relatively small changes in bond angles or quite large rearrangements in secondary structure and tertiary fold. The equilibrium between discrete structural substates on the microsecond to millisecond time scale is sometimes termed conformational exchange. Protein dynamics and conformational exchange are believed to provide the basis for many important activities, such as protein-protein and protein-ligand interactions, enzymatic activity and protein allostery; however, for many proteins, the dynamics and conformational exchange that lead to function are poorly defined. Spectroscopic methods, such as NMR, are among the most important methods to explore protein dynamics and conformational exchange; however, they are difficult to implement in some systems and with some types of exchange events. Site-directed spin labeling (SDSL) is an EPR based approach that is particularly well-suited to high molecular-weight systems such as membrane proteins. Because of the relatively fast time scale for EPR spectroscopy, it is an excellent method to examine exchange. Conformations that are in exchange are captured as distinct populations in the EPR spectrum, and this feature when combined with the use of methods that can shift the free energy of conformational substates allows one to identify regions of proteins that are in dynamic exchange. In addition, modern pulse EPR methods have the ability to examine conformational heterogeneity, resolve discrete protein states, and identify the substates in exchange. Protein crystallography has provided high-resolution models for a number of membrane proteins; but because of conformational exchange, these models do not always reflect the structures that are present when the protein is in a native bilayer environment. In the case of the Escherichia coli vitamin B12 transporter, BtuB, the energy coupling segment of this protein undergoes a substrate-dependent unfolding, which acts to couple this outer-membrane protein to the inner-membrane protein TonB. EPR spectroscopy demonstrates that the energy coupling segment is in equilibrium between ordered and disordered states, and that substrate binding shifts this equilibrium to favor an unfolded state. However, in crystal structures of BtuB, this segment is resolved and folded within the protein, and neither the presence of this equilibrium nor the substrate-induced change is revealed. This is a result of the solute environment and the crystal lattice, both of which act to stabilize one conformational substate of the transporter. Using SDSL, it can be shown that conformational exchange is present in other regions of BtuB and in other members of this transporter family. Conformational exchange has also been examined in systems such as the plasma membrane SNARE protein, syntaxin 1A, where dynamics are controlled by regulatory proteins such as munc18. Regulating the microsecond to millisecond time scale dynamics in the neuronal SNAREs is likely to be a key feature that regulates assembly of the SNAREs and neurotransmitter release.

Project description:The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca(2+)-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase C alpha (PKC alpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca(2+) binding loops and an anion binding site on beta-strands 3-4, such that the beta-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca(2+) binding loops and the beta-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKC alpha C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca(2+) activation or membrane docking trigger measurable fluorescence changes. Ca(2+) binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca(2+) binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Phi, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca(2+) binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca(2+) first and third Ca(2+) binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on beta-strands 3-4 lies near the headgroups. The data support a model in which the beta-strands are tilted toward the parallel orientation relative to the membrane surface.

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Project description:Protein kinase C isoform alpha (PKCalpha) is a ubiquitous, conventional PKC enzyme that possesses a conserved C2 domain. Upon activation by cytoplasmic Ca (2+) ions, the C2 domain specifically binds to the plasma membrane inner leaflet where it recognizes the target lipids phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP 2). The membrane penetration depth and docking angle of the membrane-associated C2 domain is not well understood. The present study employs EPR site-directed spin labeling and relaxation methods to generate a medium-resolution model of the PKCalpha C2 domain docked to a membrane of lipid composition similar to the plasma membrane inner leaflet. The approach measures EPR depth parameters for 10 function-retaining spin labels coupled to the C2 domain, and for spin labels coupled to depth calibration molecules. The resulting depth parameters, together with the known structure of the free C2 domain, provide a sufficient number of constraints to define two membrane docking geometries for C2 domain bound to physiological membranes lacking or containing PIP 2, respectively. In both the absence and presence of PIP 2, the two bound Ca (2+) ions of the C2 domain lie near the anionic phosphate plane in the headgroup region, consistent with the known ability of the Ca (2+) and membrane-binding loops (CMBLs) to bind the headgroup of the PS target lipid. In the absence of PIP 2, the polybasic lipid binding site on the beta3-beta4 hairpin is occupied with PS, but in the presence of PIP 2 this larger, higher affinity target lipid competitively displaces PS and causes the long axis of the domain to tilt 40 +/- 10 degrees toward the bilayer normal. The ability of the beta3-beta4 hairpin site to bind PS as well as PIP 2 extends the lifetime of the membrane-docked state and is predicted to enhance the kinase turnover number of PKCalpha during a single membrane docking event. In principle, PIP 2-induced tilting of the C2 domain could modulate the activity of membrane-docked PKCalpha as it diffuses between membrane regions with different local PS and PIP 2 concentrations. Finally, the results demonstrate that EPR relaxation methods are sufficiently sensitive to detect signaling-induced changes in the membrane docking geometries of peripheral membrane proteins.

Dataset Information

Structure of membrane-bound alpha-synuclein from site-directed spin labeling and computational refinement.

Publications

Structure of membrane-bound alpha-synuclein from site-directed spin labeling and computational refinement.

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