Zhiping Jiang

Biophysics of α-Synuclein Membrane Interactions

Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinsons disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. This article is part of a Special Issue entitled: Membrane protein structure and function.

Membrane remodeling by α-synuclein and effects on amyloid formation.

α-Synuclein (α-Syn), an intrinsically disordered protein, is associated with Parkinsons disease. Though molecular pathogenic mechanisms are ill-defined, mounting evidence connects its amyloid forming and membrane binding propensities to disease etiology. Contrary to recent data suggesting that membrane remodeling by α-syn involves anionic phospholipids and helical structure, we discovered that the protein deforms vesicles with no net surface charge (phosphatidylcholine, PC) into tubules (average diameter ∼20 nm). No discernible secondary structural changes were detected by circular dichroism spectroscopy upon the addition of vesicles. Notably, membrane remodeling inhibits α-syn amyloid formation affecting both lag and growth phases. Using five single tryptophan variants and time-resolved fluorescence anisotropy measurements, we determined that α-syn influences bilayer structure with surprisingly weak interaction and no site specificity (partition constant, Kp ∼ 300 M(-1)). Vesicle deformation by α-syn under a variety of different lipid/protein conditions is characterized via transmission electron microscopy. As cellular membranes are enriched in PC lipids, these results support possible biological consequences for α-syn induced membrane remodeling related to both function and pathogenesis.

Molecular Details of α-Synuclein Membrane Association Revealed by Neutrons and Photons

α-Synuclein (α-syn) is an abundant neuronal protein associated with Parkinsons disease that is disordered in solution, but it exists in equilibrium between a bent-helix and an elongated-helix on negatively charged membranes. Here, neutron reflectometry (NR) and fluorescence spectroscopy were employed to uncover molecular details of the interaction between α-syn and two anionic lipids, phosphatidic acid (PA) and phosphatidylserine (PS). Both NR and site-specific Trp measurements indicate that penetration depth of α-syn is similar for either PA- or PS-containing membranes (∼9-11 Å from bilayer center) even though there is a preference for α-syn binding to PA. However, closer examination of the individual Trp quenching profiles by brominated lipids reveals differences into local membrane interactions especially at position 39 where conformational heterogeneity was observed. The data also indicate that while W94 penetrates the bilayer as deeply as W4, W94 resides in a more polar surrounding. Taken together, we suggest the N- and C-terminal regions near positions 4 and 94 are anchored to the membrane, while the putative linker spanning residue 39 samples multiple conformations, which are sensitive to the chemical nature of the membrane surface. This flexibility may enable α-syn to bind diverse biomembranes in vivo.

Structural Features of Membrane-bound Glucocerebrosidase and α-Synuclein Probed by Neutron Reflectometry and Fluorescence Spectroscopy

Background: A specific interaction exists between α-synuclein and glucocerebrosidase on the lipid membrane, resulting in enzyme inhibition. Results: Binding glucocerebrosidase has a profound effect on α-synuclein, moving roughly half of its embedded helical region above the membrane plane. Conclusion: A model is proposed with structural insights into glucocerebrosidase inhibition by α-synuclein. Significance: α-Synuclein-glucocerebrosidase interaction provides a molecular connection between Parkinson and Gaucher diseases. Mutations in glucocerebrosidase (GCase), the enzyme deficient in Gaucher disease, are a common genetic risk factor for the development of Parkinson disease and related disorders, implicating the role of this lysosomal hydrolase in the disease etiology. A specific physical interaction exists between the Parkinson disease-related protein α-synuclein (α-syn) and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. Here, neutron reflectometry was employed as a first direct structural characterization of GCase and α-syn·GCase complex on a sparsely-tethered lipid bilayer, revealing the orientation of the membrane-bound GCase. GCase binds to and partially inserts into the bilayer with its active site most likely lying just above the membrane-water interface. The interaction was further characterized by intrinsic Trp fluorescence, circular dichroism, and surface plasmon resonance spectroscopy. Both Trp fluorescence and neutron reflectometry results suggest a rearrangement of loops surrounding the catalytic site, where they extend into the hydrocarbon chain region of the outer leaflet. Taking advantage of contrasting neutron scattering length densities, the use of deuterated α-syn versus protiated GCase showed a large change in the membrane-bound structure of α-syn in the complex. We propose a model of α-syn·GCase on the membrane, providing structural insights into inhibition of GCase by α-syn. The interaction displaces GCase away from the membrane, possibly impeding substrate access and perturbing the active site. GCase greatly alters membrane-bound α-syn, moving helical residues away from the bilayer, which could impact the degradation of α-syn in the lysosome where these two proteins interact.

Molecular origin of pH-dependent fibril formation of a functional amyloid.

Fibrils derived from Pmel17 are functional amyloids upon which melanin is deposited. Fibrils of the repeat domain (RPT) of Pmel17 form under strict melanosomal pH (4.5–5.5) and completely dissolve at pH≥6. To determine which Glu residue is responsible for this reversibility, aggregation of single, double, and quadruple Ala and Gln mutants were examined by intrinsic Trp fluorescence, circular dichroism spectroscopy, and transmission electron microscopy. Charge neutralization of E404, E422, E425, or E430, which are located in the putative amyloid‐forming region, modulated aggregation kinetics. Remarkably, the removal of a single negative charge at E422, one of 16 carboxylic acids, shifted the pH dependence by a full pH unit. Mutation at E404, E425, or E430 had little to no effect. We suggest that protonation at E422 is essential for initiating amyloid formation and that the other Glu residues play an allosteric role in fibril stability.

Lysophospholipid-Containing Membranes Modulate the Fibril Formation of the Repeat Domain of a Human Functional Amyloid, Pmel17

Pmel17 is an important protein for pigmentation in human skin and eyes. Proteolytic fragments from Pmel17 form fibrils upon which melanin is deposited in melanosomes. The repeat domain (RPT) derived from Pmel17 only forms fibrils under acidic melanosomal conditions. Here, we examined the effects of lipids on RPT aggregation to explore whether intramelanosomal vesicles can facilitate fibrillogenesis. Using transmission electron microscopy, circular dichroism, and fluorescence spectroscopy, we monitored fibril formation at the ultrastructural, secondary conformational, and local levels, respectively. Phospholipid vesicles and lysophospholipid (lysolipid) micelles were employed as membrane mimics. The surfactant-like lysolipids are particularly pertinent due to their high content in melanosomal membranes. Interestingly, RPT aggregation kinetics were influenced only by lysolipid-containing phospholipid vesicles. While both vesicles containing either anionic lysophosphatidylglycerol (LPG) or zwitterionic lysophosphatidylcholine (LPC) stimulate aggregation, LPG exerted a greater effect on reducing the apparent nucleation time. A detailed comparison showed distinct behaviors of LPG versus LPC monomers and micelles plausibly originating from their headgroup hydrogen bonding capabilities. Acceleration and retardation of aggregation were observed for LPG monomers and micelles, respectively. Because a specific interaction between LPG and RPT was identified by intrinsic W423 fluorescence and induced α-helical structure, it is inferred that binding of LPG near the C-terminal amyloid core initiates intermolecular association, whereas stabilization of α-helical conformation inhibits β-sheet formation. Contrastingly, LPC promotes RPT aggregation at both submicellar and micellar concentrations via non-specific binding with undetectable secondary structural change. Our findings suggest that protein-lysolipid interactions within melanosomes may regulate amyloid formation in vivo.

Segmental Deuteration of α-Synuclein for Neutron Reflectometry on Tethered Bilayers

Neutron reflectometry (NR) is uniquely suited for studying protein interaction with phospholipid bilayers along the bilayer normal on an angstrom scale. However, NR on its own cannot discern specific membrane-bound regions due to a lack of scattering contrast within a protein. Here we report the successful coupling of native chemical ligation (NCL) and NR to study α-synuclein (α-syn), a membrane-binding neuronal protein central in Parkinsons disease. Two α-syn variants were generated where either the first 86 or last 54 residues are deuterated, allowing for region-specific contrast within the protein and the identification of membrane interacting residues by NR. Residues 1-86 are positioned at the hydrocarbon/headgroup interface of the outer leaflet, whereas the density distribution of the 54 C-terminal residues ranges from the hydrocarbon region to the aqueous environment. Coupling of NCL and NR should have broad utility in studies of membrane protein folding.

Dissociation of glucocerebrosidase dimer in solution by its co-factor, saposin C.

Mutations in the gene for the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease and are the most common risk factor for Parkinson disease (PD). Analytical ultracentrifugation of 8 μM GCase shows equilibrium between monomer and dimer forms. However, in the presence of its co-factor saposin C (Sap C), only monomer GCase is seen. Isothermal calorimetry confirms that Sap C associates with GCase in solution in a 1:1 complex (Kd = 2.1 ± 1.1 μM). Saturation cross-transfer NMR determined that the region of Sap C contacting GCase includes residues 63-66 and 74-76, which is distinct from the region known to enhance GCase activity. Because α-synuclein (α-syn), a protein closely associated with PD etiology, competes with Sap C for GCase binding, its interaction with GCase was also measured by ultracentrifugation and saturation cross-transfer. Unlike Sap C, binding of α-syn to GCase does not affect multimerization. However, adding α-syn reduces saturation cross-transfer from Sap C to GCase, confirming displacement. To explore where Sap C might disrupt multimeric GCase, GCase x-ray structures were analyzed using the program PISA, which predicted stable dimer and tetramer forms. For the most frequently predicted multimer interface, the GCase active sites are partially buried, suggesting that Sap C might disrupt the multimer by binding near the active site.

Stimulation of α-synuclein amyloid formation by phosphatidylglycerol micellar tubules

α-Synuclein (α-Syn) is a presynaptic protein that is accumulated in its amyloid form in the brains of Parkinsons patients. Although its biological function remains unclear, α-syn has been suggested to bind to synaptic vesicles and facilitate neurotransmitter release. Recently, studies have found that α-syn induces membrane tubulation, highlighting a potential mechanism for α-syn to stabilize highly curved membrane structures which could have both functional and dysfunctional consequences. To understand how membrane remodeling by α-syn affects amyloid formation, we have studied the α-syn aggregation process in the presence of phosphatidylglycerol (PG) micellar tubules, which were the first reported example of membrane tubulation by α-syn. Aggregation kinetics, β-sheet content, and macroscopic protein-lipid structures were observed by Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy, respectively. Collectively, the presence of PG micellar tubules formed at a stochiometric (L/P = 1) ratio was found to stimulate α-syn fibril formation. Moreover, transmission electron microscopy and solid-state nuclear magnetic resonance spectroscopy revealed the co-assembly of PG and α-syn into fibril structures. However, isolated micellar tubules do not form fibrils by themselves, suggesting an important role of free α-syn monomers during amyloid formation. In contrast, fibrils did not form in the presence of excess PG lipids (≥L/P = 50), where most of the α-syn molecules are in a membrane-bound α-helical form. Our results provide new mechanistic insights into how membrane tubules modulate α-syn amyloid formation and support a pivotal role of protein-lipid interaction in the dysfunction of α-syn.

Effects of phosphatidylcholine membrane fluidity on the conformation and aggregation of N-terminally acetylated α-synuclein

Membrane association of α-synuclein (α-syn), a neuronal protein associated with Parkinsons disease (PD), is involved in α-syn function and pathology. Most previous studies on α-syn–membrane interactions have not used the physiologically relevant N-terminally acetylated (N-acetyl) α-syn form nor the most naturally abundant cellular lipid, i.e. phosphatidylcholine (PC). Here, we report on how PC membrane fluidity affects the conformation and aggregation propensity of N-acetyl α-syn. It is well established that upon membrane binding, α-syn adopts an α-helical structure. Using CD spectroscopy, we show that N-acetyl α-syn transitions from α-helical to disordered at the lipid melting temperature (Tm). We found that this fluidity sensing is a robust characteristic, unaffected by acyl chain length (Tm = 34–55 °C) and preserved in its homologs β- and γ-syn. Interestingly, both N-acetyl α-syn membrane binding and amyloid formation trended with lipid order (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) > 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin/cholesterol (2:2:1) ≥ DOPC), with gel-phase vesicles shortening aggregation kinetics and promoting fibril formation compared to fluid membranes. Furthermore, we found that acetylation enhances binding to PC micelles and small unilamellar vesicles with high curvature (r ∼16–20 nm) and that DPPC binding is reduced in the presence of cholesterol. These results confirmed that the exposure of hydrocarbon chains (i.e. packing defects) is essential for binding to zwitterionic gel membranes. Collectively, our in vitro results suggest that N-acetyl α-syn localizes to highly curved, ordered membranes inside a cell. We propose that age-related changes in membrane fluidity can promote the formation of amyloid fibrils, insoluble materials associated with PD.