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Structure of Saposin a Lipoprotein Discs

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Structure of Saposin a Lipoprotein Discs Structure of saposin A lipoprotein discs Konstantin Popovica, John Holyoakeb,c, Régis Pomèsb,d, and Gilbert G. Privéa,c,d,1 aDepartment of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 2M9; bMolecular Structure and Function, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8; cOntario Cancer Institute, Campbell Family Institute for Cancer Research, Toronto, ON, Canada M5G 1L7; and dDepartment of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8 Edited by Donald Engelman, Yale University, New Haven, CT, and approved December 17, 2011 (received for review September 23, 2011) The saposins are small, membrane-active proteins that exist in both functions to activate the sphingolipid hydrolysis reaction. How- soluble and lipid-bound states. Saposin A has roles in sphingolipid ever, structural flexibility is a crucial feature for the membrane catabolism and transport and is required for the breakdown of surface binding and lipid-solubilizing abilities of the saposin pro- galactosylceramide by β-galactosylceramidase. In the absence of teins (3, 18, 19). lipid, saposin A adopts a closed monomeric apo conformation Here, we characterize the interactions of saposin A with var- typical of this family. To study a lipid-bound state of this protein, ious amphiphiles. Saposin A undergoes a conformational change we determined the crystal structure of saposin A in the presence of in the presence of lipids and detergents and forms small lipo- detergent to 1.9 Å resolution. The structure reveals two chains of protein particles with a wide range of lipids. The 1.9 Å crystal saposin A in an open conformation encapsulating 40 internally structure of saposin A in complex with zwitterionic detergent bound detergent molecules organized in a highly ordered bilayer- lauryldimethylamine-N-oxide (LDAO) reveals two saposin chains like hydrophobic core. The complex provides a high-resolution in an open state forming an amphipathic protein belt surrounding view of a discoidal lipoprotein particle in which all of the interna- 40 ordered detergent molecules. The structure of the saposin A lized acyl chains are resolved. Saposin A lipoprotein discs exhibit lipoprotein discs is further supported by coarse-grained molecu- limited selectivity with respect to the incorporated lipid, and can lar dynamics simulations. Overall, we find that saposin A/lipid solubilize phospholipids, sphingolipids, and cholesterol into dis- assemblies form unusual lipoprotein complexes that share some crete, monodisperse particles with mass of approximately 27 kDa. similarities with discoidal high density lipoprotein particles, but These discs may be the smallest possible lipoprotein structures that are significantly smaller. are stabilized by lipid self-assembly. BIOPHYSICS AND Results and Discussion COMPUTATIONAL BIOLOGY protein-lipid complex ∣ X-ray crystallography ∣ molecular dynamics ∣ Characterization of Saposin A with Amphiphiles. To describe the Krabbe disease lipid-induced conformational changes in the structure of human saposin A, we used intrinsic tryptophan fluorescence spectro- phingolipid activator proteins (SAPs) are nonenzymatic pro- scopy and monitored amphiphile-induced spectral shifts. All Steins that are required for the lysosomal breakdown of certain solution experiments were conducted at pH 4.8, reflective of the sphingolipids by hydrolase enzymes (1). In general, the SAPs are pH within lysosomes. Saposin A contains a single tryptophan, thought to act by modifying the physical states of the target lipids W37, which is solvent exposed in the closed monomeric form. so that the substrates become accessible to the active sites of the Incubation of saposin A with liposomes resulted in an increase in enzymes. The details of how this effect is achieved are not clear, the intensity of the tryptophan emission spectrum and a 6–8nm but it is likely that different SAPs use different mechanisms in blue shift, indicative of a reduction in the solvent accessibility of activating one or more specific lipid/enzyme reactions (2–4). the residue and a shift to a less polar environment (Fig. 1A). This Human lysosomes contain five SAPs: the GM2 activator and the effect was seen with liposomes made from a wide variety of lipids saposins A, B, C, and D. The four homologous saposins are small and lipid mixtures. We also found that several nondenaturing cysteine-rich α-helical glycoproteins that are derived from the detergents, including LDAO, induced similar spectral shifts. The prosaposin precursor protein (5, 6). Saposin A activates the characteristic changes occurred at concentrations above the cri- hydrolysis of galactosylceramide by β-galactosylceramidase to tical micelle concentration of the detergent. produce ceramide and galactose (7, 8). Loss of functional saposin Saposin A interacts with liposomes and immobilized lipids A results in a variant form of Krabbe disease, which is character- (20) but does not remain bound to liposomes (21), suggesting that ized by demyelination, loss of oligodendrocytes, and infiltration the protein can form soluble lipid complexes. We incubated lipo- of globoid cells (9–12). Saposin A is also important for the load- somes of varying composition with saposin A and analyzed the ing of lipid antigens onto CD1d molecules (13, 14). solutions by size exclusion chromatography (SEC). We consis- We previously reported the crystal structure of the soluble tently observed a single sharp peak in the 35–45 kDa range of the form of saposin A in the absence of bound lipids (15). In this column, corresponding to hydrodynamic radii ranging from 3.07– structure, the small 81 residue protein adopts the compact, mono- 3.45 nm (Fig. 1B, Fig. S1, TableS1). Collectively, we refer to these meric four α-helix closed form of the fold seen in other saposins species as 3.2-nm particles. An additional absorbance peak at and saposin-like proteins (15–18). Helices 1 and 4 are disulfide the void volume of the column was occasionally detected but did linked in a stem structure, whereas helices 2 and 3 are disulfide not contain significant amounts of protein and was due to light linked to each other and form a helical hairpin. In the closed scattering from large lipid vesicles (Fig. S1). The exact size of form, the stem and hairpin segments collapse onto themselves, burying the hydrophobic surfaces of the four amphipathic helices Author contributions: K.P., J.H., R.P., and G.G.P. designed research; K.P. and J.H. performed via bends at the short turns at the α1∕α2 and α3∕α4 junctions. research; K.P., J.H., R.P., and G.G.P. analyzed data; and K.P., J.H., R.P., and G.G.P. wrote Notably, the closed form of the saposin fold buries the majority the paper. of the hydrophobic surfaces into a small hydrophobic core and The authors declare no conflict of interest. does not form a cavity that can accommodate lipids. In addition, This article is a PNAS Direct Submission. the external surface of the closed conformation of saposin A is Data deposition: Crystallography, atomic coordinates, and structure factors have been polar and does not have any extended nonpolar regions that deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4DDJ). would suggest a membrane-active protein. Overall, the lipid-free 1To whom correspondence should be addressed. E-mail: [email protected]. structure of saposin A provides only limited insight into the lipid This article contains supporting information online at www.pnas.org/lookup/suppl/ interaction properties of saposin A and does not explain how it doi:10.1073/pnas.1115743109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1115743109 PNAS Early Edition ∣ 1of5 Downloaded by guest on September 24, 2021 higher energy X-rays. Data collection and refinement statistics are 5.2 nm 232 kDa 43 kDa 6.5 kDa Vo 3.1 nm 1.35 nm given in Ta b l e S 2 . A saposin A B + lipid mix 4 The crystal structure revealed an open saposin fold in which + PC 0.5 + LDAO the stem and hairpin segments of the protein are opened in a 3 0.4 jackknife fashion relative to the closed form of the protein 0.3 2 (Fig. 2). A comparison of the lipid-free closed form of saposin 0.2 A (15) and LDAO-bound open form reveal that the main struc- 1 tural rearrangements in the protein involve residues 19–23 and 0.1 A280 nm (mAU) – 0.0 0 62 69. These two regions form the hinges that allow the stem and Relative fluorescence 320 340 360 380 400 0 5 10 15 20 25 hairpin segments of saposin A to open up into a V shape, result- Wavelength (nm) mL ing in an opening of approximately 115° between the two halves of 30 the protein. Residues 62–67 are in a coil conformation in the C D 3 6 α 25 closed form, but extend the C-terminal end of helix 3 in the open form. There are only minor conformational changes near residue 20 2 4 Y54, which is an important hinge residue in saposins B and C (3, 15 18, 19). Structures of saposin C have been observed in two open 1 % Intensity 10 2 states: as a dimeric homodimer with no associated lipid (18) and A280 nm (mAU) 5 A490 nm (mAU) in association with SDS micelles by NMR (19). In the latter 0 0 0 study, no information about the structure of the bound detergent 0.1 1 10 100 1,000 10,000 0 510152025 or the overall assembly of the saposin C/SDS complex could be mL Rh (nm) determined. Fig. 1. Solution characterization of saposin A in the presence of lipids and The open state of the protein exposes a concave hydrophobic detergent. (A) Emission spectra of saposin A in the absence of lipid or deter- inner face, and this surface is covered by LDAO acyl chains in the gent (blue), in the presence of a lipid mix designed to mimic lysosomal lipids crystal structure (Fig.
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