Saposin B is a small, nonenzymatic glycosphingolipid activator protein required for the breakdown of cerebroside sulfates (sulfatides) within the lysosome. The protein can extract target lipids from membranes, forming soluble protein-lipid complexes that are recognized by arylsulfatase A. The crystal structure of human saposin B reveals an unusual shell-like dimer consisting of a monolayer of ␣-helices enclosing a large hydrophobic cavity. Although the secondary structure of saposin B is similar to that of the known monomeric members of the saposin-like superfamily, the helices are repacked into a different tertiary arrangement to form the homodimer. A comparison of the two forms of the saposin B dimer suggests that extraction of target lipids from membranes involves a conformational change that facilitates access to the inner cavity.T he saposins are a group of four structurally related activator proteins that function in conjunction with hydrolase enzymes for the stepwise breakdown of glycosphingolipids within the lysosome (1, 2). These proteins act by modifying the local environment of target lipids, and ''activate'' the breakdown of their substrates by presenting them in a form in which the lipid scissile bonds are accessible to the active sites of the specific enzymes. Presumably, the hydrolases cannot form a catalytic complex with the glycosphingolipids when these are in unmodified membrane bilayers.Each of the four saposins activates one or more lipid exohydrolases. For example, saposin B (also known as the cerebroside sulfate activator, or CS-Act) facilitates the hydrolysis of the sulfate group from cerebroside sulfate by arylsulfatase A, resulting in the formation of galactosylceramide (3). This glycolipid is then catabolized to ceramide by ␤-galactosylceramidase in a reaction activated by saposin C. There seems to be more than one mechanism of activation by the saposins, and this may further depend on the particular target lipid. Thus, saposin B is able to extract and solubilize cerebroside sulfates from membranes, allowing arylsulfatase A to act on the small, diffusible protein-lipid complexes. Saposin B may also have a physiological role in activating the hydrolysis of the ganglioside GM1 to GM2 by lysosomal ␤-galactosidase, and in this case, the activator may act by modifying the local lipid structure at the membrane surface to allow catalysis to proceed (4).The saposins belong to a large and diverse family of small, cysteine-rich proteins that share a common ability to interact with membranes but act in a wide variety of functions. Other members of the ''saposin-like'' superfamily of proteins include the lung surfactant-associated protein B (SP-B), the tumorolytic protein NK-lysin, granulysin, the pore-forming amoebapores, and the membrane-targeting domain of some enzymes (5, 6). The saposin-like proteins are Ϸ80 residues in length and have a characteristic pattern of conserved cysteines ( Fig. 1A; refs. 7 and 8). To date, the three-dimensional modeling of the saposins and other members of the sapo...

The ability of enzymes to distinguish between fatty acyl groups can involve molecular measuring devices termed hydrocarbon rulers, but the molecular basis for acyl-chain recognition in any membrane-bound enzyme remains to be defined. PagP is an outer membrane acyltransferase that helps pathogenic bacteria to evade the host immune response by transferring a palmitate chain from a phospholipid to lipid A (endotoxin). PagP can distinguish lipid acyl chains that differ by a single methylene unit, indicating that the enzyme possesses a remarkably precise hydrocarbon ruler. We present the 1.9 Å crystal structure of PagP, an eight-stranded β-barrel with an unexpected interior hydrophobic pocket that is occupied by a single detergent molecule. The buried detergent is oriented normal to the presumed plane of the membrane, whereas the PagP β-barrel axis is tilted by approximately 25°. Acyl group specificity is modulated by mutation of Gly88 lining the bottom of the hydrophobic pocket, thus confirming the hydrocarbon ruler mechanism for palmitate recognition. A striking structural similarity between PagP and the lipocalins suggests an evolutionary link between these proteins

Summary LDL receptor-related proteins 5 and 6 (LRP5/6) are co-receptors for Wnt growth factors, and also bind Dkk proteins, secreted inhibitors of Wnt signaling. The LRP5/6 ectodomain contains four β-propeller/EGF-like domain repeats. The first two repeats (LRP6(1-2)) bind to several Wnt variants, whereas LRP6(3-4) binds other Wnts. We present the crystal structure of the Dkk1 C-terminal domain bound to LRP6(3-4), and show that the Dkk1 N-terminal domain binds to LRP6(1-2), demonstrating that a single Dkk1 molecule can bind to both portions of the LRP6 ectodomain and thereby inhibit different Wnts. Small-angle x-ray scattering analysis of LRP6(1-4) bound to a non-inhibitory antibody fragment or to full-length Dkk1 shows that in both cases the ectodomain adopts a curved conformation that places the first three repeats at a similar height relative to the membrane. Thus, Wnts bound to either portion of the LRP6 ectodomain likely bear a similar spatial relationship to Frizzled co-receptors.

Saposins Aa nd Ca re sphingolipid activator proteins requiredf or thel ysosomal breakdowno f galactosylceramide and glucosylceramide,r espectively.T he saposins interact with lipids, leading to an enhanced accessibility of thel ipid headgroups to their cognate hydrolases. We have determinedt he crystal structures of humans aposinsAandCt o2 .0 A˚and 2.4Å ,r espectively,a nd both reveal the compact, monomeric saposin fold. We confirmed that these twoproteins were monomeric in solution at pH 7.0b yanalytical centrifugation. However, at pH 4.8,inthe presence of thedetergent C 8 E 5 ,saposin Aa ssembled intod imers, while saposin Cf ormed trimers. Saposin Bw as dimeric under allc onditions tested.T he self-associationo ft he saposins is likelyt ob er elevantt oh ow theses mall proteins interact with lipids, membranes, andh ydrolase enzymes.Keywords: saposins;X -ray crystallography; analyticalu ltracentrifugation; protein-detergent interactions Saposins A, B, C, and Da re small, nonenzymatic proteins required for the breakdown of glycosphingolipids within the lysosome (Kolter and Sandhoff2005). They are derived from the proteolytic processing of the precursor protein prosaposin, producing the four individual saposins. The four saposin domains contained within prosaposin most likely arose from two tandem duplications of an ancestral gene into one single copy gene (Hazkani-Covoetal. 2002). Each saposin ''activates''the breakdown of particular lipid substrates by facilitating the access of the lipid headgroups to the active sites of cognate hydrolases. It is generally believed that in the absence of sphingolipid activator proteins, the oligosaccharide chains of the membranebound lipids do not extend far enough into the lysosomal lumen to be accessible to the active sites of the hydrolases. Mechanistically,t he saposins appear to activate lipid hydrolysis by solubilizing the lipid substrates or possibly by destabilizing the membrane structure (Vaccaro et al. 1993(Vaccaro et al. , 1995(Vaccaro et al. , 1997Wilkening et al. 1998;Salvioli et al. 2000). Article publishedonline aheado fp rint.A rticle and publicationd ate area th ttp://www.proteinscience.org/cgi

SUMMARY Wnts are secreted growth factors that have critical roles in cell fate determination and stem cell renewal. The Wnt/β-catenin pathway is initiated by binding of a Wnt protein to a Frizzled (Fzd) receptor and a co-receptor, LDL receptor-related protein 5 or 6 (LRP5/6). We report the 2.1Å resolution crystal structure of a Drosophila WntD fragment encompassing the N-terminal domain and the linker that connects it to the C-terminal domain. Differences in the structures of WntD and Xenopus Wnt8, including the positions of a receptor-binding β-hairpin and a large solvent-filled cavity in the helical core, indicate conformational plasticity in the N-terminal domain that may be important for Wnt-Frizzled specificity. Structure-based mutational analysis of mouse Wnt3a shows that the linker between the N- and C-terminal domains is required for LRP6 binding. These findings provide important insights into Wnt function and evolution.