Structure of human saposin A at lysosomal pH.

The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme β-galactocerebrosidase (GALC), which catalyzes the breakdown of β-D-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from a `closed' to an `open' conformation. However, previous structures were determined at non-lysosomal pH. This work describes a 1.8 Å resolution X-ray crystal structure determined at the physiologically relevant lysosomal pH 4.8. In the absence of lipid or detergent at pH 4.8, SapA is observeed to adopt a conformation closely resembling the previously determined `closed' conformation, showing that pH alone is not sufficient for the transition to the `open' conformation. Structural alignments reveal small conformational changes, highlighting regions of flexibility.CHH is funded by a Wellcome Trust PhD studentship, RJR is supported by a Principal Research Fellowship funded by the Wellcome Trust (Grant No. 082961/Z/07/Z) and JED is supported by a Royal Society University Research Fellowship (UF100371). The Cambridge Institute for Medical Research is supported by a Wellcome Trust Strategic Award (100140).This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1107/S2053230X1500858

Molecular models should not be published without the corresponding atomic coordinates.

In PNAS, Romero et al. (1) present models of how glucocerebrosidase (GCase) interacts with saposin C (SAPC) and membranes. Unfortunately, the authors do not publish representative atomic coordinates or molecular dynamics trajectories for their models, denying researchers the opportunity to scrutinize the data Romero et al. (1) use to draw their functional conclusions. Access to these data is an important issue for structural biologists (2), and the open release of experimentally determined structural data has been the accepted practice for many years (3). Indeed, Romero et al. rely on several such publically available structures to carry out their study.J.E.D. is supported by a Royal Society University Research Fellowship (UF100371). S.C.G. is supported by a Sir Henry Dale Fellowship co-funded by the Royal Society and Wellcome Trust (098406/Z/12/B)

The lipid transfer protein Saposin B does not directly bind CD1d for lipid antigen loading.

Background: Lipid antigens are presented on the surface of cells by the CD1 family of glycoproteins, which have structural and functional similarity to MHC class I molecules. The hydrophobic lipid antigens are embedded in membranes and inaccessible to the lumenal lipid-binding domain of CD1 molecules. Therefore, CD1 molecules require lipid transfer proteins for lipid loading and editing. CD1d is loaded with lipids in late endocytic compartments, and lipid transfer proteins of the saposin family have been shown to play a crucial role in this process. However, the mechanism by which saposins facilitate lipid binding to CD1 molecules is not known and is thought to involve transient interactions between protein components to ensure CD1-lipid complexes can be efficiently trafficked to the plasma membrane for antigen presentation. Of the four saposin proteins, the importance of Saposin B (SapB) for loading of CD1d is the most well-characterised. However, a direct interaction between CD1d and SapB has yet to be described. Methods: In order to determine how SapB might load lipids onto CD1d, we used purified, recombinant CD1d and SapB and carried out a series of highly sensitive binding assays to monitor direct interactions. We performed equilibrium binding analysis, chemical cross-linking and co-crystallisation experiments, under a range of different conditions. Results: We could not demonstrate a direct interaction between SapB and CD1d using any of these binding assays. Conclusions: This work strongly indicates that the role of SapB in lipid loading does not involve direct binding to CD1d. We discuss the implication of this for our understanding of lipid loading of CD1d and propose several factors that may influence this process

New therapeutic approaches for Krabbe disease: The potential of pharmacological chaperones.

Missense mutations in the lysosomal hydrolase β-galactocerebrosidase (GALC) account for at least 40% of known cases of Krabbe disease (KD). Most of these missense mutations are predicted to disrupt the fold of the enzyme, preventing GALC in sufficient amounts from reaching its site of action in the lysosome. The predominant central nervous system (CNS) pathology and the absence of accumulated primary substrate within the lysosome mean that strategies used to treat other lysosomal storage disorders (LSDs) are insufficient in KD, highlighting the still unmet clinical requirement for successful KD therapeutics. Pharmacological chaperone therapy (PCT) is one strategy being explored to overcome defects in GALC caused by missense mutations. In recent studies, several small-molecule inhibitors have been identified as promising chaperone candidates for GALC. This Review discusses new insights gained from these studies and highlights the importance of characterizing both the chaperone interaction and the underlying mutation to define properly a responsive population and to improve the translation of existing lead molecules into successful KD therapeutics. We also highlight the importance of using multiple complementary methods to monitor PCT effectiveness. Finally, we explore the exciting potential of using combination therapy to ameliorate disease through the use of PCT with existing therapies or with more generalized therapeutics, such as proteasomal inhibition, that have been shown to have synergistic effects in other LSDs. This, alongside advances in CNS delivery of recombinant enzyme and targeted rational drug design, provides a promising outlook for the development of KD therapeutics. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.S.J.S. is funded by an MRC PhD studentship and J.E.D. is supported by a Royal Society University Research Fellowship (UF100371). The Cambridge Institute for Medical Research is supported by Wellcome Trust Strategic Award 100140.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Wiley

Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system

A construct consisting of residues 10–310 of mature BipD, a component of the B. pseudomallei type III secretion system, has been crystallized. Native BipD crystals and SeMet and K2PtCl4 derivative crystals have undergone preliminary crystallographic analysis

The mechanism of glycosphingolipid degradation revealed by a GALC-SapA complex structure.

Sphingolipids are essential components of cellular membranes and defects in their synthesis or degradation cause severe human diseases. The efficient degradation of sphingolipids in the lysosome requires lipid-binding saposin proteins and hydrolytic enzymes. The glycosphingolipid galactocerebroside is the primary lipid component of the myelin sheath and is degraded by the hydrolase β-galactocerebrosidase (GALC). This enzyme requires the saposin SapA for lipid processing and defects in either of these proteins causes a severe neurodegenerative disorder, Krabbe disease. Here we present the structure of a glycosphingolipid-processing complex, revealing how SapA and GALC form a heterotetramer with an open channel connecting the enzyme active site to the SapA hydrophobic cavity. This structure defines how a soluble hydrolase can cleave the polar glycosyl headgroups of these essential lipids from their hydrophobic ceramide tails. Furthermore, the molecular details of this interaction provide an illustration for how specificity of saposin binding to hydrolases is encoded

TAPBPR mediates peptide dissociation from MHC class I using a leucine lever.

Tapasin and TAPBPR are known to perform peptide editing on major histocompatibility complex class I (MHC I) molecules; however, the precise molecular mechanism(s) involved in this process remain largely enigmatic. Here, using immunopeptidomics in combination with novel cell-based assays that assess TAPBPR-mediated peptide exchange, we reveal a critical role for the K22-D35 loop of TAPBPR in mediating peptide exchange on MHC I. We identify a specific leucine within this loop that enables TAPBPR to facilitate peptide dissociation from MHC I. Moreover, we delineate the molecular features of the MHC I F pocket required for TAPBPR to promote peptide dissociation in a loop-dependent manner. These data reveal that chaperone-mediated peptide editing on MHC I can occur by different mechanisms dependent on the C-terminal residue that the MHC I accommodates in its F pocket and provide novel insights that may inform the therapeutic potential of TAPBPR manipulation to increase tumour immunogenicity.Wellcome PhD studentship (109076/Z/15/A). Wellcome Senior Research Fellowship (104647/Z/14/Z) South African Medical Research Council Bosch-Forschungsstiftung. Royal Society University Research Fellowship (UF100371)

A Tetrameric Assembly of Saposin A: Increasing Structural Diversity in Lipid Transfer Proteins

Saposins are lipid transfer proteins required for the degradation of sphingolipids in the lysosome. These small proteins bind lipids by transitioning from a closed, monomeric state to an open conformation exposing a hydrophobic surface that binds and shields hydrophobic lipid tails from the aqueous environment. Saposins form a range of multimeric assemblies to encompass these bound lipids and present them to hydrolases in the lysosome. This lipid-binding property of human saposin A has been exploited to form lipoprotein nanodiscs suitable for structural studies of membrane proteins. Here we present the crystal structure of a unique tetrameric assembly of murine saposin A produced serendipitously, following modifications of published protocols for making lipoprotein nanodiscs. The structure of this new saposin oligomer highlights the diversity of tertiary arrangement that can be adopted by these important lipid transfer proteins

TAPBPR bridges UDP-glucose : glycoprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway

Funding Wellcome: Senior Research Fellowship 104647, Andreas Neerincx, Louise H Boyle Royal Society: University Research Fellowship, UF100371, Janet E Deane Cancer Research UK: Programme Grant, C7056A, Andy van Hateren, Tim Elliott Deutsche Forschungsgemeinschaft: SFB 685, Nico Trautwein, Stefan Stevanović Wellcome: PhD studentship, 089563, Clemens Hermann Wellcome: Strategic Award 100140, Robin Antrobus Wellcome: Programme grant, WT094847MA, Huan Cao Acknowledgements We are extremely grateful to Peter Cresswell and Najla Arshad (Yale University School of Medicine, New Haven, CT) for valuable advice, tapasin and TAP-specific antibody reagents, and the recombinant calreticulin proteins. We thank John Trowsdale (University of Cambridge, UK) for his mentorship and critical reading of this manuscript, and Jim Kaufman (University of Cambridge, UK) for useful discussions. We also thank Yi Cao (Cranfield University, UK) for MATLAB programming for densitometry analysis, and Mark Vickers and Sadie Henderson (Scottish National Blood Transfusion Services, UK) for permitting the use of and assistance with the Amersham WB system. The reagent ARP7099 FEC peptide pool was obtained from the Centre for AIDS Reagents, National Institute for Biological Standards and Control (NIBSC), and was donated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH.Peer reviewedPublisher PD

Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system

IpaD, the putative needle-tip protein of the S. flexneri type III secretion system, has been crystallized in a variety of crystal forms using in-drop proteolysis. Native and selenomethionine-labelled data collection and preliminary analyses are reported