Structures of the mannose-6-phosphate pathway enzyme, GlcNAc-1-phosphotransferase.

The mannose-6-phosphate (M6P) pathway is responsible for the transport of hydrolytic enzymes to lysosomes. N-acetylglucosamine-1-phosphotransferase (GNPT) catalyzes the first step of tagging these hydrolases with M6P, which when recognized by receptors in the Golgi diverts them to lysosomes. Genetic defects in the GNPT subunits, GNPTAB and GNPTG, cause the lysosomal storage diseases mucolipidosis types II and III. To better understand its function, we determined partial three-dimensional structures of the GNPT complex. The catalytic domain contains a deep cavity for binding of uridine diphosphate-N-acetylglucosamine, and the surrounding residues point to a one-step transfer mechanism. An isolated structure of the gamma subunit of GNPT reveals that it can bind to mannose-containing glycans in different configurations, suggesting that it may play a role in directing glycans into the active site. These findings may facilitate the development of therapies for lysosomal storage diseases.

Crystal structure of the mammalian lipopolysaccharide detoxifier.

LPS is a potent bacterial endotoxin that triggers the innate immune system. Proper recognition of LPS by pattern-recognition receptors requires a full complement of typically six acyl chains in the lipid portion. Acyloxyacyl hydrolase (AOAH) is a host enzyme that removes secondary (acyloxyacyl-linked) fatty acids from LPS, rendering it immunologically inert. This activity is critical for recovery from immune tolerance that follows Gram-negative infection. To understand the molecular mechanism of AOAH function, we determined its crystal structure and its complex with LPS. The substrate's lipid moiety is accommodated in a large hydrophobic pocket formed by the saposin and catalytic domains with a secondary acyl chain inserted into a narrow lateral hydrophobic tunnel at the active site. The enzyme establishes dispensable contacts with the phosphate groups of LPS but does not interact with its oligosaccharide portion. Proteolytic processing allows movement of an amphipathic helix possibly involved in substrate access at membranes.

Molecular mechanism of activation of the immunoregulatory amidase NAAA.

Palmitoylethanolamide is a bioactive lipid that strongly alleviates pain and inflammation in animal models and in humans. Its signaling activity is terminated through degradation by N-acylethanolamine acid amidase (NAAA), a cysteine hydrolase expressed at high levels in immune cells. Pharmacological inhibitors of NAAA activity exert profound analgesic and antiinflammatory effects in rodent models, pointing to this protein as a potential target for therapeutic drug discovery. To facilitate these efforts and to better understand the molecular mechanism of action of NAAA, we determined crystal structures of this enzyme in various activation states and in complex with several ligands, including both a covalent and a reversible inhibitor. Self-proteolysis exposes the otherwise buried active site of NAAA to allow catalysis. Formation of a stable substrate- or inhibitor-binding site appears to be conformationally coupled to the interaction of a pair of hydrophobic helices in the enzyme with lipid membranes, resulting in the creation of a linear hydrophobic cavity near the active site that accommodates the ligand's acyl chain.

Crystal structure of saposin D in an open conformation.

Saposins are accessory proteins that aid in the degradation of sphingolipids by hydrolytic enzymes. Their structure usually comprises four α-helices arranged in various conformations including an open, V-shaped form that is generally associated with the ability to interact with membranes and/or enzymes to accentuate activity. Saposin D is required by the lysosomal hydrolase, acid ceramidase, which breaks down ceramide into sphingosine and free fatty acid, to display optimal activity. The structure of saposin D was previously determined in an inactive conformation, revealing a monomeric, closed and compact form. Here, we present the crystal structure of the open, V-shaped form of saposin D. The overall shape is similar to the open conformation found in other saposins with slight differences in the angles between the α-helices. The structure forms a dimer that serves to stabilize the hydrophobic surface exposed in the open form, which results in an internal, hydrophobic cavity that could be used to carry extracted membrane lipids.

Crystal structure of the human alkaline sphingomyelinase provides insights into substrate recognition.

Absorption of dietary sphingomyelin (SM) requires its initial degradation into ceramide, a process catalyzed by the intestinal enzyme alkaline sphingomyelinase (alk-SMase, NPP7, ENPP7). alk-SMase belongs to the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, the members of which hydrolyze nucleoside phosphates, phospholipids, and other related molecules. NPP7 is the only paralog that can cleave SM, and its activity requires the presence of bile salts, a class of physiological anionic detergents. To elucidate the mechanism of substrate recognition, we determined the crystal structure of human alk-SMase in complex with phosphocholine, a reaction product. Although the overall fold and catalytic center are conserved relative to other NPPs, alk-SMase recognizes the choline moiety of its substrates via an NPP7-specific aromatic box composed of tyrosine residues. Mutational analysis and enzymatic activity assays identified features on the surface of the protein-a cationic patch and a unique hydrophobic loop-that are essential for accessing SM in bile salt micelles. These results shed new light on substrate specificity determinants within the NPP enzyme family.

Structural Basis for Nucleotide Hydrolysis by the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3A.

Sphingomyelin phosphodiesterase, acid-like 3A (SMPDL3A) is a member of a small family of proteins founded by the well characterized lysosomal enzyme, acid sphingomyelinase (ASMase). ASMase converts sphingomyelin into the signaling lipid, ceramide. It was recently discovered that, in contrast to ASMase, SMPDL3A is inactive against sphingomyelin and, surprisingly, can instead hydrolyze nucleoside diphosphates and triphosphates, which may play a role in purinergic signaling. As none of the ASMase-like proteins has been structurally characterized to date, the molecular basis for their substrate preferences is unknown. Here we report crystal structures of murine SMPDL3A, which represent the first structures of an ASMase-like protein. The catalytic domain consists of a central mixed ß-sandwich surrounded by α-helices. Additionally, SMPDL3A possesses a unique C-terminal domain formed from a cluster of four α-helices that appears to distinguish this protein family from other phosphoesterases. We show that SMDPL3A is a di-zinc-dependent enzyme with an active site configuration that suggests a mechanism of phosphodiester hydrolysis by a metal-activated water molecule and protonation of the leaving group by a histidine residue. Co-crystal structures of SMPDL3A with AMP and α,ß-methylene ADP (AMPCP) reveal that the substrate binding site accommodates nucleotides by establishing interactions with their base, sugar, and phosphate moieties, with the latter the major contributor to binding affinity. Our study provides the structural basis for SMPDL3A substrate specificity and sheds new light on the function of ASMase-like proteins.

Crystal Structure of the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3B Provides Insights into Determinants of Substrate Specificity.

The enzyme acid sphingomyelinase-like phosphodiesterase 3B (SMPDL3B) was shown to act as a negative regulator of innate immune signaling, affecting cellular lipid composition and membrane fluidity. Furthermore, several reports identified this enzyme as an off target of the therapeutic antibody rituximab, with implications in kidney disorders. However, structural information for this protein is lacking. Here we present the high resolution crystal structure of murine SMPDL3B, which reveals a substrate binding site strikingly different from its paralogs. The active site is located in a narrow boot-shaped cavity. We identify a unique loop near the active site that appears to impose size constraints on incoming substrates. A structure in complex with phosphocholine indicates that the protein recognizes this head group via an aromatic box, a typical choline-binding motif. Although a potential substrate for SMPDL3B is sphingomyelin, we identify other possible substrates such as CDP-choline, ATP, and ADP. Functional experiments employing structure-guided mutagenesis in macrophages highlight amino acid residues potentially involved in recognition of endogenous substrates. Our study is an important step toward elucidating the specific function of this poorly characterized enzyme.

Secondary deficiency of neuraminidase 1 contributes to CNS pathology in neurological mucopolysaccharidoses via hypersialylation of brain glycoproteins.

Mucopolysaccharidoses (MPS) are lysosomal storage diseases caused by defects in catabolism of glycosaminoglycans. MPS I, II, III and VII are associated with lysosomal accumulation of heparan sulphate and manifest with neurological deterioration. Most of these neurological MPS currently lack effective treatments. Here, we report that, compared to controls, neuraminidase 1 (NEU1) activity is drastically reduced in brain tissues of neurological MPS patients and in mouse models of MPS I, II, IIIA, IIIB and IIIC, but not of other neurological lysosomal disorders not presenting with heparan sulphate storage. We further show that accumulated heparan sulphate disrupts the lysosomal multienzyme complex of NEU1 with cathepsin A (CTSA), ß-galactosidase (GLB1) and glucosamine-6-sulfate sulfatase (GALNS) necessary to maintain enzyme activity, and that NEU1 deficiency is linked to partial deficiencies of GLB1 and GALNS in cortical tissues and iPSC-derived cortical neurons of neurological MPS patients. Increased sialylation of N-linked glycans in brain samples of human MPS III patients and MPS IIIC mice implicated insufficient processing of brain N-linked sialylated glycans, except for polysialic acid, which was reduced in the brains of MPS IIIC mice. Correction of NEU1 activity in MPS IIIC mice by lentiviral gene transfer ameliorated previously identified hallmarks of the disease, including memory impairment, behavioural traits, and reduced levels of the excitatory synapse markers VGLUT1 and PSD95. Overexpression of NEU1 also restored levels of VGLUT1-/PSD95-positive puncta in cortical neurons derived from iPSC of an MPS IIIA patient. Together, our data demonstrate that heparan sulphate-induced secondary NEU1 deficiency and aberrant sialylation of glycoproteins implicated in synaptogenesis, memory, and behaviour constitute a novel pathological pathway in neurological MPS spectrum crucially contributing to CNS pathology.

Structure of the immunoregulatory sialidase NEU1.

Sialic acids linked to glycoproteins and glycolipids are important mediators of cell and protein recognition events. These sugar residues are removed by neuraminidases (sialidases). Neuraminidase-1 (sialidase-1 or NEU1) is a ubiquitously expressed mammalian sialidase located in lysosomes and on the cell membrane. Because of its modulation of multiple signaling processes, it is a potential therapeutic target for cancers and immune disorders. Genetic defects in NEU1 or in its protective protein cathepsin A (PPCA, CTSA) cause the lysosomal storage diseases sialidosis and galactosialidosis. To further our understanding of this enzyme's function at the molecular level, we determined the three-dimensional structure of murine NEU1. The enzyme oligomerizes through two self-association interfaces and displays a wide substrate-binding cavity. A catalytic loop adopts an inactive conformation. We propose a mechanism of activation involving a conformational change in this loop upon binding to its protective protein. These findings may facilitate the development of selective inhibitor and agonist therapies.

Structural basis of carbohydrate recognition by calreticulin.

The calnexin cycle is a process by which glycosylated proteins are subjected to folding cycles in the endoplasmic reticulum lumen via binding to the membrane protein calnexin (CNX) or to its soluble homolog calreticulin (CRT). CNX and CRT specifically recognize monoglucosylated Glc(1)Man(9)GlcNAc(2) glycans, but the structural determinants underlying this specificity are unknown. Here, we report a 1.95-Å crystal structure of the CRT lectin domain in complex with the tetrasaccharide α-Glc-(1→3)-α-Man-(1→2)-α-Man-(1→2)-Man. The tetrasaccharide binds to a long channel on CRT formed by a concave ß-sheet. All four sugar moieties are engaged in the protein binding via an extensive network of hydrogen bonds and hydrophobic contacts. The structure explains the requirement for glucose at the nonreducing end of the carbohydrate; the oxygen O(2) of glucose perfectly fits to a pocket formed by CRT side chains while forming direct hydrogen bonds with the carbonyl of Gly(124) and the side chain of Lys(111). The structure also explains a requirement for the Cys(105)-Cys(137) disulfide bond in CRT/CNX for efficient carbohydrate binding. The Cys(105)-Cys(137) disulfide bond is involved in intimate contacts with the third and fourth sugar moieties of the Glc(1)Man(3) tetrasaccharide. Finally, the structure rationalizes previous mutagenesis of CRT and lays a structural groundwork for future studies of the role of CNX/CRT in diverse biological pathways.

Structure of the murine lysosomal multienzyme complex core.

The enzymes ß-galactosidase (GLB1) and neuraminidase 1 (NEU1; sialidase 1) participate in the degradation of glycoproteins and glycolipids in the lysosome. To remain active and stable, they associate with PPCA [protective protein cathepsin A (CTSA)] into a high-molecular weight lysosomal multienzyme complex (LMC), of which several forms exist. Genetic defects in these three proteins cause the lysosomal storage diseases GM1-gangliosidosis/mucopolysaccharidosis IV type B, sialidosis, and galactosialidosis, respectively. To better understand the interactions between these enzymes, we determined the three-dimensional structure of the murine LMC core. This 0.8-MDa complex is composed of three GLB1 dimers and three CTSA dimers, adopting a triangular architecture maintained through six copies of a unique GLB1-CTSA polar interface. Mutations in this contact surface that occur in GM1-gangliosidosis prevent formation of the LMC in vitro. These findings may facilitate development of therapies for lysosomal storage disorders.

Crystal Structure of the Mannose-6-Phosphate Uncovering Enzyme.

Most lysosomal hydrolytic enzymes reach their destination via the mannose-6-phosphate (M6P) pathway. The enzyme N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (NAGPA, or "uncovering enzyme") catalyzes the second step in the M6P tag formation, namely the removal of the masking N-acetylglucosamine (GlcNAc) portion. Defects in this protein are associated with non-syndromic stuttering. To gain a better understanding of the function and regulation of this enzyme, we determined its crystal structure. The propeptide binds in a groove on the globular catalytic domain, blocking active site access. High-affinity substrate binding is enabled by a conformational switch in an active site loop. The protein recognizes the GlcNAc and phosphate portions of its substrate, but not the mannose moiety of the glycan. Based on enzymatic and 1H-NMR analysis, a catalytic mechanism is proposed. Crystallographic and solution scattering analyses suggest that the C-terminal domain forms a long flexible stem that extends the enzyme away from the Golgi membrane.

Crystal structure of the nucleotide-metabolizing enzyme NTPDase4.

The ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes found on the cell surface and in the lumen of certain organelles, that are major regulators of purinergic signaling. Their intracellular roles, however, have not been clearly defined. NTPDase4 (UDPase, ENTPD4) is a Golgi protein potentially involved in nucleotide recycling as part of protein glycosylation, and is also found in lysosomes, where its purpose is unknown. To further our understanding of NTPDase4 function, we determined its crystal structure. The enzyme adopts a wide open, inactive conformation. Differences in the nucleotide-binding site relative to its homologs could account for its substrate selectivity. The putative membrane-interacting loop of cell-surface NTPDases is drastically altered in NTPDase4, potentially affecting its interdomain dynamics at the Golgi membrane.

The structure of mammalian ß-mannosidase provides insight into ß-mannosidosis and nystagmus.

ß-Mannosidase is a lysosomal enzyme from the glycosyl hydrolase family 2 that cleaves the single ß(1-4)-linked mannose at the nonreducing end of N-glycosylated proteins, and plays an important role in the polysaccharide degradation pathway. Mutations in the MANBA gene, which encodes the ß-mannosidase, can lead to the lysosomal storage disease ß-mannosidosis, as well as nystagmus, an eye condition characterized by involuntary eye movements. Here, we present the first structures of a mammalian ß-mannosidase in both the apo- and mannose-bound forms. The structure is similar to previously determined ß-mannosidase structures with regard to domain organization and fold, however, there are important differences that underlie substrate specificity between species. Additionally, in contrast to most other ligand-bound ß-mannosidases from bacterial and fungal sources where bound sugars were in a boat-like conformation, we find the mannose in the chair conformation. Evaluation of known disease mutations in the MANBA gene provides insight into their impact on disease phenotypes. Together, these results will be important for the design of therapeutics for treating diseases caused by ß-mannosidase deficiency. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6DDT and 6DDU.

Identification of Allosteric Inhibitors against Active Caspase-6.

Caspase-6 is a cysteine protease that plays essential roles in programmed cell death, axonal degeneration, and development. The excess neuronal activity of Caspase-6 is associated with Alzheimer disease neuropathology and age-dependent cognitive impairment. Caspase-6 inhibition is a promising strategy to stop early stage neurodegenerative events, yet finding potent and selective Caspase-6 inhibitors has been a challenging task due to the overlapping structural and functional similarities between caspase family members. Here, we investigated how four rare non-synonymous missense single-nucleotide polymorphisms (SNPs), resulting in amino acid substitutions outside human Caspase-6 active site, affect enzyme structure and catalytic efficiency. Three investigated SNPs were found to align with a putative allosteric pocket with low sequence conservation among human caspases. Virtual screening of 57,700 compounds against the putative Caspase-6 allosteric pocket, followed by in vitro testing of the best virtual hits in recombinant human Caspase-6 activity assays identified novel allosteric Caspase-6 inhibitors with IC50 and Ki values ranging from ~2 to 13 µM. This report may pave the way towards the development and optimisation of novel small molecule allosteric Caspase-6 inhibitors and illustrates that functional characterisation of rare natural variants holds promise for the identification of allosteric sites on other therapeutic targets in drug discovery.

Molecular Mechanism of Inhibition of Acid Ceramidase by Carmofur.

Human acid ceramidase (AC) is a lysosomal cysteine amidase, which has received a great deal of interest in recent years as a potential target for the development of new therapeutics against melanoma and glioblastoma tumors. Despite the strong interest in obtaining structural information, only the structures of the apo-AC enzyme in its zymogen and activated conformations are available. In this work, the crystal structure of AC in complex with the covalent carmofur inhibitor is presented. Carmofur is an antineoplastic drug containing an electrophilic carbonyl reactive group that targets the catalytic cysteine. This novel structural data explains the basis of the AC inhibition, provides insights into the enzymatic properties of the protein, and is a great aid toward the structure-based drug design of potent inhibitors for AC, providing the detailed mechanism, which has eluded the scientific community for more than 30 years, of carmofur's mysterious 5-fluorouracil-independent antitumor activity.

Structural basis for nucleotide recognition by the ectoenzyme CD203c.

The ecto-nucleotide pyrophosphatase/phosphodiesterase (NPP) enzyme family modulates purinergic signaling by degrading extracellular nucleotides. CD203c (NPP3, ENPP3) regulates the inflammatory response of basophils via ATP hydrolysis and is a marker for allergen sensitivity on the surface of these cells. Multiple other roles and substrates have also been proposed for this protein. In order to gain insight into its molecular functions, we determined the crystal structure of human NPP3 as well as its complex with an ATP analog. The enzyme exhibits little preference for nucleobase type, and forms specific contacts with the alpha and beta phosphate groups of its ligands. Dimerization of the protein does not affect its catalytic activity. These findings expand our understanding of substrate recognition within the NPP family. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6C01 (human NPP3) and 6C02 (human NPP3 T205A N594S with AMPCPP).

Structural basis for the activation of acid ceramidase.

Acid ceramidase (aCDase, ASAH1) hydrolyzes lysosomal membrane ceramide into sphingosine, the backbone of all sphingolipids, to regulate many cellular processes. Abnormal function of aCDase leads to Farber disease, spinal muscular atrophy with progressive myoclonic epilepsy, and is associated with Alzheimer's, diabetes, and cancer. Here, we present crystal structures of mammalian aCDases in both proenzyme and autocleaved forms. In the proenzyme, the catalytic center is buried and protected from solvent. Autocleavage triggers a conformational change exposing a hydrophobic channel leading to the active site. Substrate modeling suggests distinct catalytic mechanisms for substrate hydrolysis versus autocleavage. A hydrophobic surface surrounding the substrate binding channel appears to be a site of membrane attachment where the enzyme accepts substrates facilitated by the accessory protein, saposin-D. Structural mapping of disease mutations reveals that most would destabilize the protein fold. These results will inform the rational design of aCDase inhibitors and recombinant aCDase for disease therapeutics.

A key tyrosine substitution restricts nucleotide hydrolysis by the ectoenzyme NPP5.

The ecto-nucleotide pyrophosphatase/phosphodiesterase (NPP) family of proteins mediates purinergic signaling by degrading extracellular nucleotides and also participates in phospholipid metabolism. NPP5 (ENPP5) is the least characterized member of this group and its specific role is unknown. This enzyme does not display activity on certain nucleotides and on other typical NPP substrates. In order to gain insights into its function, we determined the crystal structure of human and murine NPP5. Structural comparison with close homologs revealed a key phenylalanine to tyrosine substitution that prevents efficient hydrolysis of nucleotide diphosphates and triphosphates; reversal of this mutation enabled degradation of these molecules. Interestingly, NPP5 is able to cleave nicotinamide adenine dinucleotide (NAD), suggesting a potential role of this enzyme in NAD-based neurotransmission. An NPP5-specific metal binding motif is found adjacent to the active site, although its significance is unclear. These findings expand our understanding of substrate specificity within the NPP family. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 5VEM, 5VEN, and 5VEO.

Crystal structure of mammalian acid sphingomyelinase.

Acid sphingomyelinase (ASMase, ASM, SMPD1) converts sphingomyelin into ceramide, modulating membrane properties and signal transduction. Inactivating mutations in ASMase cause Niemann-Pick disease, and its inhibition is also beneficial in models of depression and cancer. To gain a better understanding of this critical therapeutic target, we determined crystal structures of mammalian ASMase in various conformations. The catalytic domain adopts a calcineurin-like fold with two zinc ions and a hydrophobic track leading to the active site. Strikingly, the membrane interacting saposin domain assumes either a closed globular conformation independent from the catalytic domain, or an open conformation, which establishes an interface with the catalytic domain essential for activity. Structural mapping of Niemann-Pick mutations reveals that most of them likely destabilize the protein's fold. This study sheds light on the molecular mechanism of ASMase function, and provides a platform for the rational development of ASMase inhibitors and therapeutic use of recombinant ASMase.