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.

Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2'-O methylations.

IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2'-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-Å complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations (syn and anti) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2'-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self- versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA.

Structural basis for viral 5'-PPP-RNA recognition by human IFIT proteins.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are innate immune effector molecules that are thought to confer antiviral defence through disruption of protein-protein interactions in the host translation-initiation machinery. However, it was recently discovered that IFITs can directly recognize viral RNA bearing a 5'-triphosphate group (PPP-RNA), which is a molecular signature that distinguishes it from host RNA. Here we report crystal structures of human IFIT5, its complex with PPP-RNAs, and an amino-terminal fragment of IFIT1. The structures reveal a new helical domain that houses a positively charged cavity designed to specifically engage only single-stranded PPP-RNA, thus distinguishing it from the canonical cytosolic sensor of double-stranded viral PPP-RNA, retinoic acid-inducible gene I (RIG-I, also known as DDX58). Mutational analysis, proteolysis and gel-shift assays reveal that PPP-RNA is bound in a non-sequence-specific manner and requires a 5'-overhang of approximately three nucleotides. Abrogation of PPP-RNA binding in IFIT1 and IFIT5 was found to cause a defect in the antiviral response by human embryonic kidney cells. These results demonstrate the mechanism by which IFIT proteins selectively recognize viral RNA, and lend insight into their downstream effector function.

Structural Analysis of the Bacterial Effector AvrA Identifies a Critical Helix Involved in Substrate Recognition.

Bacterial effector proteins are essential for the infection and proliferation of pathogenic bacteria through manipulation of host immune response pathways. AvrA is a Salmonella effector that belongs to the YopJ family of acetyltransferases, which suppresses c-JUN N-terminal kinase (JNK) signaling in mammals through acetylation of mitogen-activated receptor kinase kinases 4 and 7 (MKK4/7). Interestingly, there are two paralogues of AvrA that differ by only a single internal leucine residue, which when absent (AvrAΔL140) abrogates the ability to suppress JNK signaling. Here, we present the first crystal structure of a bacterial effector from an animal pathogen, AvrAΔL140, accompanied by a thorough biophysical characterization of both AvrA variants. The structure in complex with inositol hexaphosphate and coenzyme A reveals two closely associated domains consisting of a catalytic core that resembles the CE clan peptidases and a wedge-shaped regulatory region that mediates cofactor and substrate binding. The loss of the putative function of AvrAΔL140 is due to its inability to interact with MKK4/7, which ultimately arises from an altered conformation of a critical helix adjacent to the active site that harbors L140. These results provide general insights into substrate recognition across the YopJ family of acetyltransferases.

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.

DAP5 associates with eIF2ß and eIF4AI to promote Internal Ribosome Entry Site driven translation.

Initiation is a highly regulated rate-limiting step of mRNA translation. During cap-dependent translation, the cap-binding protein eIF4E recruits the mRNA to the ribosome. Specific elements in the 5'UTR of some mRNAs referred to as Internal Ribosome Entry Sites (IRESes) allow direct association of the mRNA with the ribosome without the requirement for eIF4E. Cap-independent initiation permits translation of a subset of cellular and viral mRNAs under conditions wherein cap-dependent translation is inhibited, such as stress, mitosis and viral infection. DAP5 is an eIF4G homolog that has been proposed to regulate both cap-dependent and cap-independent translation. Herein, we demonstrate that DAP5 associates with eIF2ß and eIF4AI to stimulate IRES-dependent translation of cellular mRNAs. In contrast, DAP5 is dispensable for cap-dependent translation. These findings provide the first mechanistic insights into the function of DAP5 as a selective regulator of cap-independent translation.

Arabidopsis Argonaute MID domains use their nucleotide specificity loop to sort small RNAs.

The 5'-nucleotide of small RNAs associates directly with the MID domain of Argonaute (AGO) proteins. In humans, the identity of the 5'-base is sensed by the MID domain nucleotide specificity loop and regulates the integrity of miRNAs. In Arabidopsis thaliana, the 5'-nucleotide also controls sorting of small RNAs into the appropriate member of the AGO family; however, the structural basis for this mechanism is unknown. Here, we present crystal structures of the MID domain from three Arabidopsis AGOs, AtAGO1, AtAGO2 and AtAGO5, and characterize their interactions with nucleoside monophosphates (NMPs). In AtAGOs, the nucleotide specificity loop also senses the identity of the 5'-nucleotide but uses more diverse modes of recognition owing to the greater complexity of small RNAs found in plants. Binding analyses of these interactions reveal a strong correlation between their affinities and evolutionary conservation.

Structural basis for 5'-nucleotide base-specific recognition of guide RNA by human AGO2.

MicroRNAs (miRNAs) mediate post-transcriptional gene regulation through association with Argonaute proteins (AGOs). Crystal structures of archaeal and bacterial homologues of AGOs have shown that the MID (middle) domain mediates the interaction with the phosphorylated 5' end of the miRNA guide strand and this interaction is thought to be independent of the identity of the 5' nucleotide in these systems. However, analysis of the known sequences of eukaryotic miRNAs and co-immunoprecipitation experiments indicate that there is a clear bias for U or A at the 5' position. Here we report the crystal structure of a MID domain from a eukaryotic AGO protein, human AGO2. The structure, in complex with nucleoside monophosphates (AMP, CMP, GMP, and UMP) mimicking the 5' end of miRNAs, shows that there are specific contacts made between the base of UMP or AMP and a rigid loop in the MID domain. Notably, the structure of the loop discriminates against CMP and GMP and dissociation constants calculated from NMR titration experiments confirm these results, showing that AMP (0.26 mM) and UMP (0.12 mM) bind with up to 30-fold higher affinity than either CMP (3.6 mM) or GMP (3.3 mM). This study provides structural evidence for nucleotide-specific interactions in the MID domain of eukaryotic AGO proteins and explains the observed preference for U or A at the 5' end of miRNAs.

An immunogenic peptide in the A-box of HMGB1 protein reverses apoptosis-induced tolerance through RAGE receptor.

Apoptotic cells trigger immune tolerance in engulfing phagocytes. This poorly understood process is believed to contribute to the severe immunosuppression and increased susceptibility to nosocomial infections observed in critically ill sepsis patients. Extracellular high mobility group box 1 (HMGB1) is an important mediator of both sepsis lethality and the induction of immune tolerance by apoptotic cells. We have found that HMGB1 is sensitive to processing by caspase-1, resulting in the production of a fragment within its N-terminal DNA-binding domain (the A-box) that signals through the receptor for advanced glycation end products (RAGE) to reverse apoptosis-induced tolerance. In a two-hit mouse model of sepsis, we show that tolerance to a secondary infection and its associated mortality were effectively reversed by active immunization with dendritic cells treated with HMGB1 or the A-box fragment, but not a noncleavable form of HMGB1. These findings represent a novel link between caspase-1 and HMGB1, with potential therapeutic implications in infectious and inflammatory diseases.

An autoinhibited structure of α-catenin and its implications for vinculin recruitment to adherens junctions.

α-Catenin is an actin- and vinculin-binding protein that regulates cell-cell adhesion by interacting with cadherin adhesion receptors through ß-catenin, but the mechanisms by which it anchors the cadherin-catenin complex to the actin cytoskeleton at adherens junctions remain unclear. Here we determined crystal structures of αE-catenin in the autoinhibited state and the actin-binding domain of αN-catenin. Together with the small-angle x-ray scattering analysis of full-length αN-catenin, we deduced an elongated multidomain assembly of monomeric α-catenin that structurally and functionally couples the vinculin- and actin-binding mechanisms. Cellular and biochemical studies of αE- and αN-catenins show that αE-catenin recruits vinculin to adherens junctions more effectively than αN-catenin, partly because of its higher affinity for actin filaments. We propose a molecular switch mechanism involving multistate conformational changes of α-catenin. This would be driven by actomyosin-generated tension to dynamically regulate the vinculin-assisted linkage between adherens junctions and the actin cytoskeleton.

Isolation of short RNAs with homogeneous 3'-ends using quaternary-amine anion exchange chromatography.

Visualizing RNA-protein interactions through structural approaches requires the use of RNA molecules purified to homogeneity. We describe here a simple and effective method, free of acrylamide contamination and without using UV radiation, to separate in vitro synthesized, heterogeneous RNA transcripts (up to ∼15 nucleotides) at single-nucleotide resolution by quaternary-amine anion exchange chromatography. The quality of short RNAs isolated through this method is validated by gel electrophoresis, mass spectrometry, and crystallization with a protein-binding partner.

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.

Structural analysis of 5'-mRNA-cap interactions with the human AGO2 MID domain.

In RNA silencing, microRNA (miRNA)-mediated translational repression occurs through mechanisms that do not invoke messenger-RNA (mRNA) target cleavage by Argonaute proteins. The nature of these mechanisms is unclear, but several recent studies have proposed that a direct interaction between the mRNA-cap and the middle (MID) domain of Argonautes is involved. Here, we present crystallographic and NMR data demonstrating that cap analogues do not bind significantly to the isolated MID domain of human Argonaute 2 (hAGO2) and are found in the miRNA 5'-nucleotide binding site in an implausible binding mode. Additionally, in vitro pull-down experiments with full-length hAGO2 indicate that the interaction with cap analogues is nonspecific.

Targeting DEAD-box RNA helicases: The emergence of molecular staples.

RNA helicases constitute a large family of proteins that play critical roles in mediating RNA function. They have been implicated in all facets of gene expression pathways involving RNA, from transcription to processing, transport and translation, and storage and decay. There is significant interest in developing small molecule inhibitors to RNA helicases as some family members have been documented to be dysregulated in neurological and neurodevelopment disorders, as well as in cancers. Although different functional properties of RNA helicases offer multiple opportunities for small molecule development, molecular staples have recently come to the forefront. These bifunctional molecules interact with both protein and RNA components to lock them together, thereby imparting novel gain-of-function properties to their targets. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.