Fluorescence polarisation activity-based protein profiling for the identification of deoxynojirimycin-type inhibitors selective for lysosomal retaining alpha- and beta-glucosidases.

Lysosomal exoglycosidases are responsible for processing endocytosed glycans from the non-reducing end to produce the corresponding monosaccharides. Genetic mutations in a particular lysosomal glycosidase may result in accumulation of its particular substrate, which may cause diverse lysosomal storage disorders. The identification of effective therapeutic modalities to treat these diseases is a major yet poorly realised objective in biomedicine. One common strategy comprises the identification of effective and selective competitive inhibitors that may serve to stabilize the proper folding of the mutated enzyme, either during maturation and trafficking to, or residence in, endo-lysosomal compartments. The discovery of such inhibitors is greatly aided by effective screening assays, the development of which is the focus of the here-presented work. We developed and applied fluorescent activity-based probes reporting on either human GH30 lysosomal glucosylceramidase (GBA1, a retaining ß-glucosidase) or GH31 lysosomal retaining α-glucosidase (GAA). FluoPol-ABPP screening of our in-house 358-member iminosugar library yielded compound classes selective for either of these enzymes. In particular, we identified a class of N-alkyldeoxynojirimycins that inhibit GAA, but not GBA1, and that may form the starting point for the development of pharmacological chaperone therapeutics for the lysosomal glycogen storage disease that results from genetic deficiency in GAA: Pompe disease.

Direct capture, inhibition and crystal structure of HsaD (Rv3569c) from M. tuberculosis.

A hallmark of Mycobacterium tuberculosis (M. tb), the aetiologic agent of tuberculosis, is its ability to metabolise host-derived lipids. However, the enzymes and mechanisms underlying such metabolism are still largely unknown. We previously reported that the Cyclophostin & Cyclipostins (CyC) analogues, a new family of potent antimycobacterial molecules, react specifically and covalently with (Ser/Cys)-based enzymes mostly involved in bacterial lipid metabolism. Here, we report the synthesis of new CyC alkyne-containing inhibitors (CyCyne ) and their use for the direct fishing of target proteins in M. tb culture via bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP). This approach led to the capture and identification of a variety of enzymes, and many of them involved in lipid or steroid metabolisms. One of the captured enzymes, HsaD (Rv3569c), is required for the survival of M. tb within macrophages and is thus a potential therapeutic target. This prompted us to further explore and validate, through a combination of biochemical and structural approaches, the specificity of HsaD inhibition by the CyC analogues. We confirmed that the CyC bind covalently to the catalytic Ser114 residue, leading to a total loss of enzyme activity. These data were supported by the X-ray structures of four HsaD-CyC complexes, obtained at resolutions between 1.6 and 2.6 Å. The identification of mycobacterial enzymes directly captured by the CyCyne probes through CC-ABPP paves the way to better understand and potentially target key players at crucial stages of the bacilli life cycle.

1,6-epi-Cyclophellitol Cyclosulfamidate Is a Bona Fide Lysosomal α-Glucosidase Stabilizer for the Treatment of Pompe Disease.

α-Glucosidase inhibitors are potential therapeutics for the treatment of diabetes, viral infections, and Pompe disease. Herein, we report a 1,6-epi-cyclophellitol cyclosulfamidate as a new class of reversible α-glucosidase inhibitors that displays enzyme inhibitory activity by virtue of its conformational mimicry of the substrate when bound in the Michaelis complex. The α-d-glc-configured cyclophellitol cyclosulfamidate 4 binds in a competitive manner the human lysosomal acid α-glucosidase (GAA), ER α-glucosidases, and, at higher concentrations, intestinal α-glucosidases, displaying an excellent selectivity over the human ß-glucosidases GBA and GBA2 and glucosylceramide synthase (GCS). Cyclosulfamidate 4 stabilizes recombinant human GAA (rhGAA, alglucosidase alfa, Myozyme) in cell medium and plasma and facilitates enzyme trafficking to lysosomes. It stabilizes rhGAA more effectively than existing small-molecule chaperones and does so in vitro, in cellulo, and in vivo in zebrafish, thus representing a promising therapeutic alternative to Miglustat for Pompe disease.

Molecular basis for substrate recognition and septum cleavage by AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis.

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.45 Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved ß-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA-substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.

Carnitine is a pharmacological allosteric chaperone of the human lysosomal α-glucosidase.

Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient's enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.

First insights into the structural features of Ebola virus methyltransferase activities.

The Ebola virus is a deadly human pathogen responsible for several outbreaks in Africa. Its genome encodes the 'large' L protein, an essential enzyme that has polymerase, capping and methyltransferase activities. The methyltransferase activity leads to RNA co-transcriptional modifications at the N7 position of the cap structure and at the 2'-O position of the first transcribed nucleotide. Unlike other Mononegavirales viruses, the Ebola virus methyltransferase also catalyses 2'-O-methylation of adenosines located within the RNA sequences. Herein, we report the crystal structure at 1.8 Å resolution of the Ebola virus methyltransferase domain bound to a fragment of a camelid single-chain antibody. We identified structural determinants and key amino acids specifically involved in the internal adenosine-2'-O-methylation from cap-related methylations. These results provide the first high resolution structure of an ebolavirus L protein domain, and the framework to investigate the effects of epitranscriptomic modifications and to design possible antiviral drugs against the Filoviridae family.

Substrate binding mode and catalytic mechanism of human heparan sulfate d-glucuronyl C5 epimerase.

Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component, d-glucuronic acid (GlcA), into l-iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS)n substrate or a (IdoA-GlcNS)n product. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement of N-sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs.

Genetic and enzymatic characterization of 3-O-sulfotransferase SNPs associated with Plasmodium falciparum parasitaemia.

The HS3ST3A1/B1 genes encode two homologous 3-O-sulfotransferases involved in the late modification step during heparan sulfate (HS) biosynthesis. In addition to the single nucleotide polymorphisms (SNPs) rs28470223 (C > T) in the promoter region of both HS3ST3A1 and rs62636623 (Gly/Arg) in the stem region of HS3ST3B1, three missense mutations (rs62056073, rs61729712 and rs9906590) located within the catalytic sulfotransferase domain of 3-OST-B1 are linked and associated to Plasmodium falciparum parasitaemia. To ascertain the functional effects of these SNP associations, we investigated the regulatory effect of rs28470223 and characterized the enzymatic activity of the missense SNP rs61729712 (Ser279Asn) localized at proximity of the substrate binding cleft. The SNP rs28470223 results in decreased promoter activity of HS3ST3A1 in K562 cells, suggesting a reduced in vivo transcription activity of the target gene. A comparative kinetic analysis of wt HS3ST3B1 and the Ser269Asn variant (rs61729712) using a HS-derived oligosaccharide substrate reveals a slightly higher catalytic activity for the SNP variant. These genetic and enzymatic studies suggest that genetic variations in enzymes responsible of HS 3-O-sulfation can modulate their promoter and enzymatic activities and may influence P. falciparum parasitaemia.

Glycosylate and move! The glycosyltransferase Maf is involved in bacterial flagella formation.

The flagella of various Gram-negative bacteria are decorated with diverse glycan structures, amongst them nonulosonic acids related to the sialic acid family. Although nonulosonic sugar biosynthesis pathways have been dissected in various pathogens, the enzymes transferring the sugars onto flagellin are still poorly characterized. The deletion of genes coding for motility associated factors (Mafs) found in many pathogenic strains systematically gives rise to nonflagellated bacteria lacking specific nonulosonic sugars on the flagellins, therefore, relating Maf function to flagellin glycosylation and bacterial motility. We investigated the role of Maf from our model organism, Magnetospirillum magneticum AMB-1, in the glycosylation and formation of the flagellum. Deletion of the gene amb0685 coding for Maf produced a nonflagellated bacterium where the flagellin was still produced but no longer glycosylated. Our X-ray structure analysis revealed that the central domain of Maf exhibits similarity to sialyltransferases from Campylobacter jejuni. Glycan analysis suggested that the nonulosonic carbohydrate structure transferred is pseudaminic acid or a very close derivative. This work describes the importance of glycosylation in the formation of the bacterial flagellum and provides the first structural model for a member of a new bacterial glycosyltransferase family involved in nonulosonic acids transfer onto flagellins.

Structure of human lysosomal acid α-glucosidase-a guide for the treatment of Pompe disease.

Pompe disease, a rare lysosomal storage disease caused by deficiency of the lysosomal acid α-glucosidase (GAA), is characterized by glycogen accumulation, triggering severe secondary cellular damage and resulting in progressive motor handicap and premature death. Numerous disease-causing mutations in the gaa gene have been reported, but the structural effects of the pathological variants were unknown. Here we present the high-resolution crystal structures of recombinant human GAA (rhGAA), the standard care of Pompe disease. These structures portray the unbound form of rhGAA and complexes thereof with active site-directed inhibitors, providing insight into substrate recognition and the molecular framework for the rationalization of the deleterious effects of disease-causing mutations. Furthermore, we report the structure of rhGAA in complex with the allosteric pharmacological chaperone N-acetylcysteine, which reveals the stabilizing function of this chaperone at the structural level.

Structural basis for carbohydrate binding properties of a plant chitinase-like agglutinin with conserved catalytic machinery.

A new chitinase-like agglutinin, RobpsCRA, related to family GH18 chitinases, has previously been identified in black locust (Robinia pseudoacacia) bark. The crystal structure of RobpsCRA at 1.85Å resolution reveals unusual molecular determinants responsible for the lack of its ancestral chitinase activity. Unlike other chitinase-like proteins, which lack chitinase catalytic residues, RobpsCRA has conserved its catalytic machinery. However, concerted rearrangements of loop regions coupled to non-conservative substitutions of aromatic residues central to the chitin-binding groove explain the lack of hydrolytic activity against chitin and the switch toward recognition of high-mannose type N-glycans. Identification of close homologs in flowering plants with conservation of sequence motifs associated to the structural adaptations seen in RobpsCRA defines an emerging class of agglutinins, as emphasized by a phylogenetic analysis, that are likely to share a similar carbohydrate binding specificity for high-mannose type N-glycans. This study illustrates the recent evolution and molecular adaptation of a versatile TIM-barrel scaffold within the ancestral GH18 family.

Crystal structure of the GalNAc/Gal-specific agglutinin from the phytopathogenic ascomycete Sclerotinia sclerotiorum reveals novel adaptation of a beta-trefoil domain.

A lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum that shares only weak sequence similarity with characterized fungal lectins has recently been identified. S. sclerotiorum agglutinin (SSA) is a homodimeric protein consisting of two identical subunits of approximately 17 kDa and displays specificity primarily towards Gal/GalNAc. Glycan array screening indicates that SSA readily interacts with Gal/GalNAc-bearing glycan chains. The crystal structures of SSA in the ligand-free form and in complex with the Gal-beta1,3-GalNAc (T-antigen) disaccharide have been determined at 1.6 and 1.97 A resolution, respectively. SSA adopts a beta-trefoil domain as previously identified for other carbohydrate-binding proteins of the ricin B-like lectin superfamily and accommodates terminal non-reducing galactosyl and N-acetylgalactosaminyl glycans. Unlike other structurally related lectins, SSA contains a single carbohydrate-binding site at site alpha. SSA reveals a novel dimeric assembly markedly dissimilar to those described earlier for ricin-type lectins. The present structure exemplifies the adaptability of the beta-trefoil domain in the evolution of fungal lectins.

LppX is a lipoprotein required for the translocation of phthiocerol dimycocerosates to the surface of Mycobacterium tuberculosis.

Cell envelope lipids play an important role in the pathogenicity of mycobacteria, but the mechanisms by which they are transported to the outer membrane of these prokaryotes are largely unknown. Here, we provide evidence that LppX is a lipoprotein required for the translocation of complex lipids, the phthiocerol dimycocerosates (DIM), to the outer membrane of Mycobacterium tuberculosis. Abolition of DIM transport following disruption of the lppX gene is accompanied by an important attenuation of the virulence of the tubercle bacillus. The crystal structure of LppX unveils an U-shaped beta-half-barrel dominated by a large hydrophobic cavity suitable to accommodate a single DIM molecule. LppX shares a similar fold with the periplasmic molecular chaperone LolA and the outer membrane lipoprotein LolB, which are involved in the localization of lipoproteins to the outer membrane of Gram-negative bacteria. Based on the structure and although an indirect participation of LppX in DIM transport cannot yet be ruled out, we propose LppX to be the first characterized member of a family of structurally related lipoproteins that carry lipophilic molecules across the mycobacterial cell envelope.

Crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis.

With the aim of elucidating the biological function of hypothetical proteins unique amongst the Actynomyces sub-group of bacteria, we have solved the crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis at 1.8 A resolution. Rv1155 is a homodimer both in the crystal structure and in solution and folds into two separate domains consisting of a six-stranded anti-parallel beta-barrel fold flanked by two alpha-helices and a helix-turn-helix domain. Both domains contribute to the formation of two deep clefts at the dimer interface. The overall fold of Rv1155 strikingly resembles that of flavin mononucleotide-binding protein and pyridoxamine 5'-phosphate oxydase, but the architecture of the putative binding pocket is markedly different, consistent with the lack of color of Rv1155 and its inability to bind FMN. Rv1155 thus appears to belong to a group of proteins with stringent conservation of the binding cleft, having evolved towards a new binding function.

Structure of Escherichia coli YhdH, a putative quinone oxidoreductase.

As part of a structural genomics project on bacterial gene products of unknown function, the crystal structures of YhdH, a putative quinone oxidoreductase, and its complex with NADP have been determined at 2.25 and 2.6 A resolution, respectively. The overall fold of YhdH is very similar to that of alcohol dehydrogenases and quinone reductases despite its low sequence identity. The absence of any Zn ion indicates that YdhH is a putative quinone oxidoreductase. YhdH forms a homodimer, with each subunit composed of two domains: a catalytic domain and a coenzyme-binding domain. NADP is bound in a deep cleft formed between the two domains. Large conformational changes occur upon NADP binding, with the two domains closing up to each other and narrowing the NADP-binding cleft. Comparisons of the YdhH active site with those of the quinone oxidoreductases from Escherichia coli and Thermus thermophilus made it possible to identify essential conserved residues as being Asn41, Asp43, Asp64 and Arg318. The active-site size is very narrow and unless an induced fit occurs is accessible only to reagents the size of benzoquinone.

Crystal structure and kinetics identify Escherichia coli YdcW gene product as a medium-chain aldehyde dehydrogenase.

In the context of a medium-scaled structural genomics program aiming at solving the structures of as many as possible bacterial unknown open reading frame products from Escherichia coli (Y prefix), we have solved the structure of YdcW at 2.1A resolution, using molecular replacement. According to its sequence identity, YdcW has been classified into the betaine aldehyde dehydrogenases family (EC 1.2.1.8), catalysing the oxidation of betaine aldehyde into glycine betaine. The structure of YdcW resembles that of other aldehyde dehydrogenases: it is tetrameric and binds a NADH molecule in each monomer. The NADH molecules, bound in the active site by soaking, are revealed to be in the "hydrolysis position". Activities experiments demonstrate that YdcW is more active on medium-chains aldehyde than on betaine aldehyde. However, soaking of betaine into YdcW crystals revealed its presence in one of the subunits, in two positions, a putative resting position and a hydride transfer ready position. Analysis of kinetics data and of the active site shape suggest an optimum binding of n-alkyl aldehydes up to seven to eight carbon atoms, possibly followed by a bulky cyclic or aromatic group.

Crystal structure and reactivity of YbdL from Escherichia coli identify a methionine aminotransferase function.

The ybdL gene of Escherichia coli codes for a protein of unknown function. Sequence analysis showed moderate homology to several vitamin B(6) dependent enzymes, suggesting that it may bind pyridoxal-5'-phosphate. The structure analysis of YbdL to 2.35 A resolution by protein crystallography verifies that it is a PLP dependent enzyme of fold type I, the typical aspartate aminotransferase fold. The active site contains a bound pyridoxal-5'-phosphate, covalently attached to the conserved active site lysine residue Lys236. The pattern of conserved amino acids in the putative substrate binding pocket of the enzyme reveals that it is most closely related to a hyperthermophilic aromatic residue aminotransferase from the archeon Pyrococcus horikoshii. Activity tests with 10 amino acids as amino-donors reveal, however, a preference for Met, followed by His and Phe, results which can be rationalized by modelization studies.

The crystal structure of the Calystegia sepium agglutinin reveals a novel quaternary arrangement of lectin subunits with a beta-prism fold.

The high number of quaternary structures observed for lectins highlights the important role of these oligomeric assemblies during carbohydrate recognition events. Although a large diversity in the mode of association of lectin subunits is frequently observed, the oligomeric assemblies of plant lectins display small variations within a single family. The crystal structure of the mannose-binding jacalin-related lectin from Calystegia sepium (Calsepa) has been determined at 1.37-A resolution. Calsepa exhibits the same beta-prism fold as identified previously for other members of the family, but the shape and the hydrophobic character of its carbohydrate-binding site is unlike that of other members, consistent with surface plasmon resonance analysis showing a preference for methylated sugars. Calsepa reveals a novel dimeric assembly markedly dissimilar to those described earlier for Heltuba and jacalin but mimics the canonical 12-stranded beta-sandwich dimer found in legume lectins. The present structure exemplifies the adaptability of the beta-prism building block in the evolution of plant lectins and highlights the biological role of these quaternary structures for carbohydrate recognition.

The crystal structure of the Escherichia coli YfdW gene product reveals a new fold of two interlaced rings identifying a wide family of CoA transferases.

Because of its toxicity, oxalate accumulation from amino acid catabolism leads to acute disorders in mammals. Gut microflora are therefore pivotal in maintaining a safe intestinal oxalate balance through oxalate degradation. Oxalate catabolism was first identified in Oxalobacter formigenes, a specialized, strictly anaerobic bacterium. Oxalate degradation was found to be performed successively by two enzymes, a formyl-CoA transferase (frc) and an oxalate decarboxylase (oxc). These two genes are present in several bacterial genomes including that of Escherichia coli. The frc ortholog in E. coli is yfdW, with which it shares 61% sequence identity. We have expressed the YfdW open reading frame product and solved its crystal structure in the apo-form and in complex with acetyl-CoA and with a mixture of acetyl-CoA and oxalate. YfdW exhibits a novel and spectacular fold in which two monomers assemble as interlaced rings, defining the CoA binding site at their interface. From the structure of the complex with acetyl-CoA and oxalate, we propose a putative formyl/oxalate transfer mechanism involving the conserved catalytic residue Asp169. The similarity of yfdW with bacterial orthologs (approximately 60% identity) and paralogs (approximately 20-30% identity) suggests that this new fold and parts of the CoA transfer mechanism are likely to be the hallmarks of a wide family of CoA transferases.