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Biomedical subjects

Daan M F van Aalten

Publications and source records attributed to Daan M F van Aalten.

At least 19 recordsLinked to original sources
PubMed2025

Gfa1 (glutamine fructose-6-phosphate aminotransferase) is essential for Aspergillus fumigatus growth and virulence.

BACKGROUND: Aspergillus fumigatus, the primary etiological agent of invasive aspergillosis, causes over 1.8 million deaths annually. Targeting cell wall biosynthetic pathways offers a promising antifungal strategy. Gfa1, a rate-limiting enzyme in UDP-GlcNAc synthesis, plays a pivotal role in the hexosamine biosynthetic pathway (HBP). RESULTS: Deletion of gfa1 (Δgfa1) results in auxotrophy for glucosamine (GlcN) or N-acetylglucosamine (GlcNAc). Under full recovery (FR) conditions, where minimal medium is supplemented with 5 mM GlcN as the sole carbon source, the Δgfa1 mutant shows growth comparable to the wild-type (WT). However, when supplemented with 5 mM GlcN and 55 mM glucose, growth is partially repressed, likely due to carbon catabolite repression, a condition termed partial repression (PR). Under PR conditions, Δgfa1 exhibits compromised growth, reduced conidiation, defective germination, impaired cell wall integrity, and increased sensitivity to endoplasmic reticulum (ER) stress and high temperatures. Additionally, Δgfa1 demonstrates disruptions in protein homeostasis and iron metabolism. Transcriptomic analysis of the mutant under PR conditions reveals significant alterations in carbohydrate and amino acid metabolism, unfolded protein response (UPR) processes, and iron assimilation. Importantly, Gfa1 is essential for A. fumigatus virulence, as demonstrated in Caenorhabditis elegans and Galleria mellonella infection models. CONCLUSIONS: These findings underscore the critical role of Gfa1 in fungal pathogenicity and suggest its potential as a therapeutic target for combating A. fumigatus infections.

Aspergillus fumigatus↗
PubMed2006

GlcNAcstatin: a picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-glcNAcylation levels.

Many phosphorylation signal transduction pathways in the eukaryotic cell are modulated by posttranslational modification of specific serines/threonines with N-acetylglucosamine (O-GlcNAc). Levels of O-GlcNAc on key proteins regulate biological processes as diverse as the cell cycle, insulin signaling, and protein degradation. The two enzymes involved in this dynamic and abundant modification are the O-GlcNAc transferase and O-GlcNAcase. Structural data have recently revealed that the O-GlcNAcase possesses an active site with significant structural similarity to that of the human lysosomal hexosaminidases HexA/HexB. PUGNAc, an O-GlcNAcase inhibitor widely used to raise levels of O-GlcNAc in human cell lines, also inhibits these hexosaminidases. Here, we have exploited recent structural information of an O-GlcNAcase-PUGNAc complex to design and synthesize a glucoimidazole-based inhibitor, GlcNAcstatin, which is a 5 pM competitive inhibitor of enzymes of the O-GlcNAcase family, shows 100000-fold selectivity over HexA/B, and binds to the O-GlcNAcase active site by mimicking the transition state as revealed by X-ray crystallography. This compound is able to raise O-GlcNAc levels in human HEK 293 and SH-SY5Y neuroblastoma cell lines and thus provides a novel, potent tool for the study of the role of O-GlcNAc in intracellular signal transduction pathways.

Acylation↗
PubMed2006

Structure and mechanism of chitin deacetylase from the fungal pathogen Colletotrichum lindemuthianum.

The fungal pathogen Colletotrichum lindemuthianum secretes an endo-chitin de-N-acetylase (ClCDA) to modify exposed hyphal chitin during penetration and infection of plants. Although a significant amount of biochemical data is available on fungal chitin de-N-acetylases, no structural data exist. Here we describe the 1.8 A crystal structure of a ClCDA product complex and the analysis of the reaction mechanism using Hammett linear free energy relationships, subsite probing, and atomic absorption spectroscopy studies. The structural data in combination with biochemical data reveal that ClCDA consists of a single domain encompassing a mononuclear metalloenzyme which employs a conserved His-His-Asp zinc-binding triad closely associated with the conserved catalytic base (aspartic acid) and acid (histidine) to carry out acid/base catalysis. The data presented here indicate that ClCDA possesses a highly conserved substrate-binding groove, with subtle alterations that influence substrate specificity and subsite affinity. Strikingly, the structure also shows that the hexahistidine purification tag appears to form a tight interaction with the active site groove. The enzyme requires occupancy of at least the 0 and +1 subsites by (GlcNAc)(2) for activity and proceeds through a tetrahedral oxyanion intermediate.

Acetylglucosamine↗
PubMed2006

Siglec-7 undergoes a major conformational change when complexed with the alpha(2,8)-disialylganglioside GT1b.

The siglecs are a group of mammalian sialic acid binding receptors expressed predominantly in the immune system. The CD33-related siglecs show complex recognition patterns for sialylated glycans. Siglec-7 shows a preference for alpha(2,8)-disialylated ligands and provides a structural template for studying the key interactions that drive this selectivity. We have co-crystallized Siglec-7 with a synthetic oligosaccharide corresponding to the alpha(2,8)-disialylated ganglioside GT1b. The crystal structure of the complex offers a first glimpse into how this important family of lectins binds the structurally diverse gangliosides. The structure reveals that the C-C' loop, a region implicated in previous studies as driving siglec specificity, undergoes a dramatic conformational shift, allowing it to interact with the underlying neutral glycan core of the ganglioside. The structural data in combination with mutagenesis studies show that binding of the ganglioside is driven by extensive hydrophobic contacts together with key polar interactions and that the binding site structure is complementary to preferred solution conformations of GT1b.

Amino Acid Sequence↗
PubMed2006

The structure of siglec-7 in complex with sialosides: leads for rational structure-based inhibitor design.

Siglecs (sialic acid binding Ig-like lectins) are transmembrane receptors for sialylated glycoconjugates that modulate cellular interactions and signalling events in the haematopoietic, immune and nervous systems. Siglec-7 is a structural prototype for the recently described family of immune inhibitory CD33-related siglecs and is predominantly expressed on natural killer cells and monocytes, as well as subsets of CD8 T-cells. Siglec-specific inhibitors are desired for the detection of masked and unmasked forms of siglecs, to aid in dissection of signalling pathways and as tools to investigate siglecs as potential therapeutic targets. As a first step towards this end, we present the crystal structure of siglec-7 in complex with a sialylated ligand, the ganglioside analogue DSLc4 [alpha(2,3)/alpha(2,6) disialyl lactotetraosyl 2-(trimethylsilyl)ethyl], which allows for a detailed description of the binding site, required for structure-guided inhibitor design. Mutagenesis and binding assays were used to demonstrate a key structural role for Lys131, a residue that changes conformation upon sialic acid binding. Differences between the binding sites of siglec family members were then exploited using alpha-methyl Neu5Ac (N-acetylneuraminic acid) as a basic scaffold. A co-crystal of siglec-7 in complex with the sialoside inhibitor, oxamido-Neu5Ac [methyl alpha-9-(amino-oxalyl-amino)-9-deoxy-Neu5Ac] and inhibition data for the sialosides gives clear leads for future inhibitor design.

Animals↗
PubMed2006

Screening-based discovery and structural dissection of a novel family 18 chitinase inhibitor.

Family 18 chitinases play key roles in the life cycles of a variety of organisms ranging from bacteria to man. Very recently it has been shown that one of the mammalian chitinases is highly overexpressed in the asthmatic lung and contributes to the pathogenic process through recruitment of inflammatory cells. Although several potent natural product chitinase inhibitors have been identified, their chemotherapeutic potential or their use as cell biological tools is limited due to their size, complex chemistry, and limited availability. We describe a virtual screening-based approach to identification of a novel, purine-based, chitinase inhibitor. This inhibitor acts in the low micromolar (Ki=2.8+/-0.2 microM) range in a competitive mode. Dissection of the binding mode by x-ray crystallography reveals that the compound, which consists of two linked caffeine moieties, binds in the active site through extensive and not previously observed stacking interactions with conserved, solvent exposed tryptophans. Such exposed aromatics are also present in the structures of many other carbohydrate processing enzymes. The compound exhibits favorable chemical properties and is likely to be useful as a general scaffold for development of pan-family 18 chitinase inhibitors.

Algorithms↗
PubMed2006

Structural basis of reduction-dependent activation of human cystatin F.

Cystatins are important natural cysteine protease inhibitors targeting primarily papain-like cysteine proteases, including cathepsins and parasitic proteases like cruzipain, but also mammalian asparaginyl endopeptidase. Mammalian cystatin F, which is expressed almost exclusively in hematopoietic cells and accumulates in lysosome-like organelles, has been implicated in the regulation of antigen presentation and other immune processes. It is an unusual cystatin superfamily member with a redox-regulated activation mechanism and a restricted specificity profile. We describe the 2.1A crystal structure of human cystatin F in its dimeric "off" state. The two monomers interact in a fashion not seen before for cystatins or cystatin-like proteins that is crucially dependent on an unusual intermolecular disulfide bridge, suggesting how reduction leads to monomer formation and activation. Strikingly, core sugars for one of the two N-linked glycosylation sites of cystatin F are well ordered, and their conformation and interactions with the protein indicate that this unique feature of cystatin F may modulate its inhibitory properties, in particular its reduced affinity toward asparaginyl endopeptidase compared with other cystatins.

Amino Acid Sequence↗
PubMed2006

Structural insights into the mechanism and inhibition of eukaryotic O-GlcNAc hydrolysis.

O-linked N-acetylglucosamine (O-GlcNAc) modification of specific serines/threonines on intracellular proteins in higher eukaryotes has been shown to directly regulate important processes such as the cell cycle, insulin sensitivity and transcription. The structure, molecular mechanisms of catalysis, protein substrate recognition/specificity of the eukaryotic O-GlcNAc transferase and hydrolase are largely unknown. Here we describe the crystal structure, enzymology and in vitro activity on human substrates of Clostridium perfringens NagJ, a close homologue of human O-GlcNAcase (OGA), representing the first family 84 glycoside hydrolase structure. The structure reveals a deep active site pocket highly conserved with the human enzyme, compatible with binding of O-GlcNAcylated peptides. Together with mutagenesis data, the structure supports a variant of the substrate-assisted catalytic mechanism, involving two aspartic acids and an unusually positioned tyrosine. Insights into recognition of substrate come from a complex with the transition state mimic O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (Ki=5.4 nM). Strikingly, the enzyme is inhibited by the pseudosubstrate peptide Ala-Cys(-S-GlcNAc)-Ala, and has OGA activity against O-GlcNAcylated human proteins, suggesting that the enzyme is a suitable model for further studies into the function of human OGA.

3T3 Cells↗
PubMed2006

Kinetic, inhibition and structural studies on 3-oxoacyl-ACP reductase from Plasmodium falciparum, a key enzyme in fatty acid biosynthesis.

Type II fatty acid biosynthesis represents an attractive target for the discovery of new antimalarial drugs. Previous studies have identified malarial ENR (enoyl acyl-carrier-protein reductase, or FabI) as the target for the antiseptic triclosan. In the present paper, we report the biochemical properties and 1.5 A (1 A=0.1 nm) crystal structure of OAR (3-oxoacyl acyl-carrier-protein reductase, or FabG), the second reductive step in fatty acid biosynthesis and its inhibition by hexachlorophene. Under optimal conditions of pH and ionic strength, Plasmodium falciparum OAR displays kinetic properties similar to those of OAR from bacteria or plants. Activity with NADH is <3% of that with NADPH. Fluorescence enhancement studies indicate that NADPH can bind to the free enzyme, consistent with kinetic and product inhibition studies suggesting a steady-state ordered mechanism. The crystal structure reveals a tetramer with a sulphate ion bound in the cofactor-binding site such that the side chains of the catalytic triad of serine, tyrosine and lysine are aligned in an active conformation, as previously observed in the Escherichia coli OAR-NADP+ complex. A cluster of positively charged residues is positioned at the entrance to the active site, consistent with the proposed recognition site for the physiological substrate (3-oxoacyl-acyl-carrier protein) in E. coli OAR. The antibacterial and anthelminthic agent hexachlorophene is a potent inhibitor of OAR (IC50 2.05 microM) displaying non-linear competitive inhibition with respect to NADPH. Hexachlorophene (EC50 6.2 microM) and analogues such as bithionol also have antimalarial activity in vitro, suggesting they might be useful leads for further development.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗
PubMed2005

An efficient synthesis of argifin: a natural product chitinase inhibitor with chemotherapeutic potential.

The first synthesis of the cyclopentapeptide family 18 chitinase inhibitor argifin has been achieved by a combination of solid phase and solution chemistry. Synthetic argifin is a nanomolar inhibitor of chitinase B1 from Aspergillus fumigatus and the high-resolution X-ray structure of the synthesized material in complex with the same enzyme is identical to that previously obtained for the natural product.

Antineoplastic Agents↗
PubMed2005

Structure and metal-dependent mechanism of peptidoglycan deacetylase, a streptococcal virulence factor.

Streptococcus pneumoniae peptidoglycan GlcNAc deacetylase (SpPgdA) protects the Gram-positive bacterial cell wall from host lysozymes by deacetylating peptidoglycan GlcNAc residues. Deletion of the pgda gene has been shown to result in hypersensitivity to lysozyme and reduction of infectivity in a mouse model. SpPgdA is a member of the family 4 carbohydrate esterases, for which little structural information exists, and no catalytic mechanism has yet been defined. Here we describe the native crystal structure and product complexes of SpPgdA biochemical characterization and mutagenesis. The structural data show that SpPgdA is an elongated three-domain protein in the crystal. The structure, in combination with mutagenesis, shows that SpPgdA is a metalloenzyme using a His-His-Asp zinc-binding triad with a nearby aspartic acid and histidine acting as the catalytic base and acid, respectively, somewhat similar to other zinc deacetylases such as LpxC. The enzyme is able to accept GlcNAc(3) as a substrate (K(m) = 3.8 mM, k(cat) = 0.55 s(-1)), with the N-acetyl of the middle sugar being removed by the enzyme. The data described here show that SpPgdA and the other family 4 carbohydrate esterases are metalloenzymes and present a step toward identification of mechanism-based inhibitors for this important class of enzymes.

Amidohydrolases↗
PubMed2005

The non-catalytic chitin-binding protein CBP21 from Serratia marcescens is essential for chitin degradation.

The Gram-negative soil bacterium Serratia marcescens uses three different family 18 chitinases to degrade chitin, an abundant insoluble carbohydrate polymer composed of beta(1,4)-linked units of N-acetylglucosamine. We show that efficient chitin degradation additionally depends on the action of a small non-catalytic protein, CBP21, which binds to the insoluble crystalline substrate, leading to structural changes in the substrate and increased substrate accessibility. CBP21 strongly promoted hydrolysis of crystalline beta-chitin by chitinases A and C, while it was essential for full degradation by chitinase B. CBP21 variants with single mutations on the largely polar binding surface lost their ability to promote chitin degradation, while retaining considerable affinity for the polymer. Thus, binding alone is not sufficient for CBP21 functionality, which seems to depend on specific, mostly polar interactions between the protein and crystalline chitin. This is the first time a secreted binding protein is shown to assist in the enzymatic degradation of an insoluble carbohydrate via non-hydrolytic disruption of the substrate. Interestingly, homologues of CBP21 occur in most chitin-degrading microorganisms, suggesting a general mechanism by which chitin-binding proteins enhance chitinolytic activity. Homologues also occur in chitinase-containing insect viruses, whose infectiousness is known to depend on chitinase efficiency.

Amino Acid Sequence↗
PubMed2005

The N-acetyl-D-glucosaminylphosphatidylinositol De-N-acetylase of glycosylphosphatidylinositol biosynthesis is a zinc metalloenzyme.

The de-N-acetylation of N-acetyl-D-glucosaminylphosphatidylinositol (GlcNAc-PI) is the second step of mammalian and trypanosomal glycosylphosphatidylinositol biosynthesis. Glycosylphosphatidylinositol biosynthesis is essential for Trypanosoma brucei, the causative agent of African sleeping sickness, and GlcNAc-PI de-N-acetylase has previously been validated as a drug target. Inhibition of the trypanosome cell-free system and recombinant rat GlcNAc-PI de-N-acetylase by divalent metal cation chelators demonstrates that a tightly bound divalent metal cation is essential for activity. Reconstitution of metal-free GlcNAc-PI de-N-acetylase with divalent metal cations restores activity in the order Zn(2+) > Cu(2+) > Ni(2+) > Co(2+) > Mg(2+). Site-directed mutagenesis and homology modeling were used to identify active site residues and postulate a mechanism of action. The characterization of GlcNAc-PI de-N-acetylase as a zinc metalloenzyme will facilitate the rational design of anti-protozoan parasite drugs.

Acetylglucosamine↗
PubMed2005

Role of T-loop phosphorylation in PDK1 activation, stability, and substrate binding.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates the T-loop of several AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family protein kinases, resulting in their activation. Previous structural studies have revealed that the alpha C-helix, located in the small lobe of the kinase domain of PDK1, is a key regulatory element, as it links a substrate interacting site termed the hydrophobic motif (HM) pocket with the phosphorylated Ser-241 in the T-loop. In this study we have demonstrated by mutational analysis that interactions between the phosphorylated Ser-241 and the alpha C-helix are not required for PDK1 activity or substrate binding through the HM-pocket but are necessary for PDK1 to be activated or stabilized by a peptide that binds to this site. The structure of an inactive T-loop mutant of PDK1, in which Ser-241 is changed to Ala, was also determined. This structure, together with surface plasmon resonance binding studies, demonstrates that the PDK1(S241A)-inactive mutant possesses an intact HM-pocket as well as an ordered alpha C-helix. These findings reveal that the integrity of the alpha C-helix and HM-pocket in PDK1 is not regulated by T-loop phosphorylation.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2005

Specificity and affinity of natural product cyclopentapeptide inhibitors against A. fumigatus, human, and bacterial chitinases.

Family 18 chitinases play key roles in organisms ranging from bacteria to man. There is a need for specific, potent inhibitors to probe the function of these chitinases in different organisms. Such molecules could also provide leads for the development of chemotherapeuticals with fungicidal, insecticidal, or anti-inflammatory potential. Recently, two natural product peptides, argifin and argadin, have been characterized, which structurally mimic chitinase-chitooligosaccharide interactions and inhibit a bacterial chitinase in the nM-mM range. Here, we show that these inhibitors also act on human and Aspergillus fumigatus chitinases. The structures of these enzymes in complex with argifin and argadin, together with mutagenesis, fluorescence, and enzymology, reveal that subtle changes in the binding site dramatically affect affinity and selectivity. The data show that it may be possible to develop specific chitinase inhibitors based on the argifin/argadin scaffolds.

Aspergillus fumigatus↗
PubMed2005

Methylxanthine drugs are chitinase inhibitors: investigation of inhibition and binding modes.

Family 18 chitinases play key roles in a range of pathogenic organisms and are overexpressed in the asthmatic lung. By screening a library of marketed drug molecules, we have identified methylxanthine derivatives as possible inhibitor leads. These derivatives, theophylline, caffeine, and pentoxifylline, are used therapeutically as antiinflammatory agents, with pleiotropic mechanisms of action. Here it is shown that they are also competitive inhibitors against a fungal family 18 chitinase, with pentoxifylline being the most potent (K(i) of 37 microM). Crystallographic analysis of chitinase-inhibitor complexes revealed specific interactions with the active site, mimicking the reaction intermediate analog, allosamidin. Mutagenesis identified the key active site residues, conserved in mammalian chitinases, which contribute to inhibitor affinity. Enzyme assays also revealed that these methylxanthines are active against human chitinases.

Chitinases↗
PubMed2004

Crystal structure of the PTPL1/FAP-1 human tyrosine phosphatase mutated in colorectal cancer: evidence for a second phosphotyrosine substrate recognition pocket.

Protein-tyrosine phosphatase-L1 (PTPL1, also known as FAP-1, PTP1E, PTP-BAS, and PTPN13) is mutated in a significant number of colorectal tumors and may play a role in down-regulating signaling responses mediated by phosphatidylinositol 3-kinase, although the precise substrates are as yet unknown. In this study, we describe a 1.8 A resolution crystal structure of a fully active fragment of PTPL1 encompassing the catalytic domain. PTPL1 adopts the standard PTP fold, albeit with an unusually positioned additional N-terminal helix, and shows an ordered phosphate in the active site. Interestingly, a positively charged pocket is located near the PTPL1 catalytic site, reminiscent of the second phosphotyrosine binding site in PTP1B, which is required to dephosphorylate peptides containing two adjacent phosphotyrosine residues (as occurs for example in the activated insulin receptor). We demonstrate that PTPL1, like PTP1B, interacts with and dephosphorylates a bis-phosphorylated insulin receptor peptide more efficiently than monophosphorylated peptides, indicating that PTPL1 may down-regulate the phosphatidylinositol 3-kinase pathway, by dephosphorylating insulin or growth factor receptors that contain tandem phosphotyrosines. The structure also reveals that four out of five PTPL1 mutations found in colorectal cancers are located on solvent-exposed regions remote from the active site, consistent with these mutants being normally active. In contrast, the fifth mutation, which changes Met-2307 to Thr, is close to the active site cysteine and decreases activity significantly. Our studies provide the first molecular description of the PTPL1 catalytic domain and give new insight into the function of PTPL1.

Amino Acid Sequence↗