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African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma bruceispp., is responsible for ~30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target--T. brucei N-myristoyltransferase--leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis., Protein N-myristoylation is a ubiquitous eukaryotic co- and posttranslational modification and is required for the membrane targeting and biological activity of many important proteins (1,2). The N-myristoylation reaction--the transfer of [...]

The LKB1 tumor suppressor is a protein kinase that controls the activity of adenosine monophosphate--activated protein kinase (AMPK). LKB1 activity is regulated by the pseudokinase STRAD[alpha] and the scaffolding protein MO25[alpha] through an unknown, phosphorylation-independent, mechanism. We describe the structure of the core heterotrimeric LKB1-STRAD[alpha]-MO25[alpha] complex, revealing an unusuat allosteric mechanism of LKB1 activation. STRAD[alpha] adopts a dosed conformation typical of active protein kinases and binds LKB1 as a pseudosubstrate. STRAD[alpha] and MO25[alpha] promote the active conformation of LKB1, which is stabilized by MO25[alpha] interacting with the LKB1 activation loop. This previously undescribed mechanism of kinase activation may be relevant to understanding the evolution of other pseudokinases. The structure also reveals how mutations found in Peutz-Jeghers syndrome and in various sporadic cancers impair LKB1 function. 10.1126/science.1178377

The genome sequence of the oral pathogen Streptococcus mutans predicts the presence of two putative polysaccharide deacetylases. The first, designated PgdA in this paper, shows homology to the catalytic domains of peptidoglycan deacetylases from Streptococcus pneumoniae and Listeria monocytogenes, which are both thought to be involved in the bacterial defense mechanism against human mucosal lysozyme and are part of the CAZY family 4 carbohydrate esterases. S. mutans cells in which the pgdA gene was deleted displayed a different colony texture and a slightly increased cell surface hydrophobicity and yet did not become hypersensitive to lysozyme as shown previously for S. pneumoniae. To understand this apparent lack of activity, the high-resolution X-ray structure of S. mutans PgdA was determined; it showed the typical carbohydrate esterase 4 fold, with metal bound in a His-His-Asp triad. Analysis of the protein surface showed that an extended groove lined with aromatic residues is orientated toward the active-site residues. The protein exhibited metal-dependent de-N-acetylase activity toward a hexamer of N-acetylglucosamine. No activity was observed toward shorter chitooligosaccharides or a synthetic peptidoglycan tetrasaccharide. In agreement with the lysozyme data this would suggest that S. mutans PgdA does not act on peptidoglycan but on an as-yet-unidentified polysaccharide within the bacterial cell surface. Strikingly, the pgdA-knockout strain showed a significant increase in aggregation/agglutination by salivary agglutinin, in agreement with this gene acting as a deacetylase of a cell surface glycan.

Streptococcus pneumoniae peptidoglycan GIcNAc deacetylase (SpPgdA) protects the Gram-positive bacterial cell wall from host lysozymes by deacetylating peptidoglycan GIcNAc 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 GIcN[A.sub.c.sub.3] as a substrate ([K.sub.m] = 3.8 mM, [k.sub.cat] = 0.55 [s.sup.-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 metal-loenzymes and present a step toward identification of mechanism-based inhibitors for this important class of enzymes. crystal structure | metalloenzyme

Over the past years, family 18 chitinases have been validated as potential targets for the design of drugs against human pathogens that contain or interact with chitin during their normal life cycles. Thus far, only one potent chitinase inhibitor has been described in detail, the pseudotrisaccharide allosamidin. Recently, however, two potent natural-product cyclopentapeptide chitinase inhibitors, argifin and argadin, were reported. Here, we describe high-resolution crystal structures that reveal the details of the interactions of these cyclopeptides with a family 18 chitinase. The structures are examples of complexes of a carbohydrate-processing enzyme with high-affinity peptide-based inhibitors and show in detail how the peptide backbone and side chains mimic the interactions of the enzyme with chitooligosaccharides. Together with enzymological characterization, the structures explain why argadin shows an order of magnitude stronger inhibition than allosamidin, whereas argifin shows weaker inhibition. The peptides bind to the chitinase in remarkably different ways, which may explain the differences in inhibition constants. The two complexes provide a basis for structure-based design of potent chitinase inhibitors, accessible by standard peptide chemistry.

The dynamic properties of the [Alpha]-subunit of bovine transducin (G[[Alpha].sub.t]) were studied using molecular dynamics simulations and essential dynamics analyses. The helical domain of transducin seems to move toward the guanosine triphosphate hydrolase (GTPase) domain. Our studies suggest that this movement is facilitated by a hinge bending motion that is centered on residues Gly56 and Gly179 and that this motion may be involved in GDP release and GTP hydrolysis. The dynamic properties of the GTPase domain of G[[Alpha].sub.t]-GDP were compared to those of ras p21 and reveal a significant degree of similarity, indicating common dynamic properties for an equivalent domain in two different proteins.

X-ray crystallography, enzymology, subsite probing, inductively coupled plasma mass spectrometry, and ligand modeling is used to describe the structure and study the reaction mechanism and substrate binding of endo-chitin de-N-acetylase (CICDA). This showed that CICDA possesses a highly conserved substrate-binding groove, with slight alterations, which influence substrate specificity and subsite affinity.

A rationally designed glucoimidazole, GlcNAcstatin, which inhibits a bacterial OGA (bOGA) with a [K.sub.i] of 4.5 pM and 100000 fold selectivity over HexA/B, and is active against human OGA (hOGA) in human cell lines, and is tethered in the active site as revealed by X-ray crystallography, is reported. It is shown that both picomolar inhibition and exquisite selectivity can be achieved with rationally designed glucoimidazoles.