Glycosomes are peroxisome-like organelles essential for trypanosomatid parasites. Glycosome biogenesis is mediated by proteins called “peroxins,” which are considered to be promising drug targets in pathogenic Trypanosomatidae. The first step during protein translocation across the glycosomal membrane of peroxisomal targeting signal 1 (PTS1)-harboring proteins is signal recognition by the cytosolic receptor peroxin 5 (PEX5). The C-terminal PTS1 motifs interact with the PTS1 binding domain (P1BD) of PEX5, which is made up of seven tetratricopeptide repeats. Obtaining diffraction-quality crystals of the P1BD of Trypanosoma brucei PEX5 (TbPEX5) required surface entropy reduction mutagenesis. Each of the seven tetratricopeptide repeats appears to have a residue in the αL conformation in the loop connecting helices A and B. Five crystal structures of the P1BD of TbPEX5 were determined, each in complex with a hepta- or decapeptide corresponding to a natural or nonnatural PTS1 sequence. The PTS1 peptides are bound between the two subdomains of the P1BD. These structures indicate precise recognition of the C-terminal Leu of the PTS1 motif and important interactions between the PTS1 peptide main chain and up to five invariant Asn side chains of PEX5. The TbPEX5 structures reported here reveal a unique hydrophobic pocket in the subdomain interface that might be explored to obtain compounds that prevent relative motions of the subdomains and interfere selectively with PTS1 motif binding or release in trypanosomatids, and would therefore disrupt glycosome biogenesis and prevent parasite growth.

An actomyosin motor located underneath the plasma membrane drives motility and host-cell invasion of apicomplexan parasites such as Plasmodium falciparum and Plasmodium vivax, the causative agents of malaria. Aldolase connects the motor actin filaments to transmembrane adhesive proteins of the thrombospondin-related anonymous protein (TRAP) family and transduces the motor force across the parasite surface. The TRAP–aldolase interaction is a distinctive and critical trait of host hepatocyte invasion by Plasmodium sporozoites, with a likely similar interaction crucial for erythrocyte invasion by merozoites. Here, we describe 2.4-Å and 2.7-Å structures of P. falciparum aldolase (PfAldo) obtained from crystals grown in the presence of the C-terminal hexapeptide of TRAP from Plasmodium berghei. The indole ring of the critical penultimate Trp-residue of TRAP fits snugly into a newly formed hydrophobic pocket, which is exclusively delimited by hydrophilic residues: two arginines, one glutamate, and one glutamine. Comparison with the unliganded PfAldo structure shows that the two arginines adopt new side-chain rotamers, whereas a 25-residue subdomain, forming a helix–loop–helix unit, shifts upon binding the TRAP-tail. The structural data are in agreement with decreased TRAP binding after mutagenesis of PfAldo residues in and near the induced TRAP-binding pocket. Remarkably, the TRAP- and actin-binding sites of PfAldo seem to overlap, suggesting that both the plasticity of the aldolase active-site region and the multimeric nature of the enzyme are crucial for its intriguing nonenzymatic function in the invasion machinery of the malaria parasite.

The iron-dependent regulator IdeR is a key transcriptional regulator of iron uptake in Mycobacterium tuberculosis. In order to increase our insight into the role of the SH3-like third domain of this essential regulator, the metal-binding and DNA-binding properties of two-domain IdeR (2D-IdeR) whose SH3-like domain has been truncated were characterized. The equilibrium dissociation constants for Co2+ and Ni2+ activation of 2D-IdeR for binding to the fxbA operator and the DNA-binding affinities of 2D-IdeR in the presence of excess metal ions were estimated using fluorescence spectroscopy. 2D-IdeR binds to fxbA operator DNA with similar affinity as full-length IdeR in the presence of excess metal ion. However, the Ni2+ concentrations required to activate 2D-IdeR for DNA binding appear to be smaller than that for full-length IdeR while the concentration of Co2+ required for activation remains the same. We have determined the crystal structures of Ni2+-activated 2D-IdeR at 1.96 A resolution and its double dimer complex with the mbtA-mbtB operator DNA in two crystal forms at 2.4 A and 2.6 A, the highest resolutions for DNA complexes for any structures of iron-dependent regulator family members so far. The 2D-IdeR-DNA complex structures confirm the specificity of Ser37 and Pro39 for thymine bases and suggest preferential contacts of Gln43 to cytosine bases of the DNA. In addition, our 2D-IdeR structures reveal a remarkable property of the TEV cleavage sequence remaining after removal of the C-terminal His(6). This C-terminal tail promotes crystal contacts by forming a beta-sheet with the corresponding tail of neighboring subunits in two unrelated structures of 2D-IdeR, one with and one without DNA. The contact-promoting properties of this C-terminal TEV cleavage sequence may be beneficial for crystallizing other proteins.

The Myosin A-tail interacting protein (MTIP) of the malaria parasite links the actomyosin motor of the host cell invasion machinery to its inner membrane complex. We report here that at neutral pH Plasmodium falciparum MTIP in complex with Myosin A adopts a compact conformation, with its two domains completely surrounding the Myosin A-tail helix, dramatically different from previously observed extended MTIP structures. Crystallographic and mutagenesis studies show that H810 and K813 of Myosin A are key players in the formation of the compact MTIP:Myosin A complex. Only the unprotonated state of Myosin A-H810 is compatible with the compact complex. Most surprisingly, every side-chain atom of Myosin A-K813 is engaged in contacts with MTIP. While this side-chain was previously considered to prevent a compact conformation of MTIP with Myosin A, it actually appears to be essential for the formation of the compact complex. The hydrophobic pockets and adaptability seen in the available series of MTIP structures bodes well for the discovery of inhibitors of cell invasion by malaria parasites.

The causative agents of malaria have developed a sophisticated machinery for entering multiple cell types in the human and insect hosts. In this machinery, a critical interaction occurs between the unusual myosin motor MyoA and the MyoA-tail Interacting Protein (MTIP). Here we present one crystal structure that shows three different conformations of Plasmodium MTIP, one of these in complex with the MyoA-tail, which reveal major conformational changes in the C-terminal domain of MTIP upon binding the MyoA-tail helix, thereby creating several hydrophobic pockets in MTIP that are the recipients of key hydrophobic side chains of MyoA. Because we also show that the MyoA helix is able to block parasite growth, this provides avenues for designing antimalarials.

MICROSYMPOSIA C93more than 10,000Å 3 that shields the proteolytic site.The fact that proteolysis occurs inside a closed chamber is reminiscent of the proteasome structure and for that reason we introduced a new term: the peptidasome.The chamber has no obvious opening for the substrate to enter; yet a bound peptide is found inside and amino acids separated by almost 800 residues in sequence form the active site.The structure suggests a novel mechanism for access to the active site, involving hinge-bending motions that cause the peptidasome to open and close in response to substrate binding.

Structural biology studies on cholera toxin and the closely related heat-labile enterotoxin from enterotoxigenic Escherichia coli over the past decade have shed light on the mechanism of toxin action at molecular and atomic levels. Also, components of the extracellular protein secretion apparatus that translocate the toxins across the outer membrane are being investigated. At the same time, structure-based design has led to various classes of compounds targeting different toxin sites, including highly potent multivalent inhibitors that block the toxin receptor-binding process.

Glycosome biogenesis in trypanosomatids occurs via a process that is homologous to peroxisome biogenesis in other eukaryotes. Glycosomal matrix proteins are synthesized in the cytosol and imported posttranslationally. The import process involves a series of protein-protein interactions starting by recognition of glycosomal matrix proteins by a receptor in the cytosol. Most proteins to be imported contain so-called PTS-1 or PTS-2 targeting sequences recognized by, respectively, the receptor proteins PEX5 and PEX7. PEX14, a protein associated with the peroxisomal membrane, has been identified as a component of the docking complex and a point of convergence of the PEX5- and PEX7-dependent import pathways. In this paper, the strength of the interactions between Trypanosoma brucei PEX14 and PEX5 was studied by a fluorescence assay, using (i) a panel of N-terminal regions of TbPEX14 protein variants and (ii) a series of different peptides derived from TbPEX5, each containing one of the three WXXXF/Y motifs present in this receptor protein. On the PEX14 side, the N-terminal region of TbPEX14 including residues 1-84 appeared to be responsible for TbPEX5 binding. The results from PEX14 mutants identified specific residues in the N-terminal region of TbPEX14 involved in PEX5 binding and showed that in particular hydrophobic residues F35 and F52 are critical. On the PEX5 side, 13-mer peptides incorporating the first or the third WXXXF/Y motif bind to PEX14 with an affinity in the nanomolar range. However, the second WXXXF/Y motif peptide did not show any detectable affinity. Studies using variants of second and third motif peptides suggest that the alpha-helical content of the peptides as well as the charge of a residue at position 9 in the motif may be important for PEX14 binding. Assays with 7-, 10-, 13-, and 16-mer third motif peptides showed that 16-mers and 13-mers have comparable binding affinity for PEX14, whereas 10-mers and 7-mers have about 10- and 100-fold lower affinity than the 16-mers, respectively. The low sequence identities of PEX14 and PEX5 between parasite and its human host, and the vital importance of proper glycosome biogenesis to the parasite, render these peroxins highly promising drug targets.

The structure-based design of multivalent ligands offers an attractive strategy toward high affinity protein inhibitors. The spatial arrangement of the receptor-binding sites of cholera toxin, the causative agent of the severe diarrheal disease cholera and a member of the AB(5) bacterial toxin family, provides the opportunity of designing branched multivalent ligands with 5-fold symmetry. Our modular synthesis enabled the construction of a family of complex ligands with five flexible arms each ending with a bivalent ligand. The largest of these ligands has a molecular weight of 10.6 kDa. These ligands are capable of simultaneously binding to two toxin B pentamer molecules with high affinity, thus blocking the receptor-binding process of cholera toxin. A more than million-fold improvement over the monovalent ligand in inhibitory power was achieved with the best branched decavalent ligand. This is better than the improvement observed earlier for the corresponding nonbranched pentavalent ligand. Dynamic light scattering studies demonstrate the formation of concentration-dependent unique 1:1 and 1:2 ligand/toxin complexes in solution with no sign of nonspecific aggregation. This is in complete agreement with a crystal structure of the branched multivalent ligand/toxin B pentamer complex solved at 1.45 A resolution that shows the specific 1:2 ligand/toxin complex formation in the solid state. These results reiterate the power of the structure-based design of multivalent protein ligands as a general strategy for achieving high affinity and potent inhibition.

A number of helix-rich protein motifs are involved in a variety of critical protein-protein interactions in living cells. One of these is the tetratrico peptide repeat (TPR) motif that is involved, amongst others, in cell cycle regulation, chaperone function and post-translation modifications. So far, these helix-rich TPR motifs have always been observed to be a compact unit of two helices interacting with each other in antiparallel fashion. Here, we describe the structure of the first three TPR-motifs of the peroxin PEX5 from Trypanosoma brucei, the causative agent of sleeping sickness. Peroxins are proteins involved in peroxisome, glycosome and glyoxysome biogenesis. PEX5 is the receptor of the proteins targeted to these organelles by the “peroxisomal targeting signal-1”, a C-terminal tripeptide called PTS-1. The first two of the three TPR-motifs of T. brucei PEX5 appear to adopt the canonical antiparallel helix hairpin structure. In contrast, the third TPR motif of PEX5 has a dramatically different conformation in our crystals: the two helices that were supposed to form a hairpin are folded into one single 44 Å long continuous helix. Such a conformation has never been observed before for a TPR motif. This raises interesting questions including the potential functional importance of a “jack-knife” conformational change in TPR motifs.

Cholera toxin (CT) and the closely related heat-labile enterotoxin of Escherichia coli (LT) are responsible for numerous cases of diarrhea worldwide, leading to considerable morbidity and mortality. The B subunits of these heterohexameric AB(5) toxins form a pentameric arrangement which is responsible for binding to the receptor GM1 of the target epithelial cells of the host. Blocking these B pentamer-receptor interactions forms an avenue for therapeutic intervention. Here, the structural characterization of potential receptor-blocking compounds are described based on the previously identified inhibitor m-nitrophenyl-alpha-D-galactoside (MNPG). The structure of a CTB-MNPG complex confirms that the binding mode of this inhibitor is identical in the two homologous toxins CT and LT and is characterized by a glycosyl linkage geometry that leads to displacement of a well ordered water molecule near the amide group of Gly33 by the O1-substituent of MNPG. This glycosyl geometry is not maintained in the absence of a substituent that can displace this water, as shown by a complex of LTB with p-aminophenyl-alpha-D-galactoside (PAPG). New compounds were synthesized to investigate the feasibility of maintaining the favorable binding interactions exhibited by MNPG while gaining increased affinity through the addition of hydrophobic substituents complementary to either of two hydrophobic regions of the receptor-binding site. The structural characterization of complexes of LTB with two of these compounds, 3-benzylaminocarbonylphenyl-alpha-D-galactoside (BAPG) and 2-phenethyl-7-(2,3-dihydrophthalazine-1,4-dione)-alpha-D-galactoside (PEPG), demonstrates a partial success in this goal. Both compounds exhibit a mixture of binding modes, some of which are presumably influenced by the local packing environment at multiple crystallographically independent binding sites. The terminal phenyl ring of BAPG associates either with the phenyl group of Tyr12 or with the hydrophobic patch formed by Lys34 and Ile58. The latter interaction is also made by the terminal phenyl substituent of PEPG, despite a larger ring system linking the galactose moiety to the terminal phenyl. However, neither BAPG nor PEPG displaces the intended target water molecule. Both of the designed compounds exhibit increased affinity relative to the galactose and to PAPG notwithstanding the failure to displace a bound water, confirming that additional favorable hydrophobic interactions can be gained by extending the starting inhibitor by a hydrophobic tail. The insight gained from these structures should allow the design of additional candidate inhibitors that retain both the glycosyl geometry and water displacement exhibited by MNPG and the favorable hydrophobic interactions exhibited by BAPG and PEPG.

Ganglioside GM1 is the natural receptor for cholera toxin (CT) and heat-labile enterotoxin (LT), which are the causative agents of cholera and traveler's diarrhea, respectively. This observation suggests that small molecules interfering with this recognition process may prevent entry of the toxins into intestinal cells, thereby averting their devastating effects. Here, the terminal sugar of ganglioside GM1, galactose, was chosen as a lead in designing such receptor antagonists. Guided by the experimentally determined binding mode of galactose, we selected a "substructure" for searching the Available Chemicals Database, which led to the purchase of 35 galactose derivatives. Initial screening of these compounds in an LT ELISA revealed that 22 of them have a higher affinity for LT than galactose itself. A structurally diverse subset of these galactose derivatives was selected for determination of IC50 values in the LT ELISA and IC50 values in a CT assay, as well as for the determination of Kd's using the intrinsic fluorescence of LT. The best receptor antagonist found in this study was m-nitrophenyl alpha-galactoside with an IC50 of 0.6 (2) mM in the LT ELISA and 0.72 (4) mM in the CT assay, 100-fold lower than both IC50 values of galactose. Careful analysis of our binding data and comparison with crystal structures led to the derivation of correlations between the structure and affinity of the galactose derivatives. These characteristics will be used in the design of a second round of LT and CT receptor antagonists.

Cholera toxin (CT) produced by Vibrio cholerae and heat‐labile enterotoxin (LT‐I), produced by enterotoxigenic Escherichia coli, are AB5 heterohexamers with an ADP‐ribosylating A subunit and a GM1 receptor binding B pentamer. These toxins are among the most potent mucosal adjuvants known and, hence, are of interest both for the development of anti‐diarrheal vaccines against cholera or enterotoxigenic Escherichia coli diarrhea and also for vaccines in general. However, the A subunits of CT and LT‐I are known to be relatively temperature sensitive. To improve the thermostability of LT‐I an additional disulfide bond was introduced in the A1 subunit by means of the double mutation N40C and G166C. The crystal structure of this double mutant of LT‐I has been determined to 2.0 Å resolution. The protein structure of the N40C/G166C double mutant is very similar to the native structure except for a few local shifts near the new disulfide bond. The introduction of this additional disulfide bond increases the thermal stability of the A subunit of LT‐I by 6°C. The enhancement in thermostability could make this disulfide bond variant of LT‐I of considerable interest for the design of enterotoxin‐based vaccines.