TORC1 is an essential regulator of nutrient-controlled proliferation and differentiation in Leishmania.

Leishmania parasites undergo differentiation between various proliferating and non-dividing forms to adapt to changing host environments. The mechanisms that link environmental cues with the parasite's developmental changes remain elusive. Here, we report that Leishmania TORC1 is a key environmental sensor for parasite proliferation and differentiation in the sand fly-stage promastigotes and for replication of mammalian-stage amastigotes. We show that Leishmania RPTOR1, interacts with TOR1 and LST8, and identify new parasite-specific proteins that interact in this complex. We investigate TORC1 function by conditional deletion of RPTOR1, where under nutrient-rich conditions RPTOR1 depletion results in decreased protein synthesis and growth, G1 cell cycle arrest and premature differentiation from proliferative promastigotes to non-dividing mammalian-infective metacyclic forms. These parasites are unable to respond to nutrients to differentiate into proliferative retroleptomonads, which are required for their blood-meal induced amplification in sand flies and enhanced mammalian infectivity. We additionally show that RPTOR1-/- metacyclic promastigotes develop into amastigotes but do not proliferate in the mammalian host to cause pathology. RPTOR1-dependent TORC1 functionality represents a critical mechanism for driving parasite growth and proliferation.

Ranking Metabolite Sets by Their Activity Levels.

Related metabolites can be grouped into sets in many ways, e.g., by their participation in series of chemical reactions (forming metabolic pathways), or based on fragmentation spectral similarities or shared chemical substructures. Understanding how such metabolite sets change in relation to experimental factors can be incredibly useful in the interpretation and understanding of complex metabolomics data sets. However, many of the available tools that are used to perform this analysis are not entirely suitable for the analysis of untargeted metabolomics measurements. Here, we present PALS (Pathway Activity Level Scoring), a Python library, command line tool, and Web application that performs the ranking of significantly changing metabolite sets over different experimental conditions. The main algorithm in PALS is based on the pathway level analysis of gene expression (PLAGE) factorisation method and is denoted as mPLAGE (PLAGE for metabolomics). As an example of an application, PALS is used to analyse metabolites grouped as metabolic pathways and by shared tandem mass spectrometry fragmentation patterns. A comparison of mPLAGE with two other commonly used methods (overrepresentation analysis (ORA) and gene set enrichment analysis (GSEA)) is also given and reveals that mPLAGE is more robust to missing features and noisy data than the alternatives. As further examples, PALS is also applied to human African trypanosomiasis, Rhamnaceae, and American Gut Project data. In addition, normalisation can have a significant impact on pathway analysis results, and PALS offers a framework to further investigate this. PALS is freely available from our project Web site.

Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome.

Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/ß/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys(147), resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys(147) to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca(2+) for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.

PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast.

The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His(99) and Cys(136)), and an Asp (Asp(134)) in the potential S1 binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.

Comparative structural analysis of the caspase family with other clan CD cysteine peptidases.

Clan CD forms a structural group of cysteine peptidases, containing seven individual families and two subfamilies of structurally related enzymes. Historically, it is most notable for containing the mammalian caspases, on which the structures of the clan were founded. Interestingly, the caspase family is split into two subfamilies: the caspases, and a second subfamily containing both the paracaspases and the metacaspases. Structural data are now available for both the paracaspases and the metacaspases, allowing a comprehensive structural analysis of the entire caspase family. In addition, a relative plethora of structural data has recently become available for many of the other families in the clan, allowing both the structures and the structure-function relationships of clan CD to be fully explored. The present review compares the enzymes in the caspase subfamilies with each other, together with a comprehensive comparison of all the structural families in clan CD. This reveals a diverse group of structures with highly conserved structural elements that provide the peptidases with a variety of substrate specificities and activation mechanisms. It also reveals conserved structural elements involved in substrate binding, and potential autoinhibitory functions, throughout the clan, and confirms that the metacaspases are structurally diverse from the caspases (and paracaspases), suggesting that they should form a distinct family of clan CD peptidases.

Purification, characterization, and crystallization of Trypanosoma metacaspases.

Metacaspases are cysteine peptidases found in trypanosomes but absent in mammals, and despite being distantly related to the mammalian caspases they show significant disparity in their cellular and enzymatic functions. The genome of the parasitic protozoa Trypanosoma brucei (the causative agent of African sleeping sickness) encodes five metacaspases: TbMCA1-TbMCA5. Of these TbMCA2, TbMCA3, and TbMCA5 are active cysteine peptidases expressed in the bloodstream form of the parasite. To investigate the structure-function relationship of the trypanosome metacaspases and the structural basis for their divergence from the caspases, paracaspases, and other Clan CD cysteine peptidases (or vice versa), we purified and characterized TbMCA2 and determined the three-dimensional structure of an inactive mutant using X-ray crystallography. The methods presented in this chapter describe the recombinant expression of active TbMCA2 and inactive TbMCA2(C213A). The protocols produce large amounts of recombinant protein for use in structural, biochemical, and kinetic studies and include detailed information on how to produce diffraction quality crystals of TbMCA2(C213A).

Substrate specificity and the effect of calcium on Trypanosoma brucei metacaspase 2.

Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca(2+) on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has an unambiguous preference for basic amino acids at the P1 position of peptide substrates and that this is most probably a result of hydrogen bonding from the P1 residue to Asp95 and Asp211 in TbMCA2. In addition, TbMCA2 also has a preference for charged residues at the P2 and P3 positions and for small residues at the prime side of a peptide substrate. Studies into the effects of Ca(2+) on the enzyme revealed the presence of two Ca(2+) binding sites and a reversible structural modification of the enzyme upon Ca(2+) binding. In addition, the concentration of Ca(2+) used for activation of TbMCA2 was found to produce a differential effect on the activity of TbMCA2, but only when a series of peptides that differed in P2 were examined, suggesting that Ca(2+) activation of TbMCA2 has a structural effect on the enzyme in the vicinity of the S2 binding pocket. Collectively, these data give new insights into the substrate specificity and Ca(2+) activation of TbMCA2. This provides important functional details and leads to a better understanding of metacaspases, which are known to play an important role in trypanosomes and make attractive drug targets due to their absence in humans.

High-throughput methods for the detection of protein overexpression using fluorescence markers.

Overexpression of recombinant protein is essential for the structural or biochemical characterization of a protein, and the aim is to obtain as much correctly folded protein as possible. The use of fluorescence markers such as green fluorescent protein (GFP) to monitor protein overexpression has been established for a number of years, where the fluorescent signal is a direct measure of protein quantity and quality. In this protocol, GFP is used to quickly identify the optimal overexpression conditions for six individual protein targets, by testing four growth media, four cell lines, and two temperatures. Common examples are suggested in the text, but can be replaced by the user. Methods to visualize the fluorescence readings as a heat-map, and to perform dot-blot analysis using an antibody against common tags are also described.

Crystal structure of a Trypanosoma brucei metacaspase.

Metacaspases are distantly related caspase-family cysteine peptidases implicated in programmed cell death in plants and lower eukaryotes. They differ significantly from caspases because they are calcium-activated, arginine-specific peptidases that do not require processing or dimerization for activity. To elucidate the basis of these differences and to determine the impact they might have on the control of cell death pathways in lower eukaryotes, the previously undescribed crystal structure of a metacaspase, an inactive mutant of metacaspase 2 (MCA2) from Trypanosoma brucei, has been determined to a resolution of 1.4 Å. The structure comprises a core caspase fold, but with an unusual eight-stranded ß-sheet that stabilizes the protein as a monomer. Essential aspartic acid residues, in the predicted S1 binding pocket, delineate the arginine-specific substrate specificity. In addition, MCA2 possesses an unusual N terminus, which encircles the protein and traverses the catalytic dyad, with Y31 acting as a gatekeeper residue. The calcium-binding site is defined by samarium coordinated by four aspartic acid residues, whereas calcium binding itself induces an allosteric conformational change that could stabilize the active site in a fashion analogous to subunit processing in caspases. Collectively, these data give insights into the mechanistic basis of substrate specificity and mode of activation of MCA2 and provide a detailed framework for understanding the role of metacaspases in cell death pathways of lower eukaryotes.

High-throughput identification of purification conditions leads to preliminary crystallization conditions for three inner membrane proteins.

An important factor in the crystallization, and subsequent structural determination, of integral membrane proteins is the ability to produce a stable and monodisperse solution of the protein. Obtaining the correct purification detergent to achieve this can be laborious and is often serendipitous. In this study, high-throughput methods are used to analyze the suitability of eight different detergents on the stability of 12 inner transmembrane proteins from Escherichia coli. The best results obtained from the small-scale experiments were scaled up, the aggregation state of the proteins assessed, and all monodisperse protein solutions entered into crystallization trials. This resulted in preliminary crystallization hits for three inner membrane proteins: XylH, PgpB and YjdL and this study reports the methods, purification procedures and crystallization conditions used to achieve this.

Oligopeptidase B deficient mutants of Leishmania major.

Oligopeptidase B is a clan SC, family S9 serine peptidase found in gram positive bacteria, plants and trypanosomatids. Evidence suggests it is a virulence factor and thus therapeutic target in both Trypanosoma cruzi and T. brucei, but little is known about its function in Leishmania. In this study L. major OPB-deficient mutants (Δopb) were created. These grew normally as promastigotes, had a small deficiency in their ability to undergo differentiation to metacyclic promastigotes, were significantly less able to infect and survive within macrophages in vitro, but were virulent to mice. These data suggest that L. major OPB itself is not an important virulence factor, indicating functional differences between trypanosomes and Leishmania in their interaction with the mammalian host. The possibility that an OPB-like enzyme (designated OPB2) in L. major might compensate for the loss of OPB in Δopb was investigated via by mapping its sequence onto the 1.6Å structure of L. major OPB. This suggested that the residues involved in the S1 and S2 subsites of OPB2 are identical to OPB and hence the substrate specificity would be similar. Consequently there may be redundancy between the two enzymes.

Crystal structure of Leishmania major oligopeptidase B gives insight into the enzymatic properties of a trypanosomatid virulence factor.

Oligopeptidase B (OPB) is a serine peptidase with dibasic substrate specificity. It is found in bacteria, plants, and trypanosomatid pathogens, where it has been identified as a virulence factor and potential drug target. In this study we expressed active recombinant Leishmania major OPB and provide the first structure of an oligopeptidase B at high resolution. The crystallographic study reveals that OPB comprises two domains, a catalytic and a propeller domain, linked together by a hinge region. The structure has been determined in complex with the oligopeptide, protease-inhibitor antipain, giving detailed information on the enzyme active site and extended substrate binding pockets. It shows that Glu-621 plays a critical role in the S1 binding pocket and, along with Phe-603, is largely responsible for the enzyme substrate specificity in P1. In the S2 binding pocket, Tyr-499 was shown to be important for substrate stability. The structure also allowed an investigation into the function of residues highlighted in other studies including Glu-623, which was predicted to be involved in the S1 binding pocket but is found forming an inter-domain hydrogen bond. Additional important salt bridges/hydrogen bonds between the two domains were observed, highlighting the significance of the domain interface in OPB. This work provides a foundation for the study of the role of OPBs as virulence factors in trypanosomatids. It could facilitate the development of specific OPB inhibitors with therapeutic potential by exploiting its unique substrate recognition properties as well as providing a model for OPBs in general.

Crystal structures of all-alpha type membrane proteins.

Integral membrane proteins are involved in a wide range of essential biological functions and the determination of their three-dimensional structures plays a central role in understanding their function. This review focuses on the structures of one class of integral membrane proteins: the functionally diverse all-alpha type membrane proteins. It gives an overview of all the structures determined by X-ray crystallography, describing each system and structure in turn. It shows that the structures of all-alpha type membrane proteins have made valuable contributions to understanding structure-function relationships in membrane proteins. These range from the first insights into the function of exciting individual proteins to an in-depth knowledge of protein function from entire biological systems.

A protocol for high throughput methods for the expression and purification of inner membrane proteins.

The production of diffraction quality crystals for the structural determination of inner membrane proteins relies on obtaining large amounts of stable protein. Achieving this, by finding the correct parameters to successfully express and purify these proteins is often time-consuming and frustrating. The methods described here examine the most important parameters, in both expression and purification, quickly and simply. They take into account methods previously used in successful structural determinations of inner membrane proteins and collect and analyse data for use in further experiments and to investigate overall trends. These methods make use of histidine-tagged membrane proteins with a green fluorescent protein fusion but could be adapted easily for other proteins.

Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development.

Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, we combined a rapid-screening strategy using a folate-based library with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determined selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivatives, and nine compounds showed greater activity against parasite enzymes compared with human enzymes. Compound 6a displayed a K(i) of 100 nM toward LmPTR1, and the crystal structure of the LmPTR1:NADPH:6a ternary complex revealed a substrate-like binding mode distinct from that previously observed for similar compounds. A second round of design, synthesis, and assay produced a compound (6b) with a significantly improved K(i) (37 nM) against LmPTR1, and the structure of this complex was also determined. Biological evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T. cruzi. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs.

Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate.

The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 A resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the beta6-alpha6 loop and alpha6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.

Structure and reactivity of hydroxypropylphosphonic acid epoxidase in fosfomycin biosynthesis by a cation- and flavin-dependent mechanism.

The biosynthesis of fosfomycin, an oxirane antibiotic in clinical use, involves a unique epoxidation catalyzed by (S)-2-hydroxypropylphosphonic acid epoxidase (HPPE). The reaction is essentially dehydrogenation of a secondary alcohol. A high-resolution crystallographic analysis reveals that the HPPE subunit displays a two-domain combination. The C-terminal or catalytic domain has the cupin fold that binds a divalent cation, whereas the N-terminal domain carries a helix-turn-helix motif with putative DNA-binding helices positioned 34 A apart. The structure of HPPE serves as a model for numerous proteins, of ill-defined function, predicted to be transcription factors but carrying a cupin domain at the C terminus. Structure-reactivity analyses reveal conformational changes near the catalytic center driven by the presence or absence of ligand, that HPPE is a Zn(2+)/Fe(2+)-dependent epoxidase, proof that flavin mononucleotide is required for catalysis, and allow us to propose a simple mechanism that is compatible with previous experimental data. The participation of the redox inert Zn(2+) in the mechanism is surprising and indicates that Lewis acid properties of the metal ions are sufficient to polarize the substrate and, aided by flavin mononucleotide reduction, facilitate the epoxidation.

Initiating a crystallographic analysis of recombinant (S)-2-hydroxypropylphosphonic acid epoxidase from Streptomyces wedmorensis.

The oxirane (1R,2S)-1,2-epoxypropylphosphonic acid (fosfomycin) is a natural product antibiotic produced in Streptomyces wedmorensis by the metal-ion-dependent (S)-2-hydroxypropylphosphonic acid epoxidase. This epoxidase is highly unusual since it has no requirement for a haem prosthetic group. The gene encoding the enzyme, fom4, has been cloned and a highly efficient recombinant source of the enzyme established. Two different crystal forms, tetragonal and hexagonal, have been obtained. The hexagonal form displays symmetry consistent with space group P6(1/5)22 and unit-cell parameters a = 86.44, c = 221.56 A, gamma = 120 degrees. The Matthews coefficient, VM, of 2.7 A3 Da(-1) corresponds to two subunits, each of approximate weight 21.4 kDa, in the asymmetric unit with 55% solvent content. These crystals diffract to high resolution and experimental phases are being sought to determine the structure.

Inhibition of Leishmania major pteridine reductase by 2,4,6-triaminoquinazoline: structure of the NADPH ternary complex.

The structure of Leishmania major pteridine reductase (PTR1) in complex with NADPH and the inhibitor 2,4,6-triaminoquinazoline (TAQ) has been solved in a new crystal form by molecular replacement and refined to 2.6 A resolution. The inhibitor mimics a fragment, the pterin head group, of the archetypal antifolate drug methotrexate (MTX) and exploits similar chemical features to bind in the PTR1 active site. Despite being a much smaller molecule, TAQ displays a similar inhibition constant to that of MTX. PTR1 is a target for the development of improved therapies for infections caused by trypanosomatid parasites and this analysis provides information to assist the structure-based development of novel enzyme inhibitors.

The crystal structure of the periplasmic domain of the type II secretion system protein EpsM from Vibrio cholerae: the simplest version of the ferredoxin fold.

The terminal branch of the general secretion pathway (Gsp or type II secretion system) is used by several pathogenic bacteria for the secretion of their virulence factors across the outer membrane. In these secretion systems, a complex of 12-15 Gsp proteins spans from the pore in the outer membrane via several associated signal or energy-transducing proteins in the inner membrane to a regulating ATPase in the cytosol. The human pathogen Vibrio cholerae uses such a system, called the Eps system, for the export of the cholera toxin and other virulence factors from its periplasm into the lumen of the gastrointestinal tract of the host. Here, we report the atomic structure of the periplasmic domain of the EpsM protein from V.cholerae, which is a part of the interface between the regulating part and the rest of the Eps system. The crystal structure was determined by Se-Met MAD phasing and the model was refined to 1.7A resolution. The monomer consists of two alphabetabeta-subdomains forming a sandwich of two alpha-helices and a four-stranded antiparallel beta-sheet. In the dimer, a deep cleft with a polar rim and a hydrophobic bottom made by conserved residues is located between the monomers. This cleft contains an extra electron density suggesting that this region might serve as a binding site of an unknown ligand or part of a protein partner. Unexpectedly, the fold of the periplasmic domain of EpsM is an undescribed circular permutation of the ferredoxin fold.