TB Structural Genomics Consortium Research Articles

Structural Annotation of the Mycobacterium tuberculosis Proteome.

Efforts from the TB Structural Genomics Consortium together with those of tuberculosis structural biologists worldwide have led to the determination of about 350 structures, making up nearly a tenth of the pathogen's proteome. Given that knowledge of protein structures is essential to obtaining a high-resolution understanding of the underlying biology, it is desirable to have a structural view of the entire proteome. Indeed, structure prediction methods have advanced sufficiently to allow structural models of many more proteins to be built based on homology modeling and fold recognition strategies. By means of these approaches, structural models for about 2,877 proteins, making up nearly 70% of the Mycobacterium tuberculosis proteome, are available. Knowledge from bioinformatics has made significant inroads into an improved annotation of the M. tuberculosis genome and in the prediction of key protein players that interact in vital pathways, some of which are unique to the organism. Functional inferences have been made for a large number of proteins based on fold-function associations. More importantly, ligand-binding pockets of the proteins are identified and scanned against a large database, leading to binding site-based ligand associations and hence structure-based function annotation. Near proteome-wide structural models provide a global perspective of the fold distribution in the genome. New insights about the folds that predominate in the genome, as well as the fold combinations that make up multidomain proteins, are also obtained. This chapter describes the structural proteome, functional inferences drawn from it, and its applications in drug discovery.

The Differential Gene Expression Pattern ofMycobacterium tuberculosisin Response to Capreomycin and PA-824 versus First-Line TB Drugs Reveals Stress- and PE/PPE-Related Drug Targets

Tuberculosis is a leading infectious disease causing millions of deaths each year. How to eradicate mycobacterial persistence has become a central research focus for developing next-generation TB drugs. Yet, the knowledge in this area is fundamentally limited and only a few drugs, notably capreomycin and PA-824, have been shown to be active against non-replicating persistent TB bacilli. In this study, we performed a new bioinformatics analysis on microarray-based gene expression data obtained from the public domain to explore genes that were differentially induced by drugs between the group of capreomycin and PA-824 and the group of mainly the first-line TB drugs. Our study has identified 42 genes specifically induced by capreomycin and PA-824. Many of these genes are related to stress responses. In terms of the distribution of identified genes in a specific category relative to the whole genome, only the categories of PE/PPE and conserved hypotheticals have statistical significance. Six among the 42 genes identified in this study are on the list of the top 100 persistence targets selected by the TB Structural Genomics Consortium. Further biological elucidation of their roles in mycobacterial persistence is warranted.

An automated high-throughput screening method for the identification of high-yield, soluble protein variants using cell-free expression and systematic truncation

A highly automated method for rapidly identifying soluble protein variants with good expression yields has been developed. This method is based on a commercially available in vitro protein expression system. It consists of two polymerase chain reactions (PCR) followed by in vitro protein expression and protein quantification by dot blot. The PCR protocols have been improved and optimized to allow automation using commercial fluid handling devices. A PCR primer design program has also been implemented to streamline protein variant design. This automated protocol is highly reliable and has tremendously improved the throughput of expression screening as compared to conventional cell-based methods and manual in vitro methods. We have applied this method to 32 problematic targets from the TB Structural Genomics Consortium. Experimental results of these studies are reported.

Genetic Basis for the Synthesis of the Immunomodulatory Mannose Caps of Lipoarabinomannan in Mycobacterium tuberculosis

Lipoarabinomannan (LAM) is a high molecular weight, heterogenous lipoglycan present in abundant quantities in Mycobacterium tuberculosis and many other actinomycetes. In M. tuberculosis, the non-reducing arabinan termini of the LAM are capped with alpha1-->2 mannose residues; in some other species, the arabinan of LAM is not capped or is capped with inositol phosphate. The nature and extent of this capping plays an important role in disease pathogenesis. MT1671 in M. tuberculosis CDC1551 was identified as a glycosyltransferase that could be involved in LAM capping. To determine the function of this protein a mutant strain of M. tuberculosis CDC1551 was studied, in which MT1671 was disrupted by transposition. SDS-PAGE analysis showed that the LAM of the mutant strain migrated more rapidly than that of the wild type and did not react with concanavalin A as did wild-type LAM. Structural analysis using NMR, gas chromatography/mass spectrometry, endoarabinanase digestion, Dionex high pH anion exchange chromatography, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry demonstrated that the LAM of the mutant strain was devoid of mannose capping. Since an ortholog of MT1671 is not present in Mycobacterium smegmatis mc(2)155, a recombinant strain was constructed that expressed this protein. Analysis revealed that the LAM of the recombinant strain was larger than that of the wild type, had gained concanavalin A reactivity, and that the arabinan termini were capped with a single mannose residue. Thus, MT1671 is the mannosyltransferase involved in deposition of the first of the mannose residues on the non-reducing arabinan termini and the basis of much of the interaction between the tubercle bacillus and the host cell.

Potential drug targets in Mycobacterium tuberculosis through metabolic pathway analysis

The emergence of multidrug resistant varieties of Mycobacterium tuberculosis has led to a search for novel drug targets. We have performed an insilico comparative analysis of metabolic pathways of the host Homo sapiens and the pathogen M. tuberculosis. Enzymes from the biochemical pathways of M. tuberculosis from the KEGG metabolic pathway database were compared with proteins from the host H. sapiens, by performing a BLASTp search against the non-redundant database restricted to the H. sapiens subset. The e-value threshold cutoff was set to 0.005. Enzymes, which do not show similarity to any of the host proteins, below this threshold, were filtered out as potential drug targets. We have identified six pathways unique to the pathogen M. tuberculosis when compared to the host H. sapiens. Potential drug targets from these pathways could be useful for the discovery of broad spectrum drugs. Potential drug targets were also identified from pathways related to lipid metabolism, carbohydrate metabolism, amino acid metabolism, energy metabolism, vitamin and cofactor biosynthetic pathways and nucleotide metabolism. Of the 185 distinct targets identified from these pathways, many are in various stages of progress at the TB Structural Genomics Consortium. However, 67 of our targets are new and can be considered for rational drug design. As a case study, we have built a homology model of one of the potential drug targets MurD ligase using WHAT IF software. The model could be further explored for insilico docking studies with suitable inhibitors. The study was successful in listing out potential drug targets from the M. tuberculosis proteome involved in vital aspects of the pathogen's metabolism, persistence, virulence and cell wall biosynthesis. This systematic evaluation of metabolic pathways of host and pathogen through reliable and conventional bioinformatic methods can be extended to other pathogens of clinical interest.

Structure of pyrR (Rv1379) fromMycobacterium tuberculosis: a persistence gene and protein drug target

The Mycobacterium tuberculosis pyrR gene (Rv1379) encodes a protein that regulates the expression of pyrimidine-nucleotide biosynthesis (pyr) genes in a UMP-dependent manner. Because pyrimidine biosynthesis is an essential step in the progression of TB, the gene product pyrR is an attractive antitubercular drug target. The 1.9 A native structure of Mtb pyrR determined by the TB Structural Genomics Consortium facilities in trigonal space group P3(1)21 is reported, with unit-cell parameters a = 66.64, c = 154.72 A at 120 K and two molecules in the asymmetric unit. The three-dimensional structure and residual uracil phosphoribosyltransferase activity point to a common PRTase ancestor for pyrR. However, while PRPP- and UMP-binding sites have been retained in Mtb pyrR, a distinct dimer interaction among subunits creates a deep positively charged cleft capable of binding pyr mRNA. In silico screening of pyrimidine-nucleoside analogs has revealed a number of potential lead compounds that, if bound to Mtb pyrR, could facilitate transcriptional attenuation, particularly cyclopentenyl nucleosides.

Benefits of Automated Crystallization Plate Tracking, Imaging, and Analysis

We describe the design of a database and software for managing and organizing protein crystallization data. We also outline the considerations behind the design of a fast web interface linking protein production data, crystallization images, and automated image analysis. The database and associated interfaces underpin the Oxford Protein Production Facility (OPPF) crystallization laboratory, collecting, in a routine and automatic manner, up to 100,000 images per day. Over 17 million separate images are currently held in this database. We discuss the substantial scientific benefits automated tracking, imaging, and analysis of crystallizations offers to the structural biologist: analysis of the time course of the trial and easy analysis of trials with related crystallization conditions. Features of this system address requirements common to many crystallographic laboratories that are currently setting up (semi-)automated crystallization imaging systems.

Mycobacterium tuberculosis RmlC epimerase (Rv3465): a promising drug-target structure in the rhamnose pathway.

The Mycobacterium tuberculosis rmlC gene encodes dTDP-4-keto-6-deoxyglucose epimerase, the third enzyme in the M. tuberculosis dTDP-L-rhamnose pathway which is essential for mycobacterial cell-wall synthesis. Because it is structurally unique, highly substrate-specific and does not require a cofactor, RmlC is considered to be the most promising drug target in the pathway, and the M. tuberculosis rmlC gene was selected in the initial round of TB Structural Genomics Consortium targets for structure determination. The 1.7 A native structure determined by the consortium facilities is reported and implications for in silico screening of ligands for structure-guided drug design are discussed.

Laboratory scale structural genomics.

At Lawrence Livermore National Laboratory, the development of the TB structural genomics consortium crystallization facility has paralleled several local proteomics research efforts that have grown out of gene expression microarray and comparative genomics studies. Collective experience gathered from TB consortium labs and other centers involved in the NIH-NIGMS protein structure initiative allows us to explore the possibilities and challenges of pursuing structural genomics on an academic laboratory scale. We discuss our procedures and protocols for genomic targeting approaches, primer design, cloning, small scale expression screening, scale-up and purification, through to automated crystallization screening and data collection. The procedures are carried out by a small group using a combination of traditional approaches, innovative molecular biochemistry approaches, software automation, and a modest investment in robotic equipment.

Structural genomics of Mycobacterium tuberculosis: a preliminary report of progress at UCLA

The growing list of fully sequenced genomes, combined with innovations in the fields of structural biology and bioinformatics, provides a synergy for the discovery of new drug targets. With this background, the TB Structural Genomics Consortium has been formed. This international consortium is comprised of laboratories from 31 universities and institutes in 13 countries. The goal of the consortium is to determine the structures of over 400 potential drug targets from the genome of Mycobacterium tuberculosis and analyze their structures in the context of functional information. We summarize the efforts of the UCLA consortium members. Potential drug targets were selected using a variety of bioinformatics methods and screened for certain physical and species-specific properties to yield a starting group of protein targets for structure determination. Target determination methods include protein phylogenetic profiles and Rosetta Stone methods, and the use of related biochemical pathways to select genes linked to essential prokaryotic genes. Criteria imposed on target selection included potential protein solubility, protein or domain size, and targets that lack homologs in eukaryotic organisms. In addition, some protein targets were chosen that are specific to M. tuberculosis, such as PE and PPE domains. Thus far, the UCLA group has cloned 263 targets, expressed 171 proteins and purified 40 proteins, which are currently in crystallization trials. Our efforts have yielded 13 crystals and eight structures. Seven structures are summarized here. Four of the structures are secreted proteins: antigen 85B; MPT 63, which is one of the three major secreted proteins of M. tuberculosis; a thioredoxin derivative Rv2878c; and potentially secreted glutamate synthetase. We also report the structures of three proteins that are potentially essential to the survival of M. tuberculosis: a protein involved in the folate biosynthetic pathway (Rv3607c); a protein involved in the biosynthesis of vitamin B5 (Rv3602c); and a pyrophosphatase, Rv2697c. Our approach to the M. tuberculosis structural genomics project will yield information for drug design and vaccine production against tuberculosis. In addition, this study will provide further insights into the mechanisms of mycobacterial pathogenesis.

High‐Throughput Crystallography at an Affordable Cost: The TB Structural Genomics Consortium Crystallization Facility

AbstractFor Abstract see ChemInform Abstract in Full Text.

Maximum-likelihood crystallization

The crystallization facility of the TB Structural Genomics Consortium, one of nine NIH-sponsored structural genomics pilot projects, employs a combinatorial random sampling technique in high-throughput crystallization screening. Although data are still sparse and a comprehensive analysis cannot be performed at this stage, preliminary results appear to validate the random-screening concept. A discussion of statistical crystallization data analysis aims to draw attention to the need for comprehensive and valid sampling protocols. In view of limited overlap in techniques and sampling parameters between the publicly funded high-throughput crystallography initiatives, exchange of information should be encouraged, aiming to effectively integrate data mining efforts into a comprehensive predictive framework for protein crystallization.

High-throughput crystallography at an affordable cost: the TB Structural Genomics Consortium Crystallization Facility.

The crystallization facility of the TB Structural Genomics Consortium, one of nine P50 structural genomics centers sponsored by the National Institutes of Health, provides TB consortium members with automated crystallization, data collection, and basic molecular replacement structure solution up to bias-minimized maps. In contrast to venture capital -funded commercial enterprises, the TB consortium facilities are decentralized and aim to develop high-throughput crystallography methods and automation on a comparatively small budget. In addition to financial constraints, the logistics and organization of a production environment differ considerably from academic settings. The TB Structural Genomics Consortium crystallization facility may thus provide a model for cost-effective, efficient high-throughput crystallography. Processes and methods presented in this review should assist academic institutions planning to invest in high-throughput structural biology to assess both the rewards and risks of establishing structural genomics programs.

Structural Genomics: Foundation for the Future of Biology?

INTRODUCTION. Structural genomics is the large-scale determination of protein structures. Along with DNA sequencing and functional genomics, structural genomics promises to provide a foundation for developing a fuller understanding of biology. In the short term, structural genomics is likely to be an effective means of obtaining structural information on proteins of high medical, biological, or biophysical interest, including proteins from pathogens or those involved in human disease. M. tuberculosis causes TB, a disease that continues to result in over 3 million deaths per year worldwide. Although anti-TB drugs exist, multidrug-resistant strains are becoming increasingly common and the long course of drug therapy discourages proper treatment. Structural genomics provides a promising approach to drug development against this important pathogen. METHOD. The TB Structural Genomics Consortium was formed to target and determine structures of proteins from M. tuberculosis on a genomic scale. The Consortium is international in scope and has over 230 members around the world and includes crystallographers, biochemists, geneticists, and TB biologists. The Consortium has targeted over 1000 of the 4000 TB genes and is in the process of carrying out structure determination on those that are amenable to rapid expression, purification, and crystallization or NMR spectroscopy. Those proteins that are more difficult to work with are to be engineered to increase their solubility and expression. The Consortium has policies encouraging collaboration and sharing of data among its members and with the scientific community. The public web site of the Consortium can be viewed at http://www.doe-mbi.ucla.edu/TB. RESULTS. The TB Structural Genomics Consortium has developed technologies for engineering proteins to increase their solubility and for automated structure solution and is applying them to structure determination of TB proteins. The Consortium has a centralized database that is accessible to the public and which shows the targeting and status of structure determinations of proteins from TB. The TB Structural Genomics Consortium welcomes collaborations between its members and any TB biologists interested in structural information. The consortium approach to large-scale structure determination has been effective during the first year of the TB Structural Genomics Consortium, with the structures of 22 proteins from M. tuberculosis determined so far by Consortium members.