Abstract
Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the individual proteins but also for identifying useful drug targets. From an earlier study, we had structural models of M.tb at a proteome scale from which a set of 13,858 small molecule binding pockets were identified. We use a set of NTP binding sub-structural motifs derived from a previous study and scan the M.tb pocketome, and find that 1,768 proteins or 43% of the proteome can theoretically bind NTP ligands. Using an experimental proteomics approach involving dye-ligand affinity chromatography, we confirm NTP binding to 47 different proteins, of which 4 are hypothetical proteins. Our analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. As the list includes a number of known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets.
Highlights
Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes
The structural models of 2877 proteins amounting to 70% of the M.tb proteome, which were generated by us previously, contained a total of 13858 small molecule binding pockets
The pockets identified were of high confidence as they were detected as consensus by three independent binding site prediction methods based on energetic considerations, geometric parameters and evolutionary information (See Methods: section 4.1)
Summary
Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Chemical proteomics approaches have been explored in some organisms in which a chemical probe is used to capture ATP binding proteins, which are subsequently characterized through digestion and mass profiling[8,9,10] These methods have provided a high-throughput means of identifying ATP binding proteins. The captured proteins are subsequently digested with trypsin and labeled peptides are enriched via streptavidin affinity capture beads and subjected to LC-MS/MS for the identification of ATP-labeled proteins[24], and 539 proteins are identified through this approach Another method that has been used for the same purpose involves the use of an activity-based probe to annotate and validate ATP binding proteins. We explore two independent approaches, (a) a computational screen that identifies proteins that have NTP binding structural signatures and (b) a chemical proteomics screen that identifies NTP binding proteins in M.tb cell extract
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