Interaction of LipidII with specific residues of its extracytoplasmic domain governs appropriate localization and optimal activation of Mycobacterium tuberculosis PknB
The Mycobacterium tuberculosis kinase PknB is essential for growth and survival of the pathogen in vitro and in vivo. Here we present the results of our efforts to elucidate the mechanism of regulation of PknB activity. The specific residues in the kinases’s extracytoplasmic domain that are essential for ligand interaction and survival of the bacterium have been identified. The extracytoplasmic domain interacts with mDAP-containing LipidII, and this is abolished upon mutation of the ligand-interacting residues. Abrogation of ligand-binding or sequestration of the ligand leads to aberrant localization of PknB. Contrary to the prevailing hypothesis, abrogation of ligand-binding is linked to activation loop hyperphosphorylation, and indiscriminate hyperphosphorylation of PknB substrates as well as other proteins, ultimately causing loss of homeostasis and cell death. We propose that the ligand-kinase interaction directs the appropriate localization of the kinase, coupled to stringently controlled activation of PknB., and consequently the downstream processes thereof.
Sample Processing Protocol
250 µg WCL from each samples shown in Fig 6e, were reduced using 10 mM tris (2-carboxyethyl) phosphine at 55°C for 1 h and alkylated using 10 mM iodoacetamide for 30 min at 25°C. Samples were acetone precipitated and the pellet was resuspended in 100 l of 100 mM triethylammonium bicarbonate (TEAB) and digested with 6 g Trypsin (Promega) for 16 h at 37°C. TMT labeling (Thermo Fisher Scientific) was performed as per manufacturer’s instructions. Peptides from RvΔB::V +pristinamycin; RvΔB::V –pristinamycin; RvΔB::B - pristinamycin + IVN; and RvΔB::B-GM - pristinamycin + IVN were labeled with 126, 127, 128 and 131 reporter ions respectively. Labeled samples were pooled, dried and Strong Cation Exchange chromatography (SCX) was performed as described previously using two salt gradients, 100 and 350 mM KCl. 1/20th of each fraction was secured for the total proteome analysis. Rest of the sample was enriched for the phosphopeptide using IMAC beads as described earlier.
Data Processing Protocol
The desalted samples for the total proteome analysis as well as the enriched peptide samples for phosphoproteomics analysis were reconstituted in Buffer A (95% water, 5% Acetonitrile, 0.1% Formic Acid). All experiments were performed using EASY-nLC system (Thermo Fisher Scientific) coupled to LTQ Orbitrap-Velos mass spectrometer (Thermo Fisher Scientific) equipped with nanoelectrospray ion source. A 10 cm PicoFrit Self-Pack microcapillary column (New Objective) was used to resolve the peptide mixture and the peptides were eluted as described previously [Jagtap, 2012 #6136]. The peptides were dissociated with both HCD and CID for better MS/MS spectra. The collision energy induced dissociation of X ion precursors was performed at 35 for CID and 40 for HCD. Both MS and MS/MS data were acquired using scan range of 20-2000 M/Z ratios. The dynamic exclusion was set at 500 for both ion trap (CID) and FTMS (HCD) and the resolution was set at 7500. Spectra obtained were queried against Mtb H37Rv database (refseq database 85, release date 11 January 2018). Proteome Discoverer 1.3 was used as the search algorithm with oxidation of methionine and carbamidomethylation of cysteine as static modification. Phosphorylation of serine, threonine and tyrosine was used as dynamic modification. TMT 6 plex modification of peptide N-termini and lysine residues were set as the fixed modification. The TMT ratios were calculated with TMT126 (RvΔB::V +pristinamycin sample) in the denominator. All PSMs were identified at 1% false discovery rate (FDR). Mass tolerance for precursor ions and fragment ions were set at 10ppm and 0.1 Da respectively. Mass-spectrometry analysis was performed to obtain a list of intensities of various proteins in proteome and phospho-enriched proteome.
Bolaji F Oyeyemi, International Center for Genetic Engineering and Biotechnology (ICGEB)
Vinay K. Nandicoori, Staff Scientist VII Signal Transduction Lab-1, National Institute of Immunology, Aruna Asaf Ali Marg New Delhi-110067 ( lab head )