The domain architecture of PtkA, the first tyrosine kinase from Mycobacterium tuberculosis, differs from the conventional kinase architecture

Abstract

The discovery that MptpA (low-molecular-weight protein tyrosine phosphatase A) from () has an essential role for virulence has motivated research of tyrosine-specific phosphorylation in and other pathogenic bacteria. The phosphatase activity of MptpA is regulated via phosphorylation on Tyr and Tyr Thus far, only a single tyrosine-specific kinase, protein-tyrosine kinase A (PtkA), encoded by the gene has been identified within the genome. MptpA undergoes phosphorylation by PtkA. PtkA is an atypical bacterial tyrosine kinase, as its sequence differs from the sequence consensus within this family. The lack of structural information on PtkA hampers the detailed characterization of the MptpA-PtkA interaction. Here, using NMR spectroscopy, we provide a detailed structural characterization of the PtkA architecture and describe its intra- and intermolecular interactions with MptpA. We found that PtkA's domain architecture differs from the conventional kinase architecture and is composed of two domains, the N-terminal highly flexible intrinsically disordered domain (IDD) and the C-terminal rigid kinase core domain (KCD). The interaction between the two domains, together with the structural model of the complex proposed in this study, reveal that the IDD is unstructured and highly dynamic, allowing for a "fly-casting-like" mechanism of transient interactions with the rigid KCD This interaction modulates the accessibility of the KCD active site. In general, the structural and functional knowledge of PtkA gained in this study is crucial for understanding the MptpA-PtkA interactions, the catalytic mechanism, and the role of the kinase-phosphatase regulatory system in virulence.

Long-range replica exchange molecular dynamics guided drug repurposing against tyrosine kinase PtkA of Mycobacterium tuberculosis

“…The binding of IDD to KCD in PtkA has been shown to play an inhibitory role and to decrease the catalytic activity of KCD 12 . In a recent study, the conformation of IDD PtkA has been characterized under varying biophysical conditions and phosphorylation using NMR-spectroscopy and it was confirmed that N-terminal domain of PtkA exists as an unstructured state 12 . REMD simulations was conducted on IDD PtkA to examine the conformational behavior of the peptide at different temperatures.…”

“…inhibitory activity of iDD ptkA on KcD ptkA . Since the inhibitory effect of IDD on KCD has already been verified 12 , protein-protein docking and MD simulations of IDD PtkA -KCD PtkA were conducted to explore the www.nature.com/scientificreports www.nature.com/scientificreports/ hydrophobic and hydrogen bond interaction patterns between the two domains. The top-scoring cluster having a HADDOCK score of −58.6+/− 9.0 kcal/mol and Z-score equivalent to −1.2 was considered for MD simulations.…”

“…The catalytic activity of KCD PtkA alone is greater than the catalytic activity of KCD PtkA measured along with IDD PtkA , which suggests an inhibitory role of IDD PtkA for the catalytic activity of PtkA 12 . Here, we present a rigorous study in which we have performed Replica Exchange Molecular Dynamics (REMD) to investigate the structural conformers of the disordered domain IDD PtkA.…”

“…These exchanges allow the molecule to jump out of the minima and sample efficiently on the conformational space 17 . It has been reported that the 80-residue N-terminal domain of PtkA protein is an intrinsically disordered region which lacks a stable structure and goes through conformational exchange on changing the environmental settings such as temperature, chemical denaturant and phosphorylation 12 . In the present study, to explore the conformations of IDD PtkA , REMD was performed at varying temperatures ranging from 300-500 K.…”

“…PtkA shows two conformational states-an open state conformation in which IDD is away from Tyr262 (auto phosphorylation site), making it accessible for phosphorylation and thereby increasing the activity of PtkA; and a closed state, in which IDD masks the Tyr262, thereby decreasing the activity of PtkA. Phosphorylation of IDD by Ser/Thr kinases (PknA) induces conformational changes in IDD, which move IDD away from the autophosphorylation site and promotes an open state of PtkA 12 .…”