Structural dynamics of the activation loop in the kinase domain of E.faecalis IreK.

Abstract

IreK is a transmembrane Ser/Thr PASTA kinase found in Enterococcus faecalis, as well as most other Gram-positive bacteria, and plays a key role in resistance to cell wall-active antibiotics such as cephalosporins. IreK belongs to a family of bacterial transmembrane kinases that include multiple PASTA (Penicillin-and Ser/Thr-kinase Associated) domains in the extracellular segment and are ubiquitous in nearly all Gram-positive bacteria. PASTA kinases have conserved functions across Gram-positive bacteria involving monitoring and regulating processes associated with the cell wall. Enterococci are frequently found in the human gut and are a common cause of severe and difficult to treat hospital acquired infections, due to their intrinsic drug resistance. The underlying mechanisms of antibiotic resistance in enterococci are not well understood and are an excellent target for the creation of preventative treatments to eliminate the growth opportunity of resistant E. faecalis in the gut and transfer to other parts of the body. Based on the crystal structures of IreK kinase domain homologs, the activation loop structure is unknown and the phosphorylation sites within this loop are proposed to play a key regulatory role in IreK kinase activity. IreB has also been identified as one of the targets for phosphorylation by IreK. Here, continuous wave electron paramagnetic resonance (CW EPR) spectroscopy was applied to study the dynamics of the IreK activation loop, including the effects of phosphorylation and IreB binding on activation loop structure and dynamics. In addition, new functionally important non-phosphorylation sites in the activation loop that regulate IreK function in vivo were identified and characterized. This comprehensive investigation into the IreK activation loop structure and functional dynamics characterizes the role of this loop as a key regulatory domain in kinase function.