Faculty Collaboration Database

Conformational changes in the activation loop of a bacterial PASTA kinase. Protein Sci 2023 Jul;32(7):e4697

Date

06/14/2023

Pubmed ID

37312631

Pubmed Central ID

PMC10303680

DOI

10.1002/pro.4697

Scopus ID

2-s2.0-85163810164 (requires institutional sign-in at Scopus site)

Abstract

Many bacterial genomes encode a transmembrane protein kinase belonging to the PASTA kinase family, which controls numerous processes in diverse bacterial pathogens, including antibiotic resistance, cell division, stress resistance, toxin production, and virulence. PASTA kinases share a conserved three-part domain architecture, consisting of an extracellular PASTA domain, proposed to sense the peptidoglycan layer status, a single transmembrane helix, and an intracellular Ser/Thr kinase domain. The crystal structures of the kinase domain from two homologous PASTA kinases reveal a characteristic two-lobed structure typical of eukaryotic protein kinases with a centrally located, but unresolved, activation loop that becomes phosphorylated and regulates downstream signaling pathways. We previously identified three sites of phosphorylation on the activation loop (T163, T166, and T168) of IreK, a PASTA kinase from the pathogen Enterococcus faecalis, as well as a distal phosphorylation site (T218) that each influence IreK activity in vivo. Still, the mechanism by which loop phosphorylation regulates PASTA kinase function is yet unknown. Therefore, we utilized site-directed spin labeling (SDSL) and continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy to assess the E. faecalis IreK kinase activation loop dynamics, including the effects of phosphorylation on activation loop motion, and the IreK-IreB interaction. Our results reveal that the IreK activation loop occupies a more immobile state when dephosphorylated, and that loop autophosphorylation shifts the loop to a more mobile state that can then enable interaction with IreB, a known substrate.

Author List

Bluma MS, Schultz KM, Kristich CJ, Klug CS

Authors

Candice S. Klug PhD Professor in the Biophysics department at Medical College of Wisconsin
Christopher J. Kristich PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Bacteria
Phosphorylation
Protein Kinases
Signal Transduction