TY - JOUR
T1 - Structural and thermodynamic characterization of the Escherichia coli RelBE toxin-antitoxin system: Indication for a functional role of differential stabilityt
AU - Cherny, Izhack
AU - Overgaard, Martin
AU - Borch, Jonas
AU - Bram, Yaron
AU - Gerdes, Kenn
AU - Gazit, Ehud
PY - 2007
Y1 - 2007
N2 - The RelE and RelB proteins constitute the RNA interferase (toxin) and its cognate inhibitor (antitoxin) components of the Escherichia coli relBE toxin-antitoxin system. Despite the well-described functionality and physiological activity of this system in E. coli, no structural study was performed on the folding and stability of the protein pair in solution. Here we structurally and thermodynamically characterize the ReIBE system components from E. coli in solution, both separately and in their complexed state. The ROB antitoxin, an alpha-helical protein according to circular dichroism and infrared spectroscopy, forms oligomers in solution, exhibits high thermostability with a T(M) of 58.5 degrees C, has a considerable heat resistance, and has high unfolding reversibility. In contrasts the RelE toxin includes a large portion of antiparallel P-sheets, displays lower thermostability with a T(M) of 52.5 degrees C, and exhibits exceptional sensitivity to heat. Complex formation, accompanied by a structural transition, leads to a 12 degrees C increase in the T(M) and substantial heat resistance. Moreover, in vivo interaction and protein footprint experiments indicate that the C-terminal part of RelB is responsible for RelB-RelE interaction, being protease sensitive in its free state, while it becomes protected from proteolysis when complexed with RelE. Overall, our findings support the notion that RelB lacks a well-organized hydrophobic core in solution whereas RelE is a well-folded protein. Furthermore, our results support that RelB protein from E. coli is similar to ParD and CcdA antitoxins in both fold and thermodynamic properties. The differential folding state of the proteins is discussed in the context of their physiological activities.
AB - The RelE and RelB proteins constitute the RNA interferase (toxin) and its cognate inhibitor (antitoxin) components of the Escherichia coli relBE toxin-antitoxin system. Despite the well-described functionality and physiological activity of this system in E. coli, no structural study was performed on the folding and stability of the protein pair in solution. Here we structurally and thermodynamically characterize the ReIBE system components from E. coli in solution, both separately and in their complexed state. The ROB antitoxin, an alpha-helical protein according to circular dichroism and infrared spectroscopy, forms oligomers in solution, exhibits high thermostability with a T(M) of 58.5 degrees C, has a considerable heat resistance, and has high unfolding reversibility. In contrasts the RelE toxin includes a large portion of antiparallel P-sheets, displays lower thermostability with a T(M) of 52.5 degrees C, and exhibits exceptional sensitivity to heat. Complex formation, accompanied by a structural transition, leads to a 12 degrees C increase in the T(M) and substantial heat resistance. Moreover, in vivo interaction and protein footprint experiments indicate that the C-terminal part of RelB is responsible for RelB-RelE interaction, being protease sensitive in its free state, while it becomes protected from proteolysis when complexed with RelE. Overall, our findings support the notion that RelB lacks a well-organized hydrophobic core in solution whereas RelE is a well-folded protein. Furthermore, our results support that RelB protein from E. coli is similar to ParD and CcdA antitoxins in both fold and thermodynamic properties. The differential folding state of the proteins is discussed in the context of their physiological activities.
U2 - 10.1021/bi701037e
DO - 10.1021/bi701037e
M3 - Journal article
SN - 0006-2960
VL - 46
SP - 12152
EP - 12163
JO - Biochemistry
JF - Biochemistry
IS - 43
ER -