Crystal structure of constitutively monomeric E. coli Hsp33 mutant with chaperone activity

Abstract

a b s t r a c tHeat shock protein 33 (Hsp33) from Escherichia coli is a redox-regulated molecular chaperone that protects cells from oxidative stress. To understand the molecular basis for the monomer-dimer switch in the functional regulation of E. coli Hsp33, we generated a constitutively monomeric Hsp33 by introducing the Q151E mutation in the dimeric interface and determined its crystal structure. The overall scaffold of the monomeric Hsp33 1-235 (Q151E) mutant is virtually the same as that of the dimeric form, except that there is no domain swapping. The measurement of chaperone activity to thermally denatured luciferase showed that the constitutively monomeric Hsp33 mutant still retains chaperone activity similar to that of wild-type Hsp33 1-235 , suggesting that a Hsp33 monomer is sufficient to interact with slowly unfolded substrate.

Hsp33 Controls Elongation Factor-Tu Stability and Allows Escherichia coli Growth in the Absence of the Major DnaK and Trigger Factor Chaperones

“…S4. This last result suggests that distinct intermediates populating the multistep activation cycle of Hsp33 could possess unique functions in vivo (38,41,44). In contrast, and as expected, the Hsp33(Y12E) mutant was completely nonfunctional, behaving like the plasmid vector control (Fig.…”

“…This is supported by the synergistic effects observed among the tig, dnaK (or dnaJ), and hslO mutations. However, the redox and the oligomeric status (monomeric, dimeric, or oligomeric) of cellular Hsp33 that is capable of performing such a function in vivo remains to be determined (38,41,44). In addition, it is not yet established whether Hsp33 induces EF-Tu degradation under physiological conditions where all the chaperones are present.…”

“…It is known that EF-Tu adopts several conformations and populates various intermediate stages during its functional cycle in polypeptide chain elongation (38,41,44). At this time, the actual conformational state(s) of EF-Tu that is recognized by Hsp33 and Lon remains unknown.…”

“…Specifically, we chose the following hslO mutations: Hsp33(Y12E), which is constitutively active in vitro but mainly insoluble in vivo; Hsp33(M172S), activated by high temperature in the absence of oxidizing conditions (27); Hsp33(Q151E), constitutively monomeric and redox-regulated with preference for slowly unfolding protein substrates (38); and Hsp33(1-235), constitutively active as a dimer in vitro (39). The corresponding mutant genes were cloned in a multicopy plasmid under control of an IPTG-inducible promoter and tested for their ability to suppress the growth defects exhibited by the MC4100 ⌬tig ⌬dnaKdnaJ mutant strain.…”

Oxidation-Induced Conformational Change of a Prokaryotic Molecular Chaperone, Hsp33, Monitored by Selective Isotope Labeling

“…[7][8][9][10][11] However, a detailed process of the oxidation-induced activation of Hsp33 is still the subject of much controversy, as the crystal structures contain features that lead to doubts about their physiological relevance. Thus, in the present study, we employed NMR to monitor the oxidation-induced conformational change of Hsp33 in solution.…”