Zinc-Dependent Oligomerization of Thermus thermophilus Trigger Factor Chaperone

SIMPLE SUMMARY: Metal ions often play important roles in biological processes. Thermus thermophilus trigger factor (TtTF) is a zinc-dependent molecular chaperone where Zn(2+) has been shown to enhance its folding-arrest activity. However, the mechanisms of how Zn(2+) binds to TtTF and how Zn(2+) affects the activity of TtTF are yet to be elucidated. As a first step in understanding the mechanism, we performed in vitro biophysical experiments on TtTF to investigate the zinc-binding site on TtTF and unveil how Zn(2+) alters the physical properties of TtTF, including secondary structure, thermal stability, and oligomeric state. Our results showed that TtTF binds Zn(2+) in a 1:1 ratio, and all three domains of TtTF are involved in zinc-binding. We found that Zn(2+) does not affect the thermal stability of TtTF, whereas it does induce partial structural change and promote the oligomerization of TtTF. Given that the folding-arrest activity of Escherichia coli TF (EcTF) is regulated by its oligomerization, our results imply that TtTF exploits Zn(2+) to modulate its oligomeric state to regulate the activity. ABSTRACT: Thermus thermophilus trigger factor (TtTF) is a zinc-dependent molecular chaperone whose folding-arrest activity is regulated by Zn(2+). However, little is known about the mechanism of zinc-dependent regulation of the TtTF activity. Here we exploit in vitro biophysical experiments to investigate zinc-binding, the oligomeric state, the secondary structure, and the thermal stability of TtTF in the absence and presence of Zn(2+). The data show that full-length TtTF binds Zn(2+), but the isolated domains and tandem domains of TtTF do not bind to Zn(2+). Furthermore, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra suggested that Zn(2+)-binding induces the partial structural changes of TtTF, and size exclusion chromatography-multi-angle light scattering (SEC-MALS) showed that Zn(2+) promotes TtTF oligomerization. Given the previous work showing that the activity regulation of E. coli trigger factor is accompanied by oligomerization, the data suggest that TtTF exploits zinc ions to induce the structural change coupled with the oligomerization to assemble the client-binding site, thereby effectively preventing proteins from misfolding in the thermal environment.