Abstract
Simple SummaryMetal ions often play important roles in biological processes. Thermus thermophilus trigger factor (TtTF) is a zinc-dependent molecular chaperone where Zn2+ has been shown to enhance its folding-arrest activity. However, the mechanisms of how Zn2+ binds to TtTF and how Zn2+ 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 Zn2+ alters the physical properties of TtTF, including secondary structure, thermal stability, and oligomeric state. Our results showed that TtTF binds Zn2+ in a 1:1 ratio, and all three domains of TtTF are involved in zinc-binding. We found that Zn2+ 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 Zn2+ to modulate its oligomeric state to regulate the activity.Thermus thermophilus trigger factor (TtTF) is a zinc-dependent molecular chaperone whose folding-arrest activity is regulated by Zn2+. 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 Zn2+. The data show that full-length TtTF binds Zn2+, but the isolated domains and tandem domains of TtTF do not bind to Zn2+. Furthermore, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra suggested that Zn2+-binding induces the partial structural changes of TtTF, and size exclusion chromatography-multi-angle light scattering (SEC-MALS) showed that Zn2+ 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.
Highlights
In a crowded intracellular environment, newly synthesized polypeptide chains and metastable proteins risk misfolding or aggregation [1,2]
Given the relationship between the oligomerization and activity shown for Escherichia coli TF (EcTF) [17], our results suggest the mechanism of activity modulation of Thermus thermophilus trigger factor (TtTF), in which the Zn2+ alters structural properties to induce the oligomerization of TtTF for activity regulation
SEC-MALS experiments were performed to test if the partial structural change induced by zinc-binding affects the oligomeric state of TtTF, which can be associated with the holdase activity of TtTF
Summary
In a crowded intracellular environment, newly synthesized polypeptide chains and metastable proteins risk misfolding or aggregation [1,2]. The assembly and rearrangement of the client-binding sites on TF induced by dimerization can modulate the binding kinetics with the client proteins, which explains the mechanism of the activity modulation [17]. Another strategy to modulate the activity of molecular chaperones is the binding of metal ions [19,20]. The previous study has shown the relationship between zinc-binding and holdase activity modulation [20], the mechanism of how Zn2+ alters the activity of TtTF and whether it is related to oligomerization are unknown. Given the relationship between the oligomerization and activity shown for EcTF [17], our results suggest the mechanism of activity modulation of TtTF, in which the Zn2+ alters structural properties to induce the oligomerization of TtTF for activity regulation
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
