Contribution of Subdomain Structure to the Thermal Stability of the Cholera Toxin A1 Subunit

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• 作者：Tuhina Banerjee ; Abhay Pande ; Michael G. Jobling ; Michael Taylor ; Shane Massey ; Randall K. Holmes ; Suren A. Tatulian ; Ken Teter
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：October 19, 2010
• 年：2010
• 卷：49
• 期：41
• 页码：8839-8846
• 全文大小：929K
• 年卷期：v.49,no.41(October 19, 2010)
• ISSN：1520-4995

文摘

The catalytic A1 subunit of cholera toxin (CTA1) is an ADP-ribosyltransferase with three distinct subdomains: CTA1₁ forms the catalytic core of the toxin, CTA1₂ is an extended linker between CTA1₁ and CTA1₃, and CTA1₃ is a compact globular region. CTA1 crosses the endoplasmic reticulum (ER) membrane to enter the cytosol where it initiates a cytopathic effect. Toxin translocation involves ER-associated degradation (ERAD), a quality control system that exports misfolded proteins from the ER to the cytosol. At the physiological temperature of 37 °C, the free CTA1 subunit is in a partially unfolded conformation that triggers its ERAD-mediated translocation to the cytosol. Thus, the temperature sensitivity of CTA1 structure is an important determinant of its function. Here, we examined the contribution of CTA1 subdomain structure to the thermal unfolding of CTA1. Biophysical measurements demonstrated that the CTA1₁ subdomain is thermally unstable and that the CTA1₂ subdomain provides a degree of conformational stability to CTA1₁. The CTA1₃ subdomain does not affect the overall stability of CTA1, but the thermal unfolding of CTA1 appears to begin with a local loss of structure in the CTA1₃ subdomain: glycerol and acidic pH both inhibited the thermal disordering of full-length CTA1 but not the disordering of a CTA1 construct lacking the A1₃ subdomain. These observations provide mechanistic insight regarding the thermal unfolding of CTA1, an event which facilitates its subsequent translocation to the cytosol.