Movement of a Loop in Domain 3 of Aerolysin Is Required for Channel Formation
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- 作者：Jamie Rossjohn ; Srikumar M. Raja ; Kim L. Nelson ; Susanne C. Feil ; F. Gisou van der Goot ; Michael W. Parker ; and J. Thomas Buckley
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：January 13, 1998
- 年：1998
- 卷：37
- 期：2
- 页码：741 - 746
- 全文大小：101K
- 年卷期：v.37,no.2(January 13, 1998)
- ISSN：1520-4995
文摘
Aerolysin is a channel-forming toxin that mustoligomerize in order to become insertion-competent. Modeling based on the crystal structure of theproaerolysin dimer and electron microscopicimages of the oligomer indicated that a loop in domain 3 must move awayfrom the 
chars/beta2.gif" BORDER=0 ALIGN="middle">-sheet that formsthe main body of the protein before oligomerization can proceed.In order to determine if movementactually occurs, strategically located amino acids in the loop and inthe sheet were replaced with cysteinesby site-directed mutagenesis. A double mutant was produced inwhich the new cysteines, at position 253on the loop and position 300 in the sheet, were close enough togetherto allow formation of a disulfidebridge. The double mutant was unable to oligomerize, and it wascompletely inactive, showing not onlythat the bridge had formed but also that movement of the loop wasessential for formation of the oligomer.The existence of the bridge was confirmed by X-raycrystallography. The reduced form of the proteinand the single mutants T253C and A300C were as active as wild type,indicating that the amino acidreplacements themselves had no functional consequences. Labelingstudies using an environment-sensitivefluorescent sulfhydryl-reactive probe confirmed that the structure ofthe protein changes in the loop regionas a consequence of proteolytic activation of proaerolysin, a stepwhich also must precede oligomerization.
chars/beta2.gif" BORDER=0 ALIGN="middle">-sheet that formsthe main body of the protein before oligomerization can proceed.In order to determine if movementactually occurs, strategically located amino acids in the loop and inthe sheet were replaced with cysteinesby site-directed mutagenesis. A double mutant was produced inwhich the new cysteines, at position 253on the loop and position 300 in the sheet, were close enough togetherto allow formation of a disulfidebridge. The double mutant was unable to oligomerize, and it wascompletely inactive, showing not onlythat the bridge had formed but also that movement of the loop wasessential for formation of the oligomer.The existence of the bridge was confirmed by X-raycrystallography. The reduced form of the proteinand the single mutants T253C and A300C were as active as wild type,indicating that the amino acidreplacements themselves had no functional consequences. Labelingstudies using an environment-sensitivefluorescent sulfhydryl-reactive probe confirmed that the structure ofthe protein changes in the loop regionas a consequence of proteolytic activation of proaerolysin, a stepwhich also must precede oligomerization.