Allosteric and Electrostatic Protein-Protein Interactions Regulate the Assembly of the Heterohexameric Tim9-Tim10 Complex

Ekaterina Ivanova, Hui Lu

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    Protein-protein interactions are crucial processes in virtually all cellular events. The heterohexameric Tim9-Tim10 complex of the mitochondrial intermembrane space plays an important role during import of mitochondrial membrane proteins. It consists of three molecules of each subunit arranged alternately in a ring-shaped structure. While the individual protein Tim9 forms a homodimer, Tim10 is a monomer. Further to our previous investigation on the complex formation pathway, in this study, the assembly mechanism of Tim9-Tim10 was investigated using a stopped-flow technique coupled with mutagenesis. We show that while the initial velocity of the assembly depends on Tim9 concentration linearly, it presents a sigmoid curve on Tim10. In addition, the overall rate of assembly depends on the pH level in a bell-shaped profile, and two pKa values that are in good agreement with the respective isoelectric points of Tim9 and Tim10 were determined. Using a Tim10F70W mutant, we were able to show that there was clear salt concentration dependence in the rate of assembly at the early stages. Taken together, the results of pH and salt concentration dependence indicate that electrostatic interactions are important and provide an initial driving force for the complex formation. Thus, this study not only demonstrates that allosteric and electrostatic interactions are two key regulators for the assembly of the Tim9-Tim10 complex but also has important implications for our understanding of how proteins interact with their partners. © 2008 Elsevier Ltd. All rights reserved.
    Original languageEnglish
    Pages (from-to)609-616
    Number of pages7
    JournalJournal of molecular biology
    Issue number3
    Publication statusPublished - 6 Jun 2008


    • kinetics
    • pH dependence
    • protein-protein interactions
    • salt effect
    • stopped-flow fluorescence


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