Localization of prefoldin interaction sites in the hyperthermophilic group II chaperonin and correlations between binding rate and protein transfer rate |
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Authors: | Zako Tamotsu Murase Yosuke Iizuka Ryo Yoshida Takao Kanzaki Taro Ide Naoki Maeda Mizuo Funatsu Takashi Yohda Masafumi |
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Affiliation: | Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei-Shi, Tokyo, Japan. |
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Abstract: | Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. The manner by which prefoldin interacts with a group II chaperonin is poorly understood. Here, we have examined the prefoldin interaction site in the archaeal group II chaperonin, comparing the interaction of two Thermococcus chaperonins and their mutants with Pyrococcus prefoldin by surface plasmon resonance. We show that the mutations of Lys250 and Lys256 of Thermococcus alpha chaperonin residues to Glu residues increase the affinity to Pyrococcus prefoldin to the level of Thermococcus beta chaperonin and Pyrococcus chaperonin, indicating that their Glu250 and Glu256 residues of the helical protrusion region are responsible for relatively stronger binding to Pyrococcus prefoldin than Thermococcus alpha chaperonin. Since the putative chaperonin binding sites in the distal ends of Pyrococcus prefoldin are rich in basic residues, electrostatic interaction seems to be important for their interaction. The substrate protein transfer rate from prefoldin correlates well with its affinity for chaperonin. |
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Keywords: | PhPFD, prefoldin from Pyrococcus horikoshii T. KS-1, Thermococcus sp. strain KS-1 CPN, chaperonin CPNα, wild-type T. KS-1 α chaperonin CPNβ, wild-type T. KS-1 β chaperonin PhCPN, chaperonin from Pyrococcus horikoshii CPNβαα, T. KS-1 α chaperonin with apical domain substitution from β CPNαββ, T. KS-1 β chaperonin with apical domain substitution from α CPNαK250E, T. KS-1 α chaperonin with K250E mutation CPNαK256E, T. KS-1 α chaperonin with K256E mutation CPNαK250E/K256E, T. KS-1 α chaperonin with K250E and K256E mutations CPNβdel, T. KS-1 β chaperonin mutant lacking in the helical protrusion region PFDL69C, PhPFD with L69C mutation FL-PFD, PFDL69C modified with fluorescein GFP, green fluorescent protein SPR, surface plasmon resonance |
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