IN-VITRO DISSOCIATION OF BIP-PEPTIDE COMPLEXES REQUIRES A CONFORMATIONAL CHANGE IN BIP AFTER ATP BINDING BUT DOES NOT REQUIRE ATP HYDROLYSIS

被引:131
作者
WEI, JY
GAUT, JR
HENDERSHOT, LM
机构
[1] ST JUDE CHILDRENS RES HOSP, DEPT TUMOR CELL BIOL, MEMPHIS, TN 38105 USA
[2] UNIV TENNESSEE, DEPT BIOCHEM, MEMPHIS, TN 38163 USA
[3] UNIV MICHIGAN, DEPT BIOL SCI, ANN ARBOR, MI 48109 USA
[4] UNIV MICHIGAN, INST GERONTOL, ANN ARBOR, MI 48109 USA
关键词
D O I
10.1074/jbc.270.44.26677
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In the present study, we produced single point mutations in the ATP binding site of hamster Rip, isolated recombinant proteins, and characterized them in terms of their affinity for ATP and ADP, their ability to undergo a conformational change upon nucleotide binding, and their rate of ATP hydrolysis, These analyses allowed us to classify the mutants into three groups: ATP hydrolysis (T229G), ATP binding (G226D, G227D), and ATP-induced conformation (T37G) mutants, and to test the role of these activities in the in vitro ATP-mediated release of proteins from Rip. All three classes of mutants were still able to bind peptide demonstrating that nucleotide is not involved in this function, Addition of ATP to either wild-type BiP or the T229G mutant caused the in vitro release of bound peptide, confirming that ATP hydrolysis is not required for protein release. ATP did not dissociate G226D, G227D, or T37G mutant BiP-peptide complexes, suggesting that ATP binding to Rip is not sufficient for the release of bound peptides, but that an ATP-induced conformational change in BiP is necessary, The identification of BiP mutants that are defective in each of these steps of ATP hydrolysis will allow the in vivo dissection of the role of nucleotide in BiP's activity.
引用
收藏
页码:26677 / 26682
页数:6
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