Substrate binding induces depolymerization of the C-terminal peptide binding domain of murine GRP78/BiP
Substrate binding induces depolymerization of the C-terminal peptide binding domain of murine GRP78/BiP
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DOI：
10.1074/jbc.273.41.26827
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发表时间：
1998-10-09
影响因子：
4.8
通讯作者：
Blond, SY
中科院分区：
文献类型：
--
作者：
Chevalier, M;King, L;Blond, SY
为了研究每个结构域在BiP/GRP 78功能中的作用，我们使用了工程化的全长重组BiP，其包含两个肠激酶位点;一个位点位于N-末端FLAG表位之后，第二个位点插入N-和C-末端结构域之间的连接处（FLAG-BiP，ent），FLAG-BiP.ent寡聚成多个物种，其在缓慢的浓度和温度依赖性平衡中彼此相互转化。ATP或AMP-PNP（腺苷5 '-（β，γ-亚氨基）三磷酸）的结合，而非ADP或肽底物的结合，诱导FLAG-BiP、ent的解聚和单体物质的稳定。肠激酶切割单体、无核苷酸的BiP，ent导致44-kDa N-末端ATP酶片段（N44，ent）从30-kDa C-末端底物结合结构域物理解离（C30.ent），解离后，游离的C-末端底物结合结构域容易发生自缔合，而N44，ent保持单体，在合成肽存在下进行的肠激酶切割防止游离的C30，ent结构域的寡聚化。在肠激酶裂解期间添加ATP对C30. ent寡聚化没有影响。我们的数据清楚地表明，特定肽与C-末端结构域或ATP与N-末端结构域的结合诱导C30结构域内的内部构象变化，导致BiP解聚。
To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N- and C-terminal domains (FLAG-BiP,ent), FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration- and temperature-dependent equilibrium. Binding of ATP or AMP-PNP (adenosine 5'-(beta,gamma-imino)triphosphate), but not ADP, or of a peptidic substrate induces depolymerization of FLAG-BiP,ent and stabilization of monomeric species. Enterokinase cleavage of monomeric, nucleotide-free BiP,ent results in the physical dissociation of the 44-kDa N-terminal ATPase fragment (N44,ent) from the 30-kDa C-terminal substrate binding domain (C30.ent), Upon dissociation, the freed C-terminal substrate binding domain readily undergoes self-association while N44,ent remains monomeric, Enterokinase cleavage performed in the presence of a synthetic peptide prevents oligomerization of the freed C30,ent domain. Addition of ATP during enterokinase cleavage has no effect on C30.ent oligomerization, Our data clearly indicate that binding of a specific peptide onto the C-terminal domain, or ATP onto the N-terminal domain, induces internal conformational change(s) within the C30 domain that result(s) in BiP depolymerization.