Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation.
Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation.
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GrpE.DnaK 结合界面的能量突变分析：通过沉降速度分析超速离心的平衡关联常数。
DOI：
10.1016/j.jmb.2004.03.074
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Harrison,CeliaJ
中科院分区：
文献类型：
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作者：
Gelinas,AmyD;Toth,Joseph;Bethoney,KelleyA;Stafford,WalterF;Harrison,CeliaJ
原核Hsp 70分子伴侣DnaK需要核苷酸交换因子和热休克蛋白GrpE来释放ADP。GrpE和DnaK是具有广泛蛋白质-蛋白质界面的紧密缔合分子，并且在不存在ADP的情况下，GrpE和DnaK的解离常数在低纳摩尔范围内。GrpE降低DnaK对ADP的亲和力，并且相互联系也是如此：ADP降低DnaK对GrpE的亲和力。通过丙氨酸扫描诱变探测GrpE侧链对GrpE-DnaK结合的能量贡献。用沉降速度（SV）分析超离心（AUC）测定了ADP存在下GrpE与DnaK的ATP酶结构域结合的平衡常数（Keq）。ADP结合的DnaK是GrpE的天然靶点，在预先形成的GrpE-DnaKATP酶复合物中加入ADP（终浓度为5 μM）可使平衡缔合常数进入实验可达到的范围。在这些实验条件下，一个单一的GrpE氨基酸残基，精氨酸183丙氨酸取代，导致GrpE-DnaKATP酶复合物，弱相关（Keq=9.4× 104 M）。该残基先前已被证明是GrpE的两个结构域之间的热力学连接的一部分：热敏长螺旋和C末端β结构域。在相同的实验条件下，与野生型GrpE·DnaKATP酶相比，其他几种GrpE侧链的结合自由能发生了显著变化（ΔΔG为1.5 ~ 1.7kcalmol−1）。总的来说，GrpE和DnaK之间的强相互作用似乎由静电主导，与芽孢杆菌RNA酶和芽孢杆菌RNA酶类似，这是另一种充分表征的蛋白质-蛋白质相互作用。GrpE是一种固有的热敏元件，在细菌热激温度下其核苷酸交换功能表现出非Arrhenius行为，而位于热敏元件沿着的几个暴露于溶剂的侧链的突变表明这些残基对于GrpE-DnaK相互作用确实是重要的。
DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein–protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE–DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 μM) to the preformed GrpE–DnaKATPasecomplexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE–DnaKATPasecomplex that was weakly associated (Keq=9.4×104M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal β-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (ΔΔG∼1.5 to 1.7kcalmol−1), compared to wild-type GrpE·DnaKATPasein the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein–protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE–DnaK interactions.