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首页> 外文期刊>Biochemistry >IIAGlc inhibition of glycerol kinase: a communications network tunes protein motions at the allosteric site.
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IIAGlc inhibition of glycerol kinase: a communications network tunes protein motions at the allosteric site.

机译：IIAGlc对甘油激酶的抑制：一个通讯网络调节变构位点的蛋白质运动。

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摘要

Steady-state and time-resolved fluorescence anisotropy methods applied to an extrinsic fluorophore that is conjugated to non-native cysteine residues demonstrate that amino acids in an allosteric communication network within a protein subunit tune protein backbone motions at a distal site to enable allosteric binding and inhibition. The unphosphorylated form of the phosphocarrier protein IIAGlc is an allosteric inhibitor of Escherichia coli glycerol kinase, binding more than 25 A from the kinase active site. Crystal structures that showed a ligand-dependent conformational change and large temperature factors for the IIAGlc-binding site on E. coli glycerol kinase suggest that motions of the allosteric site have an important role in the inhibition. Three E. coli glycerol kinase amino acids that are located at least 15 A from the active site and the allosteric site were shown previously to be necessary for transplanting IIAGlc inhibition into the nonallosteric glycerol kinase from Haemophilus influenzae. These three amino acids are termed the coupling locus. The apparent allosteric site motions and the requirement for the distant coupling locus to transplant allosteric inhibition suggest that the coupling locus modulates the motions of the IIAGlc-binding site. To evaluate this possibility, variants of E. coli glycerol kinase and the chimeric, allosteric H. influenzae glycerol kinase were constructed with a non-native cysteine residue replacing one of the native residues in the IIAGlc-binding site. The extrinsic fluorophore Oregon Green 488 (2',7'-difluorofluorescein) was conjugated specifically to the non-native cysteine residue. Steady-state and time-resolved fluorescence anisotropy measurements show that the motions of the fluorophore reflect backbone motions of the IIAGlc-binding site and these motions are modulated by the amino acids at the coupling locus.
机译：应用于与非天然半胱氨酸残基偶联的外在荧光团的稳态和时间分辨荧光各向异性方法证明，蛋白质亚基内的变构通讯网络中的氨基酸调节远端位点的蛋白质骨架运动，以实现变构结合和抑制。磷酸载体蛋白IIAGlc的未磷酸化形式是大肠杆菌甘油激酶的变构抑制剂，从激酶活性位点结合超过25A。对大肠杆菌甘油激酶上的IIAGlc结合位点显示配体依赖性构象变化和较大温度因子的晶体结构表明，变构位点的运动在抑制中起重要作用。先前显示，距离活性位点和变构位点至少15 A的三个大肠杆菌甘油激酶氨基酸对于将IIAGlc抑制物移植到流感嗜血杆菌的非变构甘油激酶中是必需的。这三个氨基酸称为偶联基因座。明显的变构位点运动和对远距离耦合位点移植抑制变构作用的要求表明，该耦合位点调节IIAGlc结合位点的运动。为了评估这种可能性，用非天然半胱氨酸残基替换IIAGlc结合位点中的天然残基之一，构建了大肠杆菌甘油激酶和嵌合的变构流感嗜血杆菌甘油激酶的变体。外在荧光团俄勒冈绿色488（2'，7'-二氟荧光素）特异性地缀合至非天然半胱氨酸残基。稳态和时间分辨的荧光各向异性测量结果表明，荧光团的运动反映了IIAGlc结合位点的骨架运动，并且这些运动受到耦合位点氨基酸的调节。

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