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Analysis of ATP Binding to CheA Containing Tryptophan Substitutions near the Active Site

机译：ATP与活性位点附近含有色氨酸取代的ATP结合

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Signal transduction in the chemotaxis system of Escherichia coli involves an autophos-phorylating protein histidine kinase,CheA.At the active site of CheA,phenylalanine residues 455 and 459 occupy positions near the ATP-binding pocket,immediately adjacent to one of the hinge regions of a loop that undergoes an ATP-induced conformational change("lid closure")that has been characterized previously in X-ray crystal structures [Bilwes et al.(2001)Nat.Struct.Biol.8,353-360].We generated versions of CheA carrying F455W and F459W replacements and investigated whether the fluorescence properties of the introduced tryptophan side chains were affected by nucleotide binding in a manner that would provide a signal for investigating the dynamics of active site events,such as ATP binding and lid closure.Our results indicate that CheA~(F455W)is useful in this regard,but CheA~(F459W)is not.CheA~(F455W)retained full catalytic activity and exhibited easily monitored fluorescence changes upon binding nucleotide:we observed a 25-30% decrease in CheA~(F455W)fluorescence emission intensity at 330 nm upon binding ATP in the absence of Mg~(2+);in the presence of Mg~(2+),the emission spectrum of the CheA~(F455W):ATP complex was red-shifted by 5 nm and exhibited an increased intensity(~20% higher at 345 nm relative to that of uncomplexed CheA~(F455W)).Different fluorescence changes were observed when two ATP analogues(ADPNP and ADPCP)were used instead of ATP and when Mn~(2+)or Ca~(2+)was used in place of Mg~(2+).We exploited the fluorescence changes induced by Mg~(2+)-ATP to explore the kinetics and mechanism of nucleotide binding by CheA~(F455W).In the absence of Mg~(2+),binding appears to involve a simple one-step equilibrium(k_assn = 0.7 muM~(-1)s~(-1)and k_dissn= 270 s~(-1)at 4 deg C).In the presence of Mg~(2+),the binding mechanism involves at least two steps:(i)rapid,relatively weak binding followed by(ii)a rapid,reversible step(k_forward = 300 s~(-1)and k_reverse =15 s~(-1)at 4 deg C)that enhanced the overall affinity of the complex and generated an increase in W455 fluorescence.This second step could reflect a conformational change at the CheA active site,such as lid closure.These results provide the first insight into the dynamics of nucleotide binding and substrate-induced conformational changes at the active site of a protein histidine kinase.
机译：大肠杆菌趋化性系统中的信号转导涉及自胰蛋白酶蛋白组氨酸激酶，Chea.AT的Chea，苯丙氨酸残基455和459的活性位点，占据ATP结合口袋附近的位置，立即与其中一个铰链区域相邻经历过ATP诱导的构象变化（“盖子闭合”）的环，其先前在X射线晶体结构中进行了表征[Bilwes等，（2001）Nat.Struct.biol.8,353-360]。我们生成的版本Chea携带F455W和F459W替代品，并研究了引入的色氨酸侧链的荧光性质是否受到核苷酸结合的方式，这将提供用于研究活动场所事件的动态的信号，例如ATP绑定和盖子闭合。表明Chea〜（F455W）在这方面是有用的，但是乳酸（F459W）是Not.chea〜（F455W）保留完全催化活性，并在结合NUC时易于监测荧光变化Leotide：在没有Mg〜（2 +）的情况下，在结合ATP时，在330nm时观察到25-30％的荧光发射强度下降25-30％;在Mg〜（2 +）存在下，发射光谱乳酸〜（F455W）：ATP复合物被5nm的红色移位，并且相对于未复合的Chea〜（F455W）的增长强度（345nm以345nm高〜20％〜20％）。当两个ATP时观察到不同的荧光变化。使用类似物（ADPNP和ADPCP）代替ATP，使用Mn〜（2+）或Ca〜（2+）代替Mg〜（2 +）。我们利用Mg〜（2+）诱导的荧光变化）-ATP探讨Chea〜（F455W）的核苷酸结合的动力学和机制。在没有Mg〜（2 +）的情况下，粘合似乎涉及简单的一步平衡（K_ASSn = 0.7毫米〜（-1） S〜（-1）和k_dissn = 270 s〜（-1）在4℃）。在mg〜（2 +）的存在下，结合机制涉及至少两个步骤：（i）快速，相对较弱的结合其次是（ii）快速可逆的步骤（k_forward = 300 s〜（-1）和k_reverse = 15 s〜（-1），在4 deg c）这提高了复合物的总体亲和力并产生了W455荧光的增加。这第二步可以反映Chea Active部位的构象变化，例如盖闭合。这些结果提供了对核苷酸结合和基质动态的第一洞察力 - 蛋白质组氨酸激酶的活性位点诱导构象变化。

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  • 来源
    《Biochemistry》 |2005年第11期|共11页
  • 作者

    Richard C.Stewart;

  • 作者单位

    Department of Cell Biology &

    Molecular Genetics University of Maryland College Park Maryland 20742;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
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