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首页> 外文期刊>Journal of bacteriology >Sequence Changes in the Ton Box Region of BtuB Affect Its Transport Activities and Interaction with TonB Protein
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Sequence Changes in the Ton Box Region of BtuB Affect Its Transport Activities and Interaction with TonB Protein

机译：BtuB的Ton盒区域的序列变化影响其转运活性和与TonB蛋白的相互作用

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Uptake of cobalamins by the transporter protein BtuB in the outer membrane of Escherichia coli requires the proton motive force and the transperiplasmic protein TonB. The Ton box sequence near the amino terminus of BtuB is conserved among all TonB-dependent transporters and is the only known site of mutations that confer a transport-defective phenotype which can be suppressed by certain substitutions at residue 160 in TonB. The crystallographic structures of the TonB-dependent transporter FhuA revealed that the region near the Ton box, which itself was not resolved, is exposed to the periplasmic space and undergoes an extensive shift in position upon binding of substrate. Site-directed disulfide bonding in intact cells has been used to show that the Ton box of BtuB and residues around position 160 of TonB approach each other in a highly oriented and specific manner to form BtuB-TonB heterodimers that are stimulated by the presence of transport substrate. Here, replacement of Ton box residues with proline or cysteine revealed that residue side chain recognition is not important for function, although replacement with proline at four of the seven Ton box positions impaired cobalamin transport. The defect in cobalamin utilization resulting from the L8P substitution was suppressed by cysteine substitutions in adjacent residues in BtuB or in TonB. This suppression did not restore active transport of cobalamins but may allow each transporter to function at most once. The uncoupled proline substitutions in BtuB markedly affected the pattern of disulfide bonding to TonB, both increasing the extent of cross-linking and shifting the pairs of residues that can be joined. Cross-linking of BtuB and TonB in the presence of the BtuB V10P substitution became independent of the presence of substrate, indicating an additional distortion of the exposure of the Ton box in the periplasmic space. TonB action thus requires a specific orientation for functional contact with the Ton box, and changes in the conformation of this region block transport by preventing substrate release and repeated transport cycles.
机译：大肠杆菌外膜中转运蛋白BtuB对钴胺素的吸收需要质子动力和跨周蛋白TonB。在所有依赖于TonB的转运蛋白中，BtuB氨基末端附近的Ton盒序列是保守的，并且是唯一已知的赋予运输缺陷型表型的突变位点，该突变可被TonB残基160处的某些取代所抑制。依赖TonB的转运蛋白FhuA的晶体结构表明，Ton盒附近的区域（其本身尚未分解）暴露于周质空间，并在结合底物时发生广泛的位置变化。完整细胞中的定点二硫键已用于显示BtuB的Ton框和TonB位置160周围的残基以高度定向和特定的方式彼此接近，从而形成BtuB-TonB异二聚体，该异二聚体会受到转运的刺激基质。在这里，用脯氨酸或半胱氨酸替代Ton box残基表明，残基侧链识别对于功能并不重要，尽管在七个Ton box位置中的4个位置用脯氨酸替代会破坏钴胺素的转运。 L8P取代引起的钴胺素利用缺陷被BtuB或TonB中相邻残基的半胱氨酸取代所抑制。这种抑制不能恢复钴胺素的主动转运，但可以使每个转运蛋白最多发挥功能一次。 BtuB中未偶合的脯氨酸取代显着影响了二硫键与TonB的键合模式，既增加了交联的程度，又转移了可连接的残基对。在存在BtuB V10P取代的情况下，BtuB和TonB的交联变得独立于底物的存在，表明Ton盒在周质空间中的暴露会发生其他扭曲。因此，TonB动作需要特定的方向才能与Ton盒进行功能性接触，并且此区域的构型变化会阻止底物释放和重复的运输周期，从而阻碍运输。

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