Mutational Analysis of Hemoglobin Binding and Heme Utilization by a Bacterial Hemoglobin Receptor
Mutational Analysis of Hemoglobin Binding and Heme Utilization by a Bacterial Hemoglobin Receptor
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DOI：
10.1128/jb.00199-13
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发表时间：
2013-07-01
影响因子：
3.2
通讯作者：
Leduc, I.
中科院分区：
文献类型：
--
作者：
Fusco, W. G.;Choudhary, N. R.;Leduc, I.
铁是大多数生物体的必需营养素。为了从它们的环境中获得铁，革兰氏阴性细菌使用TonB依赖性转运蛋白，该转运蛋白在细菌表面结合宿主蛋白，并通过Ton机制将铁或血红素转运到周质。TonB依赖性转运蛋白是具有22个跨膜结构域、11个表面暴露环和堵塞孔的塞子结构域的桶形外膜蛋白。为了鉴定参与血红蛋白结合和血红素转运的TonB依赖性转运蛋白的关键残基，从而定位推定的保护性表位，杜克雷嗜血杆菌HgbA的血红蛋白受体被用作从血红蛋白获得铁/血红素的模型。虽然HgbA的所有胞外环都是H. ducreyi使用血红蛋白作为铁/血红素的来源，我们以前证明了血红蛋白结合HgbA只涉及环5和7。使用缺失、取代和定点突变，我们能够区分HgbA的血红蛋白结合和血红素获取。环5的GYEAYNRQWWA区域的缺失或取代和所选组氨酸的丙氨酸取代影响HgbA与血红蛋白的结合。相反，环7 FRAP结构域中苯丙氨酸的突变或环5的NRQWWA基序的取代显著废除了血红蛋白中血红素的利用。我们的研究结果表明，细菌血红蛋白受体的血红蛋白结合和血红素利用涉及特定的基序HgbA。
Iron is an essential nutrient for most living organisms. To acquire iron from their environment, Gram-negative bacteria use TonB-dependent transporters that bind host proteins at the bacterial surface and transport iron or heme to the periplasm via the Ton machinery. TonB-dependent transporters are barrel-shaped outer membrane proteins with 22 transmembrane domains, 11 surface-exposed loops, and a plug domain that occludes the pore. To identify key residues of TonB-dependent transporters involved in hemoglobin binding and heme transport and thereby locate putative protective epitopes, the hemoglobin receptor of Haemophilus ducreyi HgbA was used as a model of iron/heme acquisition from hemoglobin. Although all extracellular loops of HgbA are required by H. ducreyi to use hemoglobin as a source of iron/heme, we previously demonstrated that hemoglobin binding by HgbA only involves loops 5 and 7. Using deletion, substitution, and site-directed mutagenesis, we were able to differentiate hemoglobin binding and heme acquisition by HgbA. Deletion or substitution of the GYEAYNRQWWA region of loop 5 and alanine replacement of selected histidines affected hemoglobin binding by HgbA. Conversely, mutation of the phenylalanine in the loop 7 FRAP domain or substitution of the NRQWWA motif of loop 5 significantly abrogated utilization of heme from hemoglobin. Our findings show that hemoglobin binding and heme utilization by a bacterial hemoglobin receptor involve specific motifs of HgbA.