High-affinity binding of the staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded β-barrel fold
High-affinity binding of the staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded β-barrel fold
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DOI：
10.1128/jb.01366-06
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发表时间：
2007-01-01
影响因子：
3.2
通讯作者：
Nagy, Eszter
中科院分区：
文献类型：
--
作者：
Dryla, Agnieszka;Hoffmann, Bernd;Nagy, Eszter
从宿主体内清除铁对病原菌的生长至关重要。在这项研究中，我们进一步的特点是两个葡萄球菌细胞壁蛋白先前显示绑定血红素蛋白。HarA和IsdB具有同源的配体结合结构域，即所谓的NEAT结构域（"近转运蛋白"），存在于革兰氏阳性病原体的几种表面蛋白中。使用谷胱甘肽S-转移酶（GST）标记的HarAD 1（HarA的配体结合结构域之一）和GST标记的全长IsdB蛋白的表面等离子体共振测量证实了与血红蛋白和触珠蛋白-血红蛋白复合物的高亲和力结合，平衡解离常数（K-D）为5至50 nM。结合珠蛋白结合只能用HarA检测到，并且在低微摩尔范围内。为了确定这种进化上保守的配体结合结构域的折叠，对未标记的HarAD 1蛋白进行核磁共振光谱分析，其显示了一个八链，纯反平行β-桶链序（-beta 1向下箭头-beta 2向上箭头-beta 3向下箭头-beta 6向上箭头-beta 5向下箭头-beta 4向上箭头-beta 7向下箭头-beta 8向上箭头），形成了两个希腊主题基于结构同源性搜索，HarAD 1结构域的拓扑结构类似于免疫球蛋白（IG）折叠家族，其成员参与蛋白质-蛋白质相互作用，但具有不同的结构特征。因此，我们认为HarAD 1/NEAT结构域折叠是IG折叠的新变体，尚未在其他蛋白质中观察到。
Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for "near transporter") present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (K-D) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel beta-barrel with the strand order (-beta 1 down arrow -beta 2 up arrow -beta 3 down arrow -beta 6 up arrow -beta 5 down arrow- beta 4 up arrow -beta 7 down arrow -beta 8 up arrow), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.