Chakraborty Ranjan, Lemke Edward A, Cao Zenghua, Klebba Phillip E, van der Helm Dick
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA.
Biometals. 2003 Dec;16(4):507-18. doi: 10.1023/a:1023485632520.
Many gram-negative bacteria produce and excrete siderophores, which complex iron with high affinity in the environment. The ferric siderophore complexes are transported across the outer membrane by receptor proteins. This process requires energy and is TonB dependent and must involve conformational changes in the receptor proteins to allow the transport of the ferric siderophores from the extracellular binding site to the periplasm. There is a large variety in the structures, molecular weights and charges among the siderophores. It was therefore realized that when the sequences of the many different receptor proteins were compared, simultaneously, all identities and close similarities, found in this manner, could only be due to residues involved in the conformational changes and transport mechanism, common to all the proteins, and not be due to the specificity of ligand recognition. Once the crystal structures of FepA, FhuA and FecA became available, it was immediately clear that the sequence similarities which were found in the simultaneous alignment, were all localized in a few structural domains, which are identical in the three structures and can therefore be expected to be maintained in all the proteins in this family. One of these domains, tentatively named the lock region, consists of 10 residues with a central quadrupole formed by two arginines and two glutamates, from the plug region and the beta barrel. We mutated several of these residues in FepA. All showed normal binding in quantitative binding studies. Some showed normal transport as well, however, the majority showed moderate to severe defective transport with ferric enterobactin. The results therefore show the validity of the hypothesis that the simultaneous sequence alignment will select the residues involved in the transport function of the receptor proteins. In addition the results allow to relate the severity of the transport deficiency to be correlated with the structure of the lock region while it is also possible to propose a function of this region in the conformational changes of the protein during the transport of the ligand from the binding site to the periplasm.
许多革兰氏阴性菌会产生并分泌铁载体，铁载体在环境中能与铁高亲和力结合。铁载体-铁复合物通过受体蛋白穿过外膜。这一过程需要能量，且依赖TonB，并且必然涉及受体蛋白的构象变化，以使铁载体-铁复合物从细胞外结合位点转运至周质。铁载体在结构、分子量和电荷方面存在很大差异。因此人们认识到，当同时比较许多不同受体蛋白的序列时，以这种方式发现的所有相同性和紧密相似性，只能归因于所有蛋白质共有的、参与构象变化和转运机制的残基，而不是由于配体识别的特异性。一旦FepA、FhuA和FecA的晶体结构可用，立即清楚的是，在同时比对中发现的序列相似性都集中在几个结构域中，这三个结构中的这些结构域是相同的，因此可以预期在这个家族的所有蛋白质中都能保持。其中一个结构域，暂称为锁定区域，由10个残基组成，在塞子区域和β桶中有两个精氨酸和两个谷氨酸形成中心四极。我们对FepA中的几个此类残基进行了突变。在定量结合研究中，所有突变体都表现出正常结合。一些突变体也表现出正常转运，然而，大多数突变体在转运高铁肠杆菌素时表现出中度至严重的转运缺陷。因此，结果表明了这样一个假设的有效性，即同时进行序列比对将选择参与受体蛋白转运功能的残基。此外，结果还表明转运缺陷的严重程度与锁定区域的结构相关，同时也有可能提出该区域在配体从结合位点转运至周质过程中蛋白质构象变化中的功能。
