Histidine146 of Human Serum Albumin Plays a Prominent Role at the Interface of Subdomains IA and IIA in Allosteric Ligand Binding

Fatty acids are endogenous ligands of human serum albumin (HSA) that induce conformational changes and participate in allosteric ligand binding to HSA. In a previous study, we showed that, when myristate (MYR) is present, the binding of [C-14] ketoprofen (KP) to subdomain IA of HSA was increased, indicating that, when MYR binds to HSA, a new binding site in formed in that region. Meanwhile, an N-B transition has been reported to increase the binding of ligands at alkaline pH when the status of albumin is the B-conformer. Six histidine single mutants of HSA, H9A, H39A, H67A, H105A, H128A and H146A were produced and photolabeled with [C-14] KP at pH 6.5, 7.4 and 8.2 and the role of each histidine in causing the N-B transition induced allosteric ligand binding was examined. Cyanogen bromide cleavage of the photolabeled native HSA showed that subdomain IA was the site of the allosteric binding of KP at pH 8.2. From the photolabeling results, H146 was found to play a prominent role whilst H128 played little or no role in the allosteric binding. However, the remaining 4 mutants did not show a clear photolabeling pattern that was similar to either native HSA or H146A and, as a result, no firm conclusions can be made. An additional histidine mutant, H146I, was produced to confirm the results for H146A. A similar experiment using H146I showed that a benzene ring-like structure at position 146 is required for the allosteric ligand binding to occur. (C) 2011 IUBMB IUBMB Life, 63( 4): 277-285, 2011

脂肪酸是人类血清白蛋白（HSA）的内源性配体，它们诱导构象变化并参与变构配体与HSA的结合。在先前的一项研究中，我们表明，当存在肉豆蔻酸（MYR）时，[C - 14]酮洛芬（KP）与HSA的亚结构域IA的结合增加，这表明当MYR与HSA结合时，在该区域形成了一个新的结合位点。同时，据报道当白蛋白处于B构象状态时，在碱性pH下N - B转变会增加配体的结合。制备了六种HSA的组氨酸单突变体，H9A、H39A、H67A、H105A、H128A和H146A，并在pH 6.5、7.4和8.2下用[C - 14] KP进行光标记，研究了每个组氨酸在引起N - B转变诱导的变构配体结合中的作用。对光标记的天然HSA进行溴化氰裂解表明，在pH 8.2时亚结构域IA是KP变构结合的位点。从光标记结果来看，发现H146在变构结合中起显著作用，而H128在变构结合中作用很小或没有作用。然而，其余4种突变体没有显示出与天然HSA或H146A相似的清晰光标记模式，因此无法得出确定的结论。制备了另外一种组氨酸突变体H146I以确认H146A的结果。使用H146I进行的类似实验表明，位置146处的苯环样结构是发生变构配体结合所必需的。（C）2011国际生物化学与分子生物学联合会 《国际生物化学与分子生物学联合会生命》，63(4): 277 - 285，2011