RESTORING ALLOSTERISM WITH COMPENSATORY MUTATIONS IN HEMOGLOBIN
RESTORING ALLOSTERISM WITH COMPENSATORY MUTATIONS IN HEMOGLOBIN
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DOI：
10.1073/pnas.91.24.11547
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发表时间：
1994-11-22
影响因子：
11.1
通讯作者：
HO, C
中科院分区：
文献类型：
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作者：
KIM, HW;SHEN, TJ;HO, C
与正常人血红蛋白相比，α (1) β(2)界面氨基酸取代的异常人血红蛋白（Hbs）具有非常高的氧亲和力，并且在Oz结合中的协同性大大降低。在这些β 99位点突变的异常Hbs中，asp - β 99和tyr1 - α 42之间以及asp - β 99和asn - α 97之间的亚基间氢键被破坏，从而破坏了这些Hbs的去氧季元结构。利用分子动力学方法设计了这些Hbs中的代偿氨基酸替代，以恢复它们的变构特性。我们在自然发生的Hb突变Hb Kempsey （Asp-alpha 99—> Asn）中设计了一种代偿突变，并使用我们的大肠杆菌表达质粒pHE2产生了它。我们已经确定了重组双突变Hb， Hb（Asp- β 99—> Asn和tyrr - α 42—> Asp）的Oz结合特性，并使用H-1核磁共振光谱研究了血红素基团周围的三级结构和(α (1) β(2)亚基界面的四级结构。我们的研究结果清楚地表明，tyr - α 42 -> Asp取代可以有效地弥补Asp- β 99 -> Asn取代引起的Hb Kempsey功能缺陷。重组Hb的结构和功能信息提供了变构的结构基础和代偿性氨基酸取代的设计，以恢复与血红蛋白病相关的其他异常Hb的功能特性。
Abnormal human hemoglobins (Hbs) with amino acid substitutions in the alpha(1) beta(2) interface have very high oxygen affinity and greatly reduced cooperativity in Oz binding compared to normal human Hb. In such abnormal Hbs with mutations at position beta 99, the intersubunit hydrogen bonds between Asp-beta 99 and Tyr-alpha 42 and between Asp-beta 99 and Asn-alpha 97 are broken, thus destabilizing the deoxyquaternary structure of these Hbs. A molecular dynamics method has been used to design compensatory amino acid substitutions in these Hbs that can restore their allosteric properties. We have designed a compensatory mutation in a naturally occurring mutant Hb, Hb Kempsey (Asp-alpha 99 --> Asn), and have produced it using our Escherichia coli expression plasmid pHE2. We have determined the Oz binding properties of this recombinant double mutant Hb, Hb(Asp-beta 99 --> Asn and Tyr-alpha 42 --> Asp) and have used H-1 NMR spectroscopy to investigate the tertiary structures around the heme groups and the quaternary structure in the (alpha(1) beta(2) subunit interface. Our results clearly show that the Tyr-alpha 42 --> Asp replacement can substantially compensate for the functional defect of Hb Kempsey caused by the Asp-beta 99 --> Asn substitution. The structural and functional information derived from this recombinant Hb provides insights into the structural basis of allosterism and the design of compensatory amino acid substitutions to restore the functional properties of other abnormal Hbs associated with hemoglobinopathies.