Yamaguchi T, Pang J, Reddy K S, Surrey S, Adachi K
Children's Hospital of Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1998 Jun 5;273(23):14179-85. doi: 10.1074/jbc.273.23.14179.
In order to assess the role of beta112 Cys in homo- and hetero-tetrameric hemoglobin formation, we expressed four beta112 variants (beta112Cys-->Asp, beta112Cys-->Ser, beta112Cys-->Thr, and beta112Cys-->Val) and studied assembly with alpha chains in vitro. beta112 Cys is normally present at beta1 beta2 and alpha1 beta1 interaction sites in homo- (beta4) and hetero-tetramers (alpha2 beta2). beta4 formation in vitro was influenced by the amino acid at beta112. beta112 Asp completely inhibited formation of homo-tetramers, whereas beta112 Ser showed only slight inhibition. In contrast, beta112 Thr or Val enhanced homo-tetramer formation compared with betaA chains. Association constants for homo-tetramer formation increased in the order of beta112Cys-->Ser, betaA, beta112Cys-->Thr, and beta112Cys-->Val, whereas the value for beta112Cys-->Asp was zero under the same conditions. These beta112 changes also affected in vitro alpha2 beta2 hetero-tetramer formation. Order of alpha2 beta2 formation under limiting alpha-globin chain conditions showed Hb betaC112S > Hb A > Hb S = Hb betaC112T = Hb betaC112V >>> Hb betaC112D. Hb beta112D can form tetrameric hemoglobin, but this beta112 change promotes dissociation into alpha and beta chains instead of alpha beta dimer formation upon dilution. These results indicate that amino acids at alpha1 beta1 interaction sites such as beta112 on the G helix play a key role in stable alpha beta dimer formation. Our findings suggest, in addition to electrostatic interaction between alpha and beta chains, that dissociation of beta4 homo-tetramers to monomers and hydrophobic interactions of the beta112 amino acid with alpha chains governs stable alpha1 beta1 interactions, which then results in formation of functional hemoglobin tetramers. Information gained from these studies should increase our understanding of the mechanism of assembly of multi-subunit proteins.
为了评估β112位半胱氨酸在同型和异型四聚体血红蛋白形成中的作用，我们表达了四种β112变体（β112半胱氨酸→天冬氨酸、β112半胱氨酸→丝氨酸、β112半胱氨酸→苏氨酸和β112半胱氨酸→缬氨酸），并在体外研究了它们与α链的组装情况。β112半胱氨酸通常存在于同型四聚体（β4）和异型四聚体（α2β2）的β1β2和α1β1相互作用位点。体外β4的形成受β112位氨基酸的影响。β112天冬氨酸完全抑制同型四聚体的形成，而β112丝氨酸仅表现出轻微抑制。相反，与βA链相比，β112苏氨酸或缬氨酸增强了同型四聚体的形成。同型四聚体形成的缔合常数按β112半胱氨酸→丝氨酸、βA、β112半胱氨酸→苏氨酸和β112半胱氨酸→缬氨酸的顺序增加，而在相同条件下β112半胱氨酸→天冬氨酸的值为零。这些β11位点的变化也影响体外α2β2异型四聚体的形成。在α-珠蛋白链有限的条件下，α2β2形成的顺序为：HbβC112S＞Hb A＞Hb S = HbβC112T = HbβC112V＞＞HbβC112D。Hbβ112D可以形成四聚体血红蛋白，但这种β112位点的变化促进其在稀释时解离为α链和β链，而不是形成αβ二聚体。这些结果表明，G螺旋上如β112位的α1β1相互作用位点的氨基酸在稳定的αβ二聚体形成中起关键作用。我们的研究结果表明，除了α链和β链之间的静电相互作用外，β4同型四聚体解离为单体以及β112位氨基酸与α链的疏水相互作用决定了稳定的α1β1相互作用，进而导致功能性血红蛋白四聚体的形成。从这些研究中获得的信息应该会增加我们对多亚基蛋白质组装机制的理解。
