Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

Takamasa Yamaguchi,Jian Pang,Konda S Reddy,Saul Surrey,Kazuhiko Adachi

doi:10.1074/jbc.273.23.14179

Takamasa Yamaguchi, Jian Pang + Show 3 more

Open Access

https://doi.org/10.1074/jbc.273.23.14179

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Abstract

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.

Highlights

In order to assess the role of ␤112 Cys in homo- and hetero-tetrameric hemoglobin formation, we expressed four ␤112 variants (␤112Cys[3] Asp, ␤112Cys[3] Ser, ␤112Cys[3] Thr, and ␤112Cys3Val) and studied assembly with ␣ chains in vitro. ␤112 Cys is normally present at ␤1␤2 and ␣1␤1 interaction sites in homo- (␤4) and hetero-tetramers (␣2␤2). ␤4 formation in vitro was influenced by the amino acid at ␤112. ␤112 Asp completely inhibited formation of homo-tetramers, whereas ␤112 Ser showed only slight inhibition
Hb ␤112D can form tetrameric hemoglobin, but this ␤112 change promotes dissociation into ␣ and ␤ chains instead of ␣␤ dimer formation upon dilution. These results indicate that amino acids at ␣1␤1 interaction sites such as ␤112 on the G helix play a key role in stable ␣␤ dimer formation
Our present results showing absence or trace amounts of disulfide dimer formation for all four of the ␤112 chain variants in contrast to ␤A and other ␤ chain variants, suggest that ␤1112 Cys and ␤2112 Cys are physically close together and that disulfide ␤ chain dimer formation is governed in part by these two cysteine residues

Summary

Introduction

In order to assess the role of ␤112 Cys in homo- and hetero-tetrameric hemoglobin formation, we expressed four ␤112 variants (␤112Cys[3] Asp, ␤112Cys[3] Ser, ␤112Cys[3] Thr, and ␤112Cys3Val) and studied assembly with ␣ chains in vitro. ␤112 Cys is normally present at ␤1␤2 and ␣1␤1 interaction sites in homo- (␤4) and hetero-tetramers (␣2␤2). ␤4 formation in vitro was influenced by the amino acid at ␤112. ␤112 Asp completely inhibited formation of homo-tetramers, whereas ␤112 Ser showed only slight inhibition. Hb ␤112D can form tetrameric hemoglobin, but this ␤112 change promotes dissociation into ␣ and ␤ chains instead of ␣␤ dimer formation upon dilution These results indicate that amino acids at ␣1␤1 interaction sites such as ␤112 on the G helix play a key role in stable ␣␤ dimer formation. In addition to electrostatic interaction between ␣ and ␤ chains, that dissociation of ␤4 homo-tetramers to monomers and hydrophobic interactions of the ␤112 amino acid with ␣ chains governs stable ␣1␤1 interactions, which 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. We showed that ␤112 Cys located at an ␣1␤1 interaction site on the G helix is critical for facilitating formation of stable ␣␤ dimers which form functional hemoglobin tetramers. We expressed four ␤112 variants and characterized effects of these changes on ␣2␤2 and ␤4 tetramer formation in vitro in order to further assess the role of ␤112 Cys on assembly of hemoglobin

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