TY - JOUR
T1 - Interspecies hybrid HbS
T2 - Complete neutralization of Val6(β)-dependent polymerization of human β-chain by pig α-chains
AU - Rao, M. Janardhan
AU - Malavalli, Ashok
AU - Manjula, Belur N.
AU - Kumar, Ramesh
AU - Prabhakaran, Muthuchidambaran
AU - Sun, D. Philip
AU - Ho, Nancy T.
AU - Ho, Chien
AU - Nagel, Ronald L.
AU - Acharya, A. Seetharama
N1 - Funding Information:
This work was supported by NIH grants HL 38655 to R.L.N. and A.S.A. and HL-55435 to R.L.N., HL-24525 to C.H., and a Grant-in-Aid from The American Heart Association, National to A.S.A.
PY - 2000/7/28
Y1 - 2000/7/28
N2 - Interspecies hybrid HbS (α2/(P)β2/(S)), has been assembled in vitro from pig α-globin and human β(S)-chain. The (α2/(P)β2/(S)) retains normal tetrameric structure (α2β2) of human Hb and an O2 affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O2 affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The 1H-NMR spectroscopy detected distinct differences between the heme environments and α1β1 interfaces of pig Hb and HbS, while their α1β2 interfaces appear very similar. The interspecies hybrid (α2/(P)β2/(S)) resembles pig Hb; the pig β-chain dictated the conformation of the heme environment of the human α-subunit, and to the α1β1 interfaces of the hybrid. In the (α2/(P)β2/(S)) hybrid, β(S)-chain dictated the conformation of human heme environment to the pig α-chain in the hybrid; but the conformation of α1β1 interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the α1β1 interface conformation is identical to that of HbS. More important, the (α2/(P)β2/(S)) does not polymerize when deoxygenated; pig α-chain completely neutralizes the β(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig α-chain is higher when it is present in the cis α(P)β(S) dimer relative to that in a trans α(P)β(A) dimer. The semisynthetically generated chimeric pig-human and human-pig α-chains by exchanging the α1-30 segments of human and pig α-chains have established that the sequence differences of pig α31-141 segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the α-chain surfaces of HbS and α2/(P)β2(S) suggests that the differences in electrostatic potential energy surfaces on the α-chain of α2/(P)β2(S) relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig α-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric α-chains can serve as blueprints for the design of a new generation of variants of α-chain(s) suitable for the gene therapy of sickle cell disease. (C) 2000 Academic Press.
AB - Interspecies hybrid HbS (α2/(P)β2/(S)), has been assembled in vitro from pig α-globin and human β(S)-chain. The (α2/(P)β2/(S)) retains normal tetrameric structure (α2β2) of human Hb and an O2 affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O2 affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The 1H-NMR spectroscopy detected distinct differences between the heme environments and α1β1 interfaces of pig Hb and HbS, while their α1β2 interfaces appear very similar. The interspecies hybrid (α2/(P)β2/(S)) resembles pig Hb; the pig β-chain dictated the conformation of the heme environment of the human α-subunit, and to the α1β1 interfaces of the hybrid. In the (α2/(P)β2/(S)) hybrid, β(S)-chain dictated the conformation of human heme environment to the pig α-chain in the hybrid; but the conformation of α1β1 interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the α1β1 interface conformation is identical to that of HbS. More important, the (α2/(P)β2/(S)) does not polymerize when deoxygenated; pig α-chain completely neutralizes the β(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig α-chain is higher when it is present in the cis α(P)β(S) dimer relative to that in a trans α(P)β(A) dimer. The semisynthetically generated chimeric pig-human and human-pig α-chains by exchanging the α1-30 segments of human and pig α-chains have established that the sequence differences of pig α31-141 segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the α-chain surfaces of HbS and α2/(P)β2(S) suggests that the differences in electrostatic potential energy surfaces on the α-chain of α2/(P)β2(S) relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig α-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric α-chains can serve as blueprints for the design of a new generation of variants of α-chain(s) suitable for the gene therapy of sickle cell disease. (C) 2000 Academic Press.
KW - Antisickling globins
KW - Gene therapy
KW - Hybrid Hb
KW - Molecular modeling
KW - Semisynthetic chimeric α-chains
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U2 - 10.1006/jmbi.2000.3898
DO - 10.1006/jmbi.2000.3898
M3 - Article
C2 - 10903876
AN - SCOPUS:0034725526
SN - 0022-2836
VL - 300
SP - 1389
EP - 1406
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 5
ER -