Linkage of interactions in sickle hemoglobin fiber assembly: inhibitory effect emanating from mutations in the AB region of the alpha-chain is annulled by a mutation at its EF corner.
The AB and GH regions of the alpha-chain are located in spatial proximity and contain a cluster of intermolecular contact residues of the sickle hemoglobin (HbS) fiber. We have examined the role of dynamics of AB/GH region on HbS polymerization through simultaneous replacement of non-contact Ala(19) and Ala(21) of the AB corner with more flexible Gly or rigid alpha-aminoisobutyric acid (Aib) residues. The polymerization behavior of HbS with Aib substitutions was similar to the native HbS. In contrast, Gly substitutions inhibited HbS polymerization. Molecular dynamics simulation studies of alpha-chains indicated that coordinated motion of AB and GH region residues present in native (Ala) as well as in Aib mutant was disrupted in the Gly mutant. The inhibitory effect due to Gly substitutions was further explored in triple mutants that included mutation of an inter-doublestrand contact (alphaAsn(78) --> His or Gln) at the EF corner. Although the inhibitory effect of Gly substitutions in the triple mutant was unaffected in the presence of alphaGln(78), His at this site almost abrogated its inhibitory potential. The polymerization studies of point mutants (alphaGln(78) --> His) indicated that the inhibitory effect due to Gly substitutions in the triple mutant was synergistically compensated for by the polymerization-enhancing activity of His(78). Similar synergistic coupling, between alphaHis(78) and an intra-double-strand contact point (alpha16) mutation located in the AB region, was also observed. Thus, two conclusions are made: (i) Gly mutations at the AB corner inhibit HbS polymerization by perturbing the dynamics of the AB/GH region, and (ii) perturbations of AB region (through changes in dynamics of the AB/GH region or abolition of a specific fiber contact site) that influence HbS polymerization do so in concert with alpha78 site at the EF corner. The overall results provide insights about the interaction-linkage between distant regions of the HbS tetramer in fiber assembly.
Sudha
Rajamani
R
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Anantharaman
Lavanya
L
Sivaram
Mylavarapu V S
MV
Mirsamadi
Neda
N
Choudhury
Devapriya
D
Lohiya
Nirmal K
NK
Gupta
Rasik B
RB
Roy
Rajendra P
RP
D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000737:chemistry; D006451:Hemoglobin, Sickle / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006720:Homozygote; D006801:Humans; D008239:Lysine / Q000737:chemistry; D009154:Mutation; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011108:Polymers / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D001426:Bacterial Proteins; C082316:Epf protein, Streptococcus suis; D006451:Hemoglobin, Sickle; D011108:Polymers; D006639:Histidine; D014357:Trypsin; D008239:Lysine; D010100:Oxygen; D005998:Glycine