Modification of axial fiber contact residues impact sickle hemoglobin polymerization by perturbing a network of coupled interactions.
The identity of intermolecular contact residues in sickle hemoglobin (HbS) fiber is largely known. However, our knowledge about combinatorial effects of two or more contact sites or the mechanistic basis of such effects is rather limited. Lys16, His20, and Glu23 of the alpha-chain occur in intra-double strand axial contacts in the sickle hemoglobin (HbS) fiber. Here we have constructed two novel double mutants, HbS (K16Q/E23Q) and (H20Q/E23Q), with a view to delineate cumulative impact of interactions emanating from the above contact sites. Far-UV and visible region CD spectra of the double mutants were similar to the native HbS indicating the presence of native-like secondary and tertiary structure in the mutants. The quaternary structures in both the mutants were also preserved as judged by the derivative UV spectra of liganded (oxy) and unliganded (deoxy) forms of the double mutants. However, the double mutants displayed interesting polymerization behavior. The polymerization behaviour of the double mutants was found to be non-additive of the individual single mutants. While HbS (H20Q/E23Q) showed inhibitory effect similar to that of HbS (E23Q), the intrinsic inhibitory propensity of the associated single mutants was totally quelled in HbS (K16Q/E23Q) double mutant. Molecular dynamics (MD) simulations studies of the isolated alpha-chains as well as a module of the fiber containing the double and associated single mutants suggested that these contact sites at the axial interface of the fiber impact HbS polymerization through a coupled interaction network. The overall results demonstrate a subtle role of dynamics and electrostatics in the polymer formation and provide insights about interaction-linkage in HbS fiber assembly.
Banerjee
Srijita
S
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110 067, India.
Mirsamadi
Neda
N
Anantharaman
Lavanya
L
Sivaram
Mylavarapu V S
MV
Gupta
Rasik B
RB
Choudhury
Devapriya
D
Roy
Rajendra P
RP
D001665:Binding Sites; D002942:Circular Dichroism; D019281:Dimerization; D006451:Hemoglobin, Sickle / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D006451:Hemoglobin, Sickle