HbS-Savaria: The Anti-polymerization Effect of a Single Mutation in Human α-chains

Abstract

Recombinant alpha-Savaria globin (alpha(S49R)) was assembled with beta(S) chains by the alloplex intermediate pathway to generate tetrameric rHbS-Sarvaria (alpha (2) (S49R) beta (2) (E6V) ) that exhibited normal O(2) affinity and co-operatively at pH 7.4. Allosteric effectors, 2,3-DPG, L35, and NaCl increased O(2) affinity by 15%. Bohr effects were similar for rHbS-Savaria and HbS (0.38 +/- 0.025 vs. 0.46 +/- 0.03, respectively). The C(SAT) of HbS increased from 16.7 +/- 0.8 to 27.0 +/- 1.0 g/dL. Co-polymerization demonstrated inhibition predominantly by the Cis-dimer. Molecular modeling indicated that the positive charge at alpha-49 generated a strong anion-binding site and reduced flexibility of the CD-region by restricting movement in the E and F helices. The molecular distance between Arg-49 and Asn-78 in the neighboring double strand decreased, and electrostatic repulsion between the inter-double strands increased, resulting in inhibition of polymerization. The Savaria mutation may be useful for the design of super-inhibitory alpha-chains and gene therapy of sickle cell anemia.

Structural basis for the antipolymer activity of Hbζ2βs2trapped in a tense conformation

“…The data anticipate that the polymer-stabilizing interactions mediated by amino acids in these two positions will be fully ablated by α→ζ subunit exchange (Table 2). These predictions are consistent with the properties of the naturally occurring variant a Memphis (α Glu →Glu ; acidic→neutral polar), which significantly mitigates the phenotype of patients with SCD, lowers their whole-blood viscosity, and reduces gelling of hemoglobin purified from their red-blood cells [54,55]; and also with the properties of α Savaria (α Ser49→Arg ; neutral→basic), which nearly doubles the saturation concentration (C Sat ) for Hb S in solution [19]. While the antipolymer activities of both the α23 and α49 substitutions might be more substantial if they were not repositioned (juxtaposing hydrophobic and hydrophilic side chains), it is clear that these two amino-acid changes ablate separate intermolecular contacts that are critical to deoxyHb S polymer stability.…”

“…The α→ζ substitution that creates Hb ζ 2 β s 2 alters both the position as well as the biochemical identities of two residues that have been implicated in deoxyHb S polymer structure: α1/α2Glu23→ζ1/ζ2Thr23 (acidic→neutral polar; 1.3Å) and α2Ser49→ζ2His49 (neutral→basic; 1.7Å) [9,19,54,55]. The data anticipate that the polymer-stabilizing interactions mediated by amino acids in these two positions will be fully ablated by α→ζ subunit exchange (Table 2).…”

Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility

“…McCune et al (62) generated an Hb S mutant with the addition of ␤Glu-22 3 Ala and ␤Asn-80 3 Lys substitutions. This mutant is better than Hb A but less effective than Hb F in inhibiting the polymerization of Hb S. Srinivasulu et al (63) substituted the ␣Ser-49 on Hb S with Arg and observed a 60% increase in solubility. However, the contribution of ␣ 2 His-50 in stabilizing the Hb S polymer has never been tested.…”

An α-chain modification rivals the effect of fetal hemoglobin in retarding the rate of sickle cell fiber formation

“…A number of other α chain mutants are known that inhibit fiber formation, including Hb Twin Peaks (L113H) 14 , Hb Stanleyville II (N78K) 15 , Hb Sealy (D47H) 16 , Hb Savaria (S49R) 17 , Hb J-Oxford (G15D) 16 and Hb La Lamentin (H20G) 18 . A subtle but important feature of the Chiapas mutation site is that it interferes with polymer formation in either of the 2 possible arrangements.…”