Inhibition of hemoglobin S polymerization in vitro by a novel 15-mer EF-helix beta73 histidine-containing peptide.

Hb S is a naturally occurring mutation of human tetrameric hemoglobin in which the β subunits have a hydrophobic Val in place of a negatively charged Glu at the β6 position. The consequence of this mutation is that solubility of Hb S decreases when oxy Hb S loses oxygen. When deoxy Hb S becomes oversaturated deoxy Hb S assembles into long, multi-stranded fibers under physiological conditions (1, 2). Fiber formation is characterized by a delay time prior to polymerization, which is explained by homogeneous and heterogeneous nucleation. During oversaturated Hb S conditions deoxy Hb S monomers form very small polymers by homogeneous nucleation, and these polymers grow by the end addition of hemoglobin molecules in solution. The surface of these growing fibers also can serve as heterogeneous nucleation sites for further growth of additional polymers (1, 3). The polymer then assembles into 14-stranded fibers, which finally form a viscous gel. Intracellular polymers or fibers cause reduction in red blood cell deformability (sickling), leading to obstruction of flow in the microcirculation, thus creating vasooclusion and a wide array of physiological problems including episodes of painful crises (1, 4). Computational refinements of x-ray-determined crystal structures clarified the details of many of the axial and lateral contacts in Hb S polymers (5). These results and properties of the βS chain and of βS peptides (6, 7) show a slight hinge-like motion of the A helix in the βS-globin subunits that makes intermolecular contact with the adjacent Hb S tetramer. Crystal structural analysis of Hb S in the deoxy form also showed not only β6Val in a largely hydrophobic acceptor pocket, but other contact sites involving a hydrogen bond between β4Thr and β73Asp in Hb S which play a critical role in protein interactions. The β4 and β73 positions are located near β6Val and the EF helix acceptor sites in the β subunit, respectively, which are critical lateral contact regions in Hb S polymerization (1, 5, 8). In addition, recent advances in understanding the molecular and cellular pathophysiology of sickle cell disease, coupled with new insights into the developmental regulation of human globin-gene expression as well as characterization of Hb S polymerization, have provided the scientific impetus and clinical rationale to attempt augmentation of the production of Hb F (4). Furthermore, rational drug design (also known as structure-based drug design) through the use of computer modeling and results of x-ray diffraction is an emerging approach that is revolutionizing the practice of drug discovery. The first anti-sickling molecules designed from receptor-based molecular modeling, the substituted benzaldehyde BW12C (now referred to as 12C79), were described by Beddell et al (9). This drug has reached clinical trials, but analysis of the covalent complex of 12C79 and Hb A showed that this agent binds to the N-terminal amino group rather than the direct interaction sites of Hb S polymers including the β6Val donor site, β85Phe and β88Leu hydrophobic acceptor sites or the β4Thr-β73Asp hydrogen bond. Our mutational studies on Hb S polymerization showed that the Hb S β73His variant (β6Val and β73His) promoted polymerization compared to deoxy Hb S, while Hb S β73Leu (β6Val and β73Leu) inhibited polymerization like naturally occurring deoxy Hb C-Harlem (β6Val and β73Asn) (10). These results suggest that the β73 position (Asp in Hb S) serves as a unique site to promote or inhibit polymerization by amino acid replacement (10). Kinetics of polymerization, solubility and minimum concentration required for polymerization of the Hb S β73 variants were affected by β73 amino acid (inhibition of polymerization: His < <Asp <<Asn < Leu). Inhibition of Hb S β73Leu polymerization compared to Hb S may be caused by weakening of the hydrogen bond interaction between the β4Thr hydroxyl group and the β73 amino acid like Hb C-Harlem (10). Furthermore, kinetics of polymerization of 1:1 Hb S/Hb A β73His mixtures were enhanced like Hb S β73His compared to AS mixtures. These results suggest that the Hb A β73His variant promotes Hb S polymerization almost as efficiently as Hb S β73His, and that β73His in Hb A and Hb S variants strengthens the hydrogen bond with β4Thr, which facilitates formation of domains and 14-stranded fibers. In addition, our results suggested that altering hydrogen bond formation between β73Asp and β4Thr produces Hb S molecules with different properties, and that decreasing hydrogen bond formation inhibited polymerization without changing significantly tetramer solubility (11). Based on these results, we hypothesized that EF-helix peptides containing β73His would interact with β4Thr in Hb S and disturb protein–protein interactions of Hb S tetramers, resulting in inhibition of Hb S polymerization. We, therefore, synthesized several EF-helix peptides containing β73His of different length, and then evaluated their effects on Hb S polymerization. We also studied effects of these peptides on domain formation, elongation of Hb S polymers using differential interference contrast (DIC) microscopy and performed kinetics, thermodynamic and computer docking studies, comparing and contrasting results of the β73His peptide to those of the β73Leu peptide.