Inhibition of Hemoglobin S Polymerization in Vitro by a Novel 15-mer EF-Helix β73 Histidine-Containing Peptide

Abstract

Our mutational studies on HbS showed that the HbS β73His variant (β6Val and β73His) promoted polymerization, while HbS β73Leu (β6Val and β73Leu) inhibited polymerization. Based on these results, we speculated that EF-helix peptides containing β73His interact with β4Thr in HbS and compete with HbS, resulting in inhibition of HbS polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-and 3-mer EF-helix peptides containing β73His on HbS polymerization. The delay time prior to HbS polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed fiber elongation rate for HbS polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer-peptide containing β73Leu instead of His and peptides shorter than 11 amino acids containing β73His including His alone showed little effect on kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer β73His peptide interacted with HbS. CD spectra of the 15-mer β73His peptide did not show a specific helical structure, however, computer docking analysis suggested a lower energy for interaction of HbS with the 15-mer β73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer β73His peptide interacts with HbS via the β4Thr in the β S -globin chain in HbS. This interaction may influence hydrogen bond interaction between β73Asp and β4Thr in HbS polymers and interfere in hydrophobic interactions of β6 Val leading to inhibition of HbS polymerization.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 th...

Role of the β4Thr−β73Asp Hydrogen Bond in HbS Polymer and Domain Formation from Multinucleate-Containing Clusters

“…An understanding of the initiation of polymerization, including the process of multifiber formation in a single domain containing double- or 14-stranded fibers of HbS or its variants, based on protein−protein interactions of HbS polymers is critical to gaining a full understanding of polymer formation in SCD and in designing structure-based anti-HbS polymerization hemoglobins and peptides , . This knowledge also may improve our understanding of diseases in which protein fibers are formed (e.g., cataracts, Alzheimer’s, Huntington’s, and prion diseases) ,− .…”

Relationship between β4 hydrogen bond and β6 hydrophobic interactions during aggregate, fiber or crystal formation in oversaturated solutions of hemoglobin A and S

“…Such knowledge will lead to a better understanding of deoxy-HbS fiber formation with domains and oxy-HbC crystal formation in vivo , which may aid in the design of structure-based anti-HbS polymerization molecules, including hemoglobin variants and peptides based on protein-protein interactions of HbS polymers [30, 31]. …”

Rational Drug Design of Peptide-Based Therapies for Sickle Cell Disease

“…The β73His 15-mer peptide is believed to interact with β4Thr and thus disrupt the hydrogen bonding between β4Thr and β'73Asp, and also hydrophobic interactions involving β6Val due to its spatial proximity. However, it should be mentioned that a peptide/HbS ratio of 3:1 was needed to obtain a noteworthy delay in HbS polymerization [131]. While it is likely that different hemoglobin binding sites were employed by these peptides, they represent about 70% improvement in potency over the peptides studied in earlier works [121,122,127].…”

“…Akbar et al studied the effects of 15-, 11-, 7-, and 3-mer peptides derived from one of the helices of the β-globin chain of hemoglobin. In the case of the 15-mer peptide, the sequence comprised the β-globin residues 65−79 with sequence KKVLGAFS[H/L]GLAHLD, where, at position 73, the β73His and β73Leu mutations were included instead of the native β73Asp, as, in HbS, these mutations were previously observed to inhibit HbS aggregation [131]. The shorter peptides with 3, 7, and 11 residues failed to inhibit polymerization, suggesting the importance of secondary structure and multiple contact points for the observed inhibitory activity.…”