High Resolution Tertiary and Quaternary Structural Changes in Deoxy and Liganded Hemoglobin E

Hemoglobin (Hb) E (β26 Glu→Lys), known to be more unstable than HbA in vitro, remains an enigma in terms of its contributions to red blood cell (RBC) pathophysiological mechanisms. EE individuals exhibit a mild, chronic anemia while HbE-β thalassemia individuals show a range of clinical manifestations, including high morbidity and death, often resulting from cardiac disease. To determine pathophysiological consequences originating from HbE, we created a transgenic mouse expressing exclusively human HbE with equivalent α- and β-chains [Chen et al., Blood 106(11):892a, 2005]. This mice model exhibited hematological characteristics similar to human EE individuals: microcytic RBC with low MCV, low MCH but normal MCHC; target RBC; mild anemia with low Hb, low HCT, elevated reticulocytes; and decreased osmotic fragility. These RBC parameters correlated with indications of mild RBC oxidative stress. Importantly, the transgenic mouse model allows us to obtain purified HbE for crystal growth and dynamic studies, without the presence of human HbA₂ (co-expressed in human HbE RBC and difficult to separate). We now present the low [PDB 1YVQ] and high salt [PDB 1YVT] liganded COHbE structures (1.8Å resolution) at or near physiological pH. The known in vitro instability of oxy HbE and its propensity for oxidation may be explained by the substitution of the β26 side-chain with Lys resulting in a positive charge repulsion with consequential changes to β117, and other key stabilizing residues at the α1β1 interface that impart normal association and oxidative stability to HbA. The specific alterations observed in this microenvironment may be the key to HbE instability, given the findings by Adachi et al. (