Crystal Structure of Deoxy-Human Hemoglobin β6 Glu → Trp

Abstract

An atomic-level understanding of the interactions between hemoglobin molecules that contribute to the formation of pathological fibers in sickle cell disease remains elusive. By exploring crystal structures of mutant hemoglobins with altered polymerization properties, insight can be gained into sickle cell hemoglobin (HbS) polymerization. We present here the 2.0-Å resolution deoxy crystal structure of human hemoglobin mutated to tryptophan at the ␤6 position, the site of the glutamate 3 valine mutation in HbS. Unlike leucine and isoleucine, which promote polymerization relative to HbS, tryptophan inhibits polymerization. Our results provide explanations for the altered polymerization properties and reveal a fundamentally different double strand that may provide a model for interactions within a fiber and/or interactions leading to heterogeneous nucleation.Sickle cell disease is caused by the mutation of human hemoglobin (HbA) 1 at the sixth position of the ␤ chain from glutamate to valine (1). The consequence of this single amino acid substitution is a drastically reduced solubility of deoxygenated sickle cell hemoglobin (HbS) leading to the formation of long polymers that distort and rigidify the normally pliable erythrocytes. Such sickled cells can occlude the capillaries of the microcirculation and decrease the oxygen supply to the surrounding tissue, which is believed to be what is responsible for the clinical manifestations of sickle-cell disease.Structural analysis of HbS fibers using single crystal x-ray diffraction, fiber x-ray diffraction, and electron microscopy, as well as complementary information from gelation experiments, has established the basic fiber architecture (2). Crystals of deoxy-HbS grown in low salt at low pH (5.0 to 6.0) display a monoclinic space group in which hemoglobin molecules are assembled in double strands (3). Axial contacts within each strand and lateral contacts between strands that involve the mutated ␤6 Val stabilize these "Wishner-Love" double strands (3, 4). Structural details of the double strand interactions have been elucidated from the refined structure of HbS at 2.05-Å resolution (5). X-ray fiber diffraction data, from both extracellular gels and sickled erythrocytes, are consistent with a similar arrangement of molecules in the crystal and fiber (6). Electron microscopy with image reconstruction has revealed that the basic HbS fiber is 210-Å thick and formed from 14 filament strands that associate as half-staggered pairs (7). Although some disagreements exist as to the arrangement of the double strands within the HbS fiber (8, 9), there is broad agreement that the basic building block of the fiber is a Wishner-Love double strand with a slight helical twist. Additional convincing evidence that the crystalline double strand is physiologically relevant comes from copolymerization studies of HbS with naturally occurring variants (10, 11). These experiments show significant changes in polymerization properties as a result of altering residues that participate in the cr...

Structure of Sickle Cell Hemoglobin Fibers Probed with UV Resonance Raman Spectroscopy

“…Polymerization studies using engineered Hb S mutants, in which β85 Phe and β88 Leu have been mutated to Glu and Ala, have shown that hydrophobicity and steric constraints of the donor−acceptor interaction are important factors in the polymerization process − (Figure ). In the current investigation the relative hydrophobicity of the acceptor pocket is directly monitored through the β85 Phe residue.…”

In-Silico structural analysis of wild-type human hemoglobin and its mutation resulting in sickle cell anemia

“…The unique contacts were formed by tetramer 1 and 2 of HbA. 16 The three-dimensional visualization of ribbon structure of hemoglobin protein and its mutated version is displayed using RasMol (figure. 5a and 5b).…”

Aryloxyalkanoic Acids as Non-Covalent Modifiers of the Allosteric Properties of Hemoglobin

“…The polymerization process is exacerbated by the low oxygen affinity of Hb S, presumably as a result of unusually high concentration of 2,3-diphosphoglycerate and/or sphingosine phosphate in sickle red blood cells (RBC) [7–10]. The polymer is initiated by a primary interaction between the pathological β2Val6 from one Hb S molecule and a hydrophobic acceptor pocket in the region of β1Ala70, β1Phe85 and β1Leu88 of another Hb molecule, and further stabilized by several secondary contacts between the Hb molecules [11–15]. Disruption or weakening of the secondary contacts is known to reduce Hb S polymerization and RBC sickling as demonstrated by a large number of naturally occurring mutations that are known to mitigate the severity of SCD [16,17].…”