Deoxyhemoglobin S
Created by Jessica Mackey
Deoxyhemoglobin S (2HBS) in Homo sapiens is a mutant form of human hemoglobin A that has a substitution at the sixth position of the Beta chain from a glutamic acid residue to a valine residue. The protein is also called sickle cell hemoglobin because the mutation causes Sickle Cell Disease. This mutation replaces a polar, acidic residue with a non-polar, hydrophobic residue (1). Wild type human hemoglobin is a soluble protein with both polar and non-polar characteristics. The resulting change in polarity created by the valine mutation reduces the proteins’ solubility in the deoxygenated state and promotes the polymerization of deoxyhemoglobin S tetramers. Hemoglobin is a globular transport protein; however, the mutation in Hemoglobin S leads to a change in the globular nature of the protein due to the formation of polymers, which is a fibrous protein characteristic.
Deoxyhemoglobin S is physiologically found in its polymerized structure in which hemoglobin tetramers bind in double stranded form. These polymers form long, rigid rods that result in the sickle cell phenotype. The polymer chains distort the wild type pliability of erythrocytes, which in turn hinders erythrocyte movement through the microcirculation (See Image 1). The rigidity of the erythrocytes can cause occlusion of the capillaries, leading to hypoxia and resulting in phenotypic sickle cell disease. These findings led researchers to categorize sickle cell disease as a “molecular disease” (1). Further understanding of the important residues involved in creating the double stranded polymers and the nature of the molecular interactions that stabilize mutant polymers can lead to important therapeutic insights in treating sickle cell disease (2). The molecular weight of Deoxyhemoglobin S is 123,728.25 Da, and the isoelectric point is 8.57.
Deoxyhemoglobin S is a tetramer with four main subunits, two alpha globin subunits and two beta globin subunits (1). The two alpha subunits are structurally similar to normal human hemoglobin while the two β subunits, which are the sites of the glutamic acid to valine residue substitution, have significant differences (2). The alpha subunits have identical primary structures of 141 amino acids and the β subunits have identical primary structures of 146 amino acids (8). Each subunit has one associated ligand, protoporphyrin IX containing Fe, or Heme. All four subunits contain a single Heme binding site that is responsible for oxygen transport; therefore, there are four heme molecules per hemoglobin tetramer. On the beta chain of deoxyhemoglobin S, the heme binding pocket doubles in function as the hydrophobic acceptor pocket for βVal-6 (1).
Deoxyhemoglobin S has a secondary structure that consists almost entirely of alpha helices separated and connected by random coils. No β sheets are present in the secondary structure. Each subunit, both alpha and beta, is made up of eight alpha helices (lettered A though H). The four subunits are bound tightly together through salt bridges and numerous hydrogen bonds and have significantly similar tertiary structures. The quaternary structure of deoxyhemoglobin S has two main conformations, similar to wild type human hemoglobin A. These conformations are defined as the T and R protein states. The R conformation is the open, mobile state that keeps the heme binding pocket open and unhindered by salt bridges and hydrogen bonds. The T state is constricted and is stabilized by salt bridges between the heme binding pockets at the distal end of the alpha chain. It is this T conformation that is responsible for decreased oxygen affinity in normal Hemoglobin A and promotes the polymerization of mutant hemoglobin S (8).
Two types of beta subunits are distinguished in Deoxyhemoglobin S: mutant valine acceptors or mutant valine donors. A beta subunit cannot act as both a donor and acceptor (1). Donating subunits are the subunits that have the βVal-6 while the accepting β subunits are the subunits that receive the βVal-6 residue in the hydrophobic binding pocket. The interaction between beta subunits drives and stabilizes polymerization of deoxyhemoglobin S physiologically. The βGlu-6 to βVal-6 mutation in Deoxyhemoglobin S leads to the reversible association of deoxyhemoglobin S tetramers into seven double strands with a slight helical twists, or fourteen-stranded polymers (3). The fourteen-stranded polymers associate as half-staggered pairs and twist around one another with an average helical pitch of 2,900 Å(5). The basic deoxyhemoglobin S fiber is 210 Å thick (1). These strands are extremely rigid and have the potential to form cross links which leads to the phenotypic “gelation” of sickle cell erythrocytes. Polymerization is driven by the association of βVal-6 on the A helix of a donor Beta subunit of one tetramer with a hydrophobic “binding pocket” on an adjacent tetramer’s E and F helices of the acceptor beta subunit (2).
Polymerization begins via homogenous nucleation in which deoxyhemoglobin S tetramers randomly form aggregates. As aggregates spontaneously form physiologically, the concentration of mutant deoxyhemoglobin S reaches a critical concentration that, once passed, leads to the formation of polymers. Once these polymers are formed, the rate of polymerization increases through heterogeneous nucleation. New nuclei (deoxyhemoglobin S tetramer aggregates) form preferentially on the polymer strands that already exist from homogeneous nucleation. Heterogeneous nucleation significantly increases the rate of polymerization, which is 80 times faster than homogenous nucleation, and leads to the severe phenotypes of sickle cell disease. If deoxyhemoglobin S tetramers could only polymerize through homogenous nucleation, the rigidity and cross linking found in sickle cell erythrocytes would be greatly reduced (3).
Tetramers interact through the βVal-6 residue of the A helix of one Beta chain and the hydrophobic binding pocket of the acceptor Beta chain on the E and F helices of an adjacent deoxyhemoglobin S molecule (5). The binding pocket of the acceptor Beta chain has two critical residues: βPhe-85 and βLeu-88. These two critical residues, as well as βVal-6, facilitate deoxyhemoglobin S polymerization. The stereospecificty and hydrophobicity of βPhe-85 and βLeu-88 in the βVal-6 binding pocket, as well as the βVal-6 residue, are critical for polymerization of deoxyhemoglobin S. Substitutions involving either of these residues lead to decreased binding affinity of the mutant βVal-6 residue or block its ability to bind completely (4). Association of these residues begins the polymerization process.
The basic building block of the deoxyhemoglobin S fiber is a “Wishner-Love” double strand with a single helical twist (1). These double strands are stabilized by two kinds of contacts: axial and lateral. Axial contacts occur within each strand (intra-strand contacts) while lateral contacts occur between strands (inter-strand contacts) and are the contacts that involve the βVal-6 residue. Both axial and lateral contacts provide stabilization for the polymer structure although lateral contacts provide greater stabilization than axial contacts (1). Lateral contacts define two important areas of structural difference between donor Beta subunits and acceptor Beta subunits. The structural differences are seen in the donor A helix and the acceptor EF corner region (1). The polymerization of deoxyhemoglobin S allows the acceptor pocket of one tetramer to bury the mutant βVal-6 within the lateral contact. This removes the hydrophobic residue from most solvent interactions. This lateral contact site has great hydrophobicity but also has a well-established network of ordered water molecules. These water molecules provide stabilization through hydrogen bonds by forming bridging hydrogen bonds between tetramers within the lateral contact. This illustrates that extensive hydrophilic interactions can also occur between deoxyhemoglobin S tetramers, regardless of the hydrophobic nature of the lateral contact. Although the major lateral contact is between βVal-6 and the corresponding acceptor pocket, other important lateral contacts exist.
βAsn-80 is an important residue in the polymerization process of deoxyhemoglobin S. In the deoxyhemoglobin S polymer, βAsn-80 and βAsp-79 form a lateral contact with αHis-50 and αSer-49. Disruption of the β80 position results in disrupted polymerization of deoxyhemoglobin S tetramers. βLys-95 is also involved in inter-double strand contacts; its terminal amino group is close to the terminal amino group of βLys-17 from the donor tetramer of another lateral contact (1). Finally, the pivot points for the movements of the A helices in the deoxyhemoglobin S polymer, which promotes its slight helical twist, are thought to be around the αLys-17 and βTrp-15 lateral contact (2).
The main βVal-6 lateral contacts which create heterogeneous nucleation are a subset of the fundamental contacts within the double strand. The double strand is created by two tetramers coming together and contacting one another through this lateral contact; however, it is now known that these contacts are also the same residues that are important for inter-fiber association and cross-linking. The externally available contact sites, the same contact sites that make up the internal lateral contacts, are able to bind double strands together and form more complex polymer aggregates. These inter-double strand bonds are implicated in being responsible for cross linking that creates the rigidity of the sickle cell erythrocyte (3).
Axial contacts in deoxyhemoglobin S polymers are similar to those found in other hemoglobin forms. These contacts are important for intra-strand binding, which creates the backbone for the more complex inter-strand polymerization. The two main axial contacts are located between tetramers translated vertically with respect to one another in the polymer strands (1). Each deoxyhemoglobin S tetramer has two axial contacts with its adjacent tetramer. The axial contact interface is made up of α-AB, α-GH, and β-GH corners from the bottom tetramer and the β-AB, β-GH, and α-GH corners from the tetramer on top (1). A corner is defined as the meeting points between two alpha helices within a subunit. This contact is defined by non polar interactions between αPro-114 and αAla-115 of one tetramer and the βHis-116, βHis-117 and βPhe-118 of the other. Two other residues, βLys-17 and βGlu-121, display different conformations depending on which tetramer interface they are found to be a part of. Recently, αLeu-113 has also been implicated in being involved in this axial contact. A study focused on αLeu-113 have shown that it has a significant impact on deoxyhemoglobin S polymerization rates. This study also reestablished that the ability of a single residue involved in inter-double strand (lateral) contact sites to inhibit polymerization is likely to be greater than those residues involved in intra-double strand (axial) contacts (6).
As mentioned previously, the acceptor pocket for βVal-6 occurs in the Heme binding pocket of deoxyhemoglobin S, which is composed of hydrophobic residues such as βPhe-85 and βLue-88. On the alpha subunits, the heme binding pocket is found at the distal histidine residue of the chain (1). Deoxyhemoglobin S also binds a hetertropic effector molecule, (2,3)-diphosphoglycerate (DPG). DPG binds more frequently to the T state of deoxyhemoglobin S, which is the state in which the tetramers form polymers. This effecter molecule is known to reduce oxygen binding affinity in the Hemoglobin S molecule, further elucidating the phenotypic effects of deoxyhemoglobin S structure in relation to sickle cell disease (8). The DPG binding pocket is able to form hydrogen bonds with water molecules associated with the alpha and beta subunits, further constricting the tetramer’s quaternary structure (1).
Extensive comparative studies have been conducted between the structures of wild type Hemoglobin A (PDB ID 4HBB) and deoxyhemoglobin S (2). Deoxyhemoglobin S has nearly identical alpha subunits when compared to Hemoglobin A, but has significant differences in beta subunits, primarily in the areas involved in lateral contacts (1). These differences are due to the mutant Val-6 residue on the beta subunit and the conformational changes it creates in the resulting polymerization. The similarity in primary structure is quantified by an E score of 3.0 x 10-100; this number indicates that the two hemoglobins' primary structures are significantly similar. Both Hemoglobin A and Hemoglobin S contain four subunits, each made up of eight alpha helices (2). HbA and HbS have significantly similar tertiary structures with a comparative Z score of 28.9 (9). Both hemoglobin proteins have T and R quaternary structural states, but only deoxyhemoglobin S forms fourteen stranded semi-helical polymer chains (8). Rather than resume the discussion of Hemoglobin A and Deoxyhemoglobin S, a more obscure protein will be used for comparison.
Myoglobin of Caretta caretta (PDB ID 1LHS) has a similar structure to the alpha subunit of deoxyhemoglobin S, but also has important differences. To have significant tertiary structural similarities, two comparative proteins must have a Z score greater than 2; Caretta caretta myoglobin has a Z score of 21.4 when compared with Deoxyhemoglobin S, indicating that the two have significantly similar tertiary structures (9). To indicate significant similarities in primary structure two proteins must have a comparative E value less than 0.5; myoglobin as an E score of 1.0 x 10-13, which indicates that it has significant similarities in primary structure compared to deoxyhemoglobin S. Myoglobin and Deoxyhemoglobin S have 48% sequence similarity. Myoglobin is made up of a single polypeptide chain with a primary structure of 153 amino acids. Like Deoxyhemoglobin S, myoglobin is entirely alpha helical in secondary structure. Myoglobin also contains a binding pocket for the protoporphyrin IX containing Fe ligand that is structurally identical to the protoporphyrin IX found in deoxyhemoglobin S. The myoglobin protein in Caretta caretta is responsible for oxygen transport throughout the turtle’s circulatory system as well as oxygen storage. Unlike Deoxyhemoglobin S, myoglobin from Caretta caretta contains only one subunit (7). It is much less complex in both structure and function when compared to deoxyhemoglobin S. Myoglobin is not a tetramer and does not form aggregates or complex polymers.
Deoxyhemoglobin S is an important protein for clinical research on the “molecular disease” of Sickle Cell anemia (1). The single mutation that changes a glutamic acid to a valine at the sixth position of the beta subunits causes an increase in hydrophobicity, a decrease in solubility and the creation of long aggregates of harmful polymers. Deoxyhemoglobin S is a tetramer that forms double stranded rod-like polymers through the interactions of the mutant beta subunits in two adjacent molecules. These polymers form due to the newly hydrophobic region of the beta chain and the resulting hydrophobic interactions with an adjacent beta subunit’s hydrophobic heme-binding pocket. The polymers also act to bury the mutant BVal-6 within the protein structure to prevent it from reacting with the surrounding solvent (1). The three most important residues in this process are βVal-6, βLeu-88 and βPhe-85. Polymerization is started through homogenous nucleation and then continued through heterogeneous nucleation. Because of heterogeneous nucleation, thick aggregates of deoxyhemoglobin S polymers form quickly; this leads to the “gelation” of deoxyhemoglobin S in erythrocytes (1). The polymer strands present in the erythrocyte cause its structural warping into the phenotypic “sickle” cell. These cells wreck havoc in the microcirculation and lead to severe occlusions, depriving tissue of oxygen. Specific residues important in polymerization are targeted by researchers to understand how to inhibit the formation of deoxyhemoglobin polymers and prevent erythrocyte deformation and Sickle Cell Disease course (6).