Enhanced polymerization of recombinant human deoxyhemoglobin beta 6 Glu----Ile.

Abstract

Polymerization ofthe deoxy form ofsickle cell hemoglobin (Hb S; 136 Glu-*Val) involves both hydrophobic and electrostatic intermolecular contacts. These interactions drive the mutated molecules into long fibrous rods composed of seven pairs of strands. X-ray crystallography of Hb S and electron microscopy image reconstruction of the fibers have revealed the remarkable complementarity between one of the 186 valines ofeach molecule (the donor site) and an acceptor site at the EF corner of a neighboring tetramer. This interaction constitutes the major lateral contact between the two strands in a pair. To estimate the relative importance of this key hydrophobic contact in polymer formation we have generated a modeling of the donor-acceptor interaction shows that the presence of an isoleucine side chain at the donor site induces increased contacts with the receptor site and an increased buried surface area, in agreement with the higher hydrophobicity of the isoleucine residue. The agreement between the predicted and experimental differences in solubility suggests that the transfer of the 136 valine or isoleucine side chain from water to a hydrophobic environment is sufficient to explain the observations.Sickle cell hemoglobin (Hb S) has been the subject of intense interest because the replacement of the charged glutamate residue at the sixth position (A3) in the 13 chains by an uncharged valine leads to a drastic decrease in the solubility of the mutant Hb in its deoxy form. This drives the mutated molecules into long fibrous rods, distorting the erythrocytes into their characteristic sickle shape (1, 2). The thermodynamic behavior and kinetics of polymerization have been studied in detail (for recent reviews see refs. 3 and 4). The long rods in sickled cells possess a highly ordered structure involving 14 helical strands of the mutated tetramers. X-ray analyses of the structure of the polymers (5) as well as molecular imaging studies (6-8) have provided a clear understanding of the mechanisms responsible for the formation of the polymers, which include both hydrophobic and electrostatic interactions between the abnormal tetramers.Three other natural mutations at the 136 position have been described: Hb C (Glu--Lys), Hb Machida (Glu-oGln) (9), and Hb G-Makassar (Glu--Ala) (10). None of these Hbs exhibit gelling properties similar to those of Hb S (10, 11). These reports strongly suggested that the localized change in hydrophobicity at the surface of the A helix in the 13 chains of Hb S is a major determinant in producing the oxygenlinked decrease in solubility of deoxy Hb S, leading to the formation of the polymers. Structural analyses of the fibers and computer molecular modeling have revealed the remarkable complementarity between the side chain of the 136 valine of one tetramer with an acceptor site constituted by the 185 phenylalanine and ,B88 leucine side chains of another tetramer, thereby explaining the primary intermolecular interaction in Hb S fibers. The solubility of the Hb S tetramers may be furth...

Properties of a recombinant human hemoglobin double mutant: Sickle hemoglobin with Leu‐88 (β) at the primary aggregation site substituted by Ala

“…Substitution of Leu by Ala apparently introduces a considerable degree of flexibility so that the interaction of the Val-6 on the donor tetramer of sickle Hb with the acceptor region between Phe-85 and Leu-88 are considerably weakened. In accord with this concept are the results of Baudin-Chich et al (1990) who found that substitution of the hydrophobic side chain of Val-6(/3) by an Ile residue led to an enhancement in gelation. The slope of the initial stage of the aggregation is steeper for HbS than for the double mutant (Fig.…”

“…There are several systems currently available for producing normal or sickle recombinant Hb (Nagai et al, 1985;Baudin-Chich et al, 1990;Wagenbach et al, 1991;Adachi et al, 1992Bihoreau et al, 1992Coghlan et al, 1992;Looker et al, 1992;Martin de Llano et al, 1993a, 1993bShen et al, 1993;Vallone et al, 1993). Previous reports have shown that the recombinant sickle Hb produced in yeast closely resembles natural Hb, i.e., , 1993a, 1993b).…”

Recombinant human sickle hemoglobin expressed in yeast.

“…In the bacterial system, expression of a stable a-globin chain has been difficult because it is apparently degraded (20). Therefore, in most cases (19,20) native a-chains are used to reconstitute hybrid tetramers. In the E. coli system, the ,3-globin is produced as a fusion protein that forms insoluble inclusion bodies and stringent conditions are required for their solubilization.…”

“…The synthetic human a-and 3-globin genes for normal Hb (17) and for a few other mutant Hbs (18)(19)(20)(21)(22)27) have been expressed in E. coli. However, the Hb produced in this system is not processed at its N terminus in the same way that human Hb is processed in human reticulocytes.…”

Mutational analysis of phenylalanine β85 in the valine β6 acceptor pocket during hemoglobin S polymerization

“…The acceptor pocket for the Val-06 is lined at the bottom with the side chains of Phe-085 and Leu-088, and has Asp-073, Thr-084, and Thr-087 around the pocket peReprint requests to: Kazuhiko Adachi, Division of Hematology, The Children's Hospital of Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, Pennsylvania 19104;e-mail: adachi@email.chop.edu. rimeter. The hydrophobic Val-, Leu-, or Ile-06 side chain appears to fit into the hydrophobic pocket between the E and F helices on an adjacent molecule, whereas Glu-06 does not because it may interact with water molecules (Baudin-Chich et al, 1990;Adachi et al, 1993). Neither Phe-nor Trp-06 appears to fit into the pocket because of their larger-sized side chains (Bihoreau et al, 1992;Adachi et al, 1993).…”