A cis-Proline in α-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of α-Hemoglobin

Abstract

␣-Hemoglobin (␣Hb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with ␣Hb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting ␣Hb to a conformation in which the heme is coordinated at both axial positions by histidine side chains (bis-histidyl coordination). Currently, the detailed mechanism by which AHSP induces structural changes in ␣Hb has not been determined. Here, we present x-ray crystallography, NMR spectroscopy, and mutagenesis data that identify, for the first time, the importance of an evolutionarily conserved proline, Pro 30 , in loop 1 of AHSP. Mutation of Pro 30 to a variety of residue types results in reduced ability to convert ␣Hb. In complex with ␣Hb, AHSP Pro 30 adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in ␣Hb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the ␣Hb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of ␣Hb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops.Mammalian adult hemoglobin (HbA) 5 is a tetramer of two ␣Hb and two ␤Hb subunits, which is produced to extremely high concentrations (ϳ340 mg/ml) in red blood cells. Numerous mechanisms exist to balance and coordinate HbA synthesis in normal erythropoiesis, and problems with the production of either HbA subunit give rise to thalassemia, a common cause of anemia worldwide. Previously, we identified ␣-hemoglobin stabilizing protein (AHSP) as an accessory factor in normal HbA production (1). AHSP forms a dimeric complex with ␣Hb (see Fig. 1A) (2) but does not interact with ␤Hb or HbA. AHSP also binds heme-free (apo) ␣Hb (3) and may serve functions in both the folding of nascent ␣Hb (4) and the detoxification of excess ␣Hb that remains following HbA assembly (2, 5). Mice carrying an Ahsp gene knock-out display mild anemia, ineffective erythropoiesis, and enhanced sensitivity to oxidative stress (1, 6), features also observed in ␤-thalassemia patients due to the cytotoxic effects of free ␣Hb.Free ␣Hb promotes the formation of harmful reactive oxygen species as a result of reduction/oxidation reactions involving the heme iron (7,8). Reactive oxygen species can damage heme, ␣Hb, and other cellular structures, resulting in hemoglobin precipitates and death of erythroid precursor cells (9 -12). The presence of AHSP may explain how cells tolerate the slight excess of ␣Hb that is observed in normal erythropoiesis, which is postulated to inhibit the formation of non-functional ␤Hb tetramers, thus providing a robust mechanism for achieving the correct subunit stoichiometry during HbA assembly (13).Structural and biochemical studies have begun to elucidate the molecular mechanism by which AHSP detoxifies ␣Hb. AHSP binds to oxygenated ␣Hb to generate an initial complex that retains the oxy-hem...

Insights into Hemoglobin Assembly through in Vivo Mutagenesis of α-Hemoglobin Stabilizing Protein

“…1). To examine the function of this amino acid, Gell et al (18) made a series of mutations that convert AHSP Pro 30 to Gly, Ala, Val, Phe, and Trp and screened the resultant protein interactions with ␣-globin. Except for the P30G mutant, all AHSP variants showed enhanced affinity (lower K D values) for ferrous ␣CO or ␣O 2 as measured by isothermal titration calorimetry.…”

“…AHSP binding to ␣O 2 accelerates spontaneous heme iron oxidation (autooxidation) from an Fe 2ϩ (ferrous) to an Fe 3ϩ (or met) state. Autooxidation is followed immediately by internal coordination of His 58 (E7) to the hemin iron, resulting in a hexacoordinate bishistidyl or hemichrome structure (18,21,22,28). Bishistidyl hemoglobins occur as ␣-globin degradation products in ␤-thalassemia (4,6,29,30), during normal degradation of hemoglobin, and in some naturally occurring globins in mammalian tissues, plants, certain bacteria, and cold water fish (31)(32)(33)(34)(35)(36)(37)(38)(39)(40).…”

“…An evolutionarily conserved proline residue within loop 1 of AHSP regulates binding to ferrous ␣O 2 and its subsequent autooxidation (18). In the structure of the ASHP⅐met-␣ complex, the cis conformation at the AHSP Asp 29 -Pro 30 peptide bond projects loop 1 toward ␣-globin helix G where AHSP Pro 30 promotes backbone hydrogen bonding interactions.…”

“…In the structure of the ASHP⅐met-␣ complex, the cis conformation at the AHSP Asp 29 -Pro 30 peptide bond projects loop 1 toward ␣-globin helix G where AHSP Pro 30 promotes backbone hydrogen bonding interactions. Pro 30 amino acid substitutions in AHSP convert the 29-30 peptide bond to a completely trans conformation, which generally enhances the affinity of AHSP for ferrous ␣CO and ␣O 2 and reduces the rates of autooxidation and subsequent conversion to the bishistidyl form (18).…”

“…Because of these large effects on AHSP affinity for reduced ␣ subunits and on the redox and coordination properties of bound ␣ subunits, we chose to examine the in vivo phenotypes of P30A and P30W variants of murine AHSP (18). Although these mutations significantly altered AHSP/␣O 2 interactions in vitro, they surprisingly produced no detectable consequences in mice.…”