α-Hemoglobin (αHb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with αHb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting αHb 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 αHb 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 αHb. In complex with αHb, AHSP Pro
30 adopts a
cis-peptidyl conformation and makes contact with the N terminus of helix G in αHb. Mutations that stabilize the
cis-peptidyl conformation of free AHSP, also enhance the αHb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of αHb, 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 αHb and two βHb subunits, which is produced to extremely high concentrations (∼340 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 α-hemoglobin stabilizing protein (AHSP) as an accessory factor in normal HbA production (
1). AHSP forms a dimeric complex with αHb (see
A) (
2) but does not interact with βHb or HbA. AHSP also binds heme-free (apo) αHb (
3) and may serve functions in both the folding of nascent αHb (
4) and the detoxification of excess αHb 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 β-thalassemia patients due to the cytotoxic effects of free αHb.
Open in a separate windowSummary of αHb·AHSP interactions.
A, the αHb·AHSP complex(PDB code
1Z8U) (
2). The interface is formed from helices 1 and 2 and the intervening loop 1 (
green) of AHSP, together with helices G-H and the B-C corner of αHb (
cyan).
B, detailed views of the heme binding site of αHb as it appears in oxy-HbA (PDB code
1GZX) (
69) and the final bis-histidyl αHb·AHSP complex (PDB code
1Z8U) with two histidine ligands to the iron. Typical visible absorption spectra in the region 450–700 nm are shown.Free αHb 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, αHb, 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 αHb that is observed in normal erythropoiesis, which is postulated to inhibit the formation of non-functional βHb 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 αHb. AHSP binds to oxygenated αHb to generate an initial complex that retains the oxy-heme, as evidenced by a characteristic visible absorption spectrum (see
B,
middle) and resonance Raman spectrum (
5). This initial oxy-αHb·AHSP complex then converts to a low spin Fe
3+ complex (
2), in which the heme iron is bound at both axial positions by the side chains of His
58 and His
87 from αHb (see
B,
right). The formation of this complex inhibits αHb peroxidase activity and heme loss (
2). Bis-histidyl heme coordination is becoming increasingly recognized as a feature of numerous vertebrate and non-vertebrate globins (
14) and has been shown previously to confer a relative stabilization of the Fe
3+ over the Fe
2+ oxidation state (
15–
17). Although bis-histidyl heme coordination has previously been detected in solutions of met-Hb, formed through spontaneous autoxidation of Hb (
18–
21), the bishis-αHb·AHSP complex provides the first evidence that the bis-histidyl heme may play a positive functional role in Hb biochemistry by inhibiting the production of harmful reactive oxygen species.Despite its potential importance, the mechanism by which AHSP influences heme coordination in its binding partner is still unknown. As shown in
A, AHSP binds αHb at a surface away from the heme pocket, and thus structural changes must somehow be transmitted through the αHb protein. It is intriguing that the free AHSP protein switches between two alternative conformations linked to
cis/
trans isomerization of the Asp
29-Pro
30 peptide bond in loop 1 (
22) and that, in complex with αHb, this loop is located at the αHb·AHSP interface (see
A). Peptide bonds preceding proline residues are unique in that the
cis or
trans bonding conformations have relatively similar stabilities (
23), allowing an interconversion between these conformations that can be important for protein function (
24,
25). Previous x-ray crystal structures of αHb·AHSP complexes have been obtained only with a P30A mutant of AHSP, in which isomerization is abolished and the Asp
29-Ala
30 peptide bond adopts a
trans conformation, leaving the potential structural and functional significance of the evolutionarily conserved Pro
30 undisclosed. Here, we demonstrate a functional role for AHSP Pro
30 in conversion of oxy-αHb to the bis-histidyl form and identify a specific structural role for a
cis Asp
29-Pro
30 peptide bond in this process. From a mechanistic understanding of how AHSP promotes formation of bis-histidyl αHb, we may eventually be able to engineer AHSP function as a tool in new treatments for Hb diseases such as β-thalassemia.
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