α-Hemoglobin Stabilizing Protein (AHSP), a Kinetic Scheme of the Action of a Human Mutant, AHSPV56G

Abstract

A kinetic analysis has been made of the interaction of ␣-Hb chains with a mutant ␣-hemoglobin stabilizing protein, AHSP V56G , which is the first case of an AHSP mutation associated with clinical symptoms of mild thalassemia syndrome. The chaperone AHSP is thought to protect nascent ␣ chains until final binding to the partner ␤-Hb. Rather than protecting ␣ chains, the mutant chaperone is partially unfolded but recovers its secondary structure via interaction with ␣-Hb. For both AHSP WT and AHSP V56G , the binding to ␣-Hb is quite rapid relative to the ␣-␤ reaction, as expected because the chaperone binding must be quite competitive to complete the ␣ chain folding process before ␣-Hb binds irreversibly to ␤-Hb. The main kinetic difference is a dissociation rate of AHSP V56G ⅐␣-Hb some four times faster relative to AHSP⅐␣-Hb. Considering a role of protein folding, the AHSP V56G apparently does not bind long enough (0.5 s versus 2 s for the WT) to complete the structural modifications. The overall replacement reaction (AHSP⅐␣-Hb ؉ ␤-Hb 3 AHSP ؉ ␣␤) can be quite long, especially if there is an excess of AHSP relative to ␤-Hb monomers.During the investigation of a child presenting with a mild thalassemia syndrome, normal globin gene pattern was observed while a structural abnormality of the ␣-hemoglobin stabilizing protein (AHSP) 2 gene (AHSP Val 56 3 Gly or AHSP V56G ) was found at the homozygous state (1). The child suffered from a microcytic, hypochromic anemia, which required a blood transfusion at the age of 1 month. This clinical observation was the first evidence that a homozygous defect of AHSP may be the etiology of thalassemia syndrome in human. AHSP is the ␣-hemoglobin (␣-Hb) chaperone that binds the holo or apo␣-Hb chain but not ␤-Hb or Hb tetramers (2). The steady state red blood cell showed values below the normal range for total Hb (11 g/decaliter), mean corpuscular volume (60 femtoliter), and mean corpuscular hemoglobin (20 pg).AHSP is a small 102-residue protein expressed only in erythroid cells, which adopts a three helix bundle (3). Helices 1 and 2 and the intervening segment of AHSP specifically recognize the G and H helices of ␣-Hb, which also are involved in the ␣1␤1 interface of Hb dimers (4). dos Santos et al. (5) have shown that, in human erythroid cells, the AHSP gene expression is related to that of the ␣-globin gene. AHSP concentration is estimated to be ϳ0.1 mM at the end of erythropoiesis (2), whereas that of Hb is ϳ5 mM. Nascent ␣ chains are protected by the chaperone before binding to the partner ␤ chains. Within the developing red blood cell precursors, the chaperone must first bind the ␣ chain to complete its correct folding. Evidence that AHSP facilitates folding of the ␣-globin subunits for Hb synthesis has been brought by Yu et al. (6), which adds a key element to the reaction scheme of Hb synthesis.If the chaperone simply acts as a buffer for an excess of ␣ chains, an appropriate affinity is necessary, but there would be freedom in the protein-protein interaction rates. This rol...

Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate

“…We confirmed and extended their work using a stopped-flow fluorometer to measure the rates of association (kЈ AHSP ) and dissociation (k AHSP ) (19), and our initial kinetic measurements were subsequently confirmed by Brillet et al (35).…”

“…Notably, x-ray crystal structures for both ferric and ferrous ␣⅐AHSP complexes have been reported (14,15 as free AHSP is generated. The rate of this process allows measurement of k AHSP (19,32,35). The observed time courses for the reaction of ␤ with ferrous ␣⅐AHSP complexes are monophasic at high concentrations, and the rate of displacement increases with increasing [␤].…”

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

“…This role is supported by kinetic studies showing that both ferrous and ferric holo-␣ bind ϳ20 times more rapidly to AHSP than to ferrous ␤ subunits (14,25,42). However, ␤ subunits have a 10,000-fold higher affinity for ␣-globin than AHSP due to an extremely small ␣1␤1 dissociation rate constant, which is on the order of 10 Ϫ5 s Ϫ1 (25,65).…”

“…Loss of AHSP also aggravates both ␤-thalassemia (11) and ␣-thalassemia (12) in mice. In humans, several naturally occurring ␣-globin missense mutations and AHSP variants appear to cause anemia by inhibiting the ability of ␣-globin to interact with AHSP (13)(14)(15)(16)(17).…”

“…AHSP helix 1, helix 2, and the interconnecting loop interact with helices G and H of ␣-globin at a surface that overlaps with the interface for ␤-globin binding. However, ␤ subunits bind ␣ sub-* This work was supported, in whole or in part, by National Institutes of Health units with higher affinity and displace ␣ from AHSP complexes in solution and in vivo (9,10,14,24,25). These observations indicate that AHSP can act as a molecular chaperone to bind nascent ␣-globin and stabilize its folding prior to incorporation into HbA during normal erythropoiesis (12,14,24,25).…”