N-terminal contributions of the gamma-subunit of fetal hemoglobin to its tetramer strength: remote effects at subunit contacts.
The greatly increased tetramer strength of liganded fetal hemoglobin compared with adult hemoglobin is shown by its 70-fold smaller tetramer-dimer dissociation constant. This property has been shown previously to be only partially caused by the 5-amino-acid differences at both types of interfaces in each hemoglobin. A major contributor to tetramer strengthening is the 18-amino-acid N-terminal A helix of the gamma-subunit of fetal hemoglobin, which differs from the beta-subunit of adult hemoglobin at eight amino acid residues. This long-distance communication between the A helix and the distant C helix and FG helical corner comprising the subunit contacts at the allosteric interface represents internal signaling. Physiologically, its greater tetramer strength endows fetal hemoglobin with the capacity to abstract oxygen from maternal adult hemoglobin. It also leads to resistance of fetal red cells to the malaria parasite because the HbF tetramer does not dissociate to dimers as readily as HbA; dimers are digested by malaria proteases but tetramers are not. In this communication, we report which sites on the A helix of the gamma-subunit are important for tetramer strengthening in HbF by substituting certain amino acids in the beta-subunit by the corresponding residues in the gamma-subunit. The recombinant hemoglobins containing up to five replacements together have been extensively characterized. Mass values were within 1 unit of theory. Gly 1 (gamma) of HbF with its high pK(a) of 8.1 compared with a 7.1 value for Val 1 (beta) of HbA creates a highly electropositive N terminus that may couple with the electronegative sequence just after it on the gamma-subunit. The Leu 3 to Phe replacement has no apparent role; however, position 5 is important because replacement of Pro 5 (beta) by Glu 5 (gamma) promotes tetramer strengthening. The Glu --> Asp replacement at position 7 enhances this effect because of the lower pK(a) of Asp but the Val --> Ile substitution at position 11 has no effect. Thus, the three positive/negative sites at positions 1, 5, and 7 account for practically all of the tetramer strength of HbF, as illustrated by an electrostatic surface potential analysis. The pathway by which information is transmitted to the distant allosteric subunit interfaces is currently under study. Oxygen-binding properties of the hemoglobins with charged substitutions more closely resemble those of HbA rather than those of HbF. Thus, whereas the A helix has a major role in controlling the strength of interactions at the tetramer-dimer allosteric interface, oxygen-binding properties of HbA and HbF are influenced by sequences in the C helix and at the FG helical corner constituting the allosteric interface.
Yagami
Takeshi
T
Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Ballard
Barry T
BT
Padovan
Julio Cesar
JC
Chait
Brian T
BT
Popowicz
Anthony M
AM
Manning
James M
JM
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D002942:Circular Dichroism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005319:Fetal Hemoglobin / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014633:Valine / Q000737:chemistry
D011994:Recombinant Proteins; D007532:Isoleucine; D001224:Aspartic Acid; D018698:Glutamic Acid; D010649:Phenylalanine; D005319:Fetal Hemoglobin; D007930:Leucine; D014633:Valine; D010100:Oxygen