title: NMR structure of the alpha-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding.
abstract: The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.
authors.affiliation: Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, United Kingdom.
authors.forename: Clara M
authors.initials: CM
authors.lastname: Santiveri
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Pérez-Cañadillas
authors.forename: Murali K
authors.initials: MK
authors.lastname: Vadivelu
authors.forename: Mark D
authors.initials: MD
authors.lastname: Allen
authors.forename: Trevor J
authors.initials: TJ
authors.lastname: Rutherford
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Watkins
authors.forename: Mark
authors.initials: M
authors.lastname: Bycroft
mesh_terms: D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D015723:Gene Library; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000097:blood / Q000737:chemistry*; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000737:chemistry
chemical_list: C468768:AHSP protein, human; D001798:Blood Proteins; D018076:DNA, Complementary; D008024:Ligands; D018832:Molecular Chaperones; D020033:Protein Isoforms; D014443:Tyrosine; D004247:DNA; D011392:Proline; D000409:Alanine