title: A cis-proline in alpha-hemoglobin stabilizing protein directs the structural reorganization of alpha-hemoglobin.
abstract: alpha-Hemoglobin (alphaHb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with alphaHb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting alphaHb 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 alphaHb 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 alphaHb. In complex with alphaHb, AHSP Pro(30) adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in alphaHb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the alphaHb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of alphaHb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops.
authors.affiliation: School of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Australia. dagell@mail.usyd.edu.au
authors.forename: David A
authors.initials: DA
authors.lastname: Gell
authors.forename: Liang
authors.initials: L
authors.lastname: Feng
authors.forename: Suiping
authors.initials: S
authors.lastname: Zhou
authors.forename: Philip D
authors.initials: PD
authors.lastname: Jeffrey
authors.forename: Katerina
authors.initials: K
authors.lastname: Bendak
authors.forename: Andrew
authors.initials: A
authors.lastname: Gow
authors.forename: Mitchell J
authors.initials: MJ
authors.lastname: Weiss
authors.forename: Yigong
authors.initials: Y
authors.lastname: Shi
authors.forename: Joel P
authors.initials: JP
authors.lastname: Mackay
mesh_terms: D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D006441:Hemoglobin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D020836:Protein Structure, Quaternary / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: C468768:AHSP protein, human; D001798:Blood Proteins; D018832:Molecular Chaperones; D006441:Hemoglobin A; D011392:Proline
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