title: Homodimerization of the PAS-B domains of hypoxia-inducible factors.
abstract: The Per-Arnt-Sim (PAS) domains of hypoxia-inducible transcription factors (HIF) mediate heterodimer formation between the HIF-α forms that are induced in the event of cellular hypoxia and the constitutive HIF-β variants. Previous efforts toward structural characterization of the HIF-1α PAS domains were limited by protein stability. Using homology modeling based on the published crystal structure of the PAS-B domain of the homologous protein HIF-2α in complex with the partner HIF-β (also known as ARNT), we have identified a variant of HIF-1α with improved solubility, monodispersity, and stability. Purified solutions of the PAS-B domains of HIF-1α and HIF-2α differ in their propensity for homodimer formation. In an attempt to understand the structural basis for this difference, and to document the structural changes that accompany homodimer formation, we have undertaken a comparative NMR study of the PAS-B domains of HIF-1α and HIF-2α and mutants of HIF-1α that mimic the behavior of HIF-2α. The NMR spectra of all of these domains are very similar, consistent with the similarity of their amino acid sequences. However, the greater propensity of the HIF-1α PAS-B domain to form dimers as the concentration was increased allowed us to determine the site of homodimerization and pointed toward possible sequence changes in HIF-1α that might discourage the formation of homodimers.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.forename: Maria
authors.initials: M
authors.lastname: Martinez-Yamout
authors.forename: Rosa
authors.initials: R
authors.lastname: Cardoso
authors.forename: Jiangli
authors.initials: J
authors.lastname: Yan
authors.forename: Robert A
authors.initials: RA
authors.lastname: Love
authors.forename: Neil
authors.initials: N
authors.lastname: Grodsky
authors.forename: Alexei
authors.initials: A
authors.lastname: Brooun
authors.forename: H Jane
authors.initials: HJ
authors.lastname: Dyson
mesh_terms: D000595:Amino Acid Sequence; D019281:Dimerization; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D012995:Solubility
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C116037:PAS domain kinases; D017346:Protein Serine-Threonine Kinases