title: The crystal structure of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt): Roles of the axial ligand Tyr75 and two distal arginines in heme binding.
abstract: Some Gram-negative pathogens utilize an extracellular heme-binding protein called hemophore to satisfy their needs for iron, a metal element essential for most living things. We report here crystal structures of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt) and its Y75A mutant. The wild-type HasAypt structure revealed that the heme iron is coordinated with Tyr75 and a water molecule. The heme-bound water molecule makes extensive hydrogen bond network that includes Arg40 and Arg144 on the distal heme pocket. Arg40, highly conserved for HasAs from Yersinia species, forms a salt bridge with the propionate side chain of heme, and makes π-π stacking and hydrophobic interactions with porphyrin plane. Interestingly, similar Arg-heme interactions are also found for periplasmic heme transporter, PhuT, suggesting that this is an example of a convergent evolution and one of the important interactions for bacterial heme transportation. Heme titration, heme binding kinetics, and the crystal structures of wild-type and Y75A proteins show that, although Tyr75 is primarily important for heme capturing, other interactions with Arg40, Arg144, and hydrophobic residues also contribute for heme acquisition. We also found that HasAypt can form a dimer in solution. The structure of the domain-swapped Y75A HasAypt dimer shows the presence of two low-spin heme molecules coordinated with His84 and His140, and displacement of the Arg40 loop of dimeric Y75A HasAypt results in deformation of the heme-binding pocket. A similar rearrangement of the distal heme loop might occur in heme transfer from HasAypt to HasRypt.
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Koyama Minami, Tottori 680-8552, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kanadani
authors.affiliation: Department of Biological Sciences, Yamaguchi University, Yoshida, Yamaguchi 753-8515, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Sato
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Koyama Minami, Tottori 680-8552, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Hino
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Koyama Minami, Tottori 680-8552, Japan. Electronic address: snagano@bio.tottori-u.ac.jp.
authors.forename: Shingo
authors.initials: S
authors.lastname: Nagano
authors.affiliation: Department of Biological Sciences, Yamaguchi University, Yoshida, Yamaguchi 753-8515, Japan. Electronic address: ozakis@yamaguchi-u.ac.jp.
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Ozaki
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000737:chemistry*; D008024:Ligands; D014867:Water / Q000737:chemistry; D015011:Yersinia pseudotuberculosis / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D008024:Ligands; D014867:Water; D006418:Heme; D001120:Arginine
keywords: Crystal structures; Heme uptake; Hemophore; Yersinia pseudotuberculosis