title: High-affinity binding of the staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded beta-barrel fold.
abstract: Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for "near transporter") present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (K(D)) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel beta-barrel with the strand order (-beta1 -beta2 -beta3 -beta6 -beta5 -beta4 -beta7 -beta8), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.
authors.affiliation: Intercell AG, Vienna Biocenter 6, A-1030 Vienna, Austria.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Dryla
authors.forename: Bernd
authors.initials: B
authors.lastname: Hoffmann
authors.forename: Dieter
authors.initials: D
authors.lastname: Gelbmann
authors.forename: Carmen
authors.initials: C
authors.lastname: Giefing
authors.forename: Markus
authors.initials: M
authors.lastname: Hanner
authors.forename: Andreas
authors.initials: A
authors.lastname: Meinke
authors.forename: Annaliesa S
authors.initials: AS
authors.lastname: Anderson
authors.forename: Walter
authors.initials: W
authors.lastname: Koppensteiner
authors.forename: Robert
authors.initials: R
authors.lastname: Konrat
authors.forename: Alexander
authors.initials: A
authors.lastname: von Gabain
authors.forename: Eszter
authors.initials: E
authors.lastname: Nagy
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D006242:Haptoglobins / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D006242:Haptoglobins; D006454:Hemoglobins; C510010:IsdB protein, Staphylococcus aureus; D008565:Membrane Proteins