High-affinity binding of the staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded beta-barrel fold.
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.
Dryla
Agnieszka
A
Intercell AG, Vienna Biocenter 6, A-1030 Vienna, Austria.
Hoffmann
Bernd
B
Gelbmann
Dieter
D
Giefing
Carmen
C
Hanner
Markus
M
Meinke
Andreas
A
Anderson
Annaliesa S
AS
Koppensteiner
Walter
W
Konrat
Robert
R
von Gabain
Alexander
A
Nagy
Eszter
E
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
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D006242:Haptoglobins; D006454:Hemoglobins; C510010:IsdB protein, Staphylococcus aureus; D008565:Membrane Proteins