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Journal of Bacteriology, January 2007, p. 254-264, Vol. 189, No. 1
0021-9193/07/$08.00+0     doi:10.1128/JB.01366-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

High-Affinity Binding of the Staphylococcal HarA Protein to Haptoglobin and Hemoglobin Involves a Domain with an Antiparallel Eight-Stranded ß-Barrel Fold{triangledown}

Agnieszka Dryla,1,{ddagger} Bernd Hoffmann,2,{dagger},{ddagger} Dieter Gelbmann,1 Carmen Giefing,1 Markus Hanner,1 Andreas Meinke,1 Annaliesa S. Anderson,4 Walter Koppensteiner,3 Robert Konrat,2 Alexander von Gabain,1 and Eszter Nagy1*

Intercell AG, Vienna Biocenter 6, A-1030 Vienna, Austria,1 Department of Biomolecular Structural Chemistry, University of Vienna, Vienna Biocenter 5, A-1030, Vienna, Austria,2 Biovertis AG, Vienna Biocenter 6, A-1030 Vienna, Austria,3 Merck and Co. Incorporated, 440 Sumneytown Pike, WP16 100, Westpoint, Pennsylvania 194864

Received 29 August 2006/ Accepted 3 October 2006

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 (KD) 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 ß-barrel with the strand order (-ß1{downarrow}-ß2{uparrow}-ß3{downarrow}-ß6{uparrow}-ß5{downarrow}-ß4{uparrow}-ß7{downarrow}-ß8{uparrow}), 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.

* Corresponding author. Mailing address: Intercell AG, Vienna Biocenter 6, A-1030 Vienna, Austria. Phone: 43-1-20620-115. Fax: 43-1-20620-805. E-mail: ENagy{at}intercell.com.

{triangledown} Published ahead of print on 13 October 2006.

{dagger} Present address: Biovertis AG, Campus Vienna Biocenter 6, A-1030, Vienna, Austria.

{ddagger} A.D. and B.H. made equal contributions to this study.

Journal of Bacteriology, January 2007, p. 254-264, Vol. 189, No. 1
0021-9193/07/$08.00+0     doi:10.1128/JB.01366-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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