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J. Bacteriol. doi:10.1128/JB.01366-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

High affinity binding of the staphylococcal HarA protein to haptoglobin and haemoglobin involves a domain having an anti-parallel eight-stranded -barrel fold

Intercell AG, Vienna Biocentrer 6, A-1030 Vienna, Austria, Dept. of Biomolecular Structural Chemistry, University of Vienna, Vienna Biocenter 5, A-1030, Vienna, Austria, Biovertis AG, Vienna Biocentrer 6, A-1030 Vienna, Austria, and Merck and Co. Inc, 440 Sumneytown Pike, WP16 100, Westpoint, PA, 19486, USA

^* To whom correspondence should be addressed. Email: ENagy{at}intercell.com.

	Abstract

Iron-scavenging from the host is essential for growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind haemoproteins. HarA and IsdB harbour homologous ligand binding domains, the so called NEAT domain (‘near transporter’) present in several surface proteins of Gram positive pathogens. Surface plasmon resonance measurements using GST-tagged HarAD1, one of the ligand binding domain of HarA and GST-tagged full length IsdB proteins confirmed high affinity binding to haemoglobin and haptoglobin-haemoglobin complexes with K_D values of 5-50 nM. Haptoglobin-binding could be detected only with HarA, and was in the low micromolar range. In order to determine the fold of this evolutionary conserved ligand binding domain, the untagged HarAD1 protein was subjected to NMR spectroscopy that revealed an eight-stranded, purely antiparallel -barrel with the strand order (-1-2-3-6-5-4-7-8) forming two Greek key motifs. Based on structural homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin fold family whose members are involved in protein-protein interactions, however 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.

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