HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 41, 30051-30061, October 12, 2007

This Article Full Text Full Text (PDF) All Versions of this Article: 282/41/30051    most recent M704247200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hammel, M. Articles by Geisbrecht, B. V. Search for Related Content PubMed PubMed Citation Articles by Hammel, M. Articles by Geisbrecht, B. V. Social Bookmarking                      What's this?

Characterization of Ehp, a Secreted Complement Inhibitory Protein from Staphylococcus aureus^*

Michal Hammel¹², Georgia Sfyroera¹, Serapion Pyrpassopoulos¹, Daniel Ricklin¹, Kasra X. Ramyar^¶, Mihai Pop^||, Zhongmin Jin^**, John D. Lambris, and Brian V. Geisbrecht³

From the School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110, the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19105, the ^¶Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21224, the ^||Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland 20742, and the ^**Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439

We report here the discovery and characterization of Ehp, a new secreted Staphylococcus aureus protein that potently inhibits the alternative complement activation pathway. Ehp was identified through a genomic scan as an uncharacterized secreted protein from S. aureus, and immunoblotting of conditioned S. aureus culture medium revealed that the Ehp protein was secreted at the highest levels during log-phase bacterial growth. The mature Ehp polypeptide is composed of 80 residues and is 44% identical to the complement inhibitory domain of S. aureus Efb (extracellular fibrinogen-binding protein). We observed preferential binding by Ehp to native and hydrolyzed C3 relative to fully active C3b and found that Ehp formed a subnanomolar affinity complex with these various forms of C3 by binding to its thioester-containing C3d domain. Site-directed mutagenesis demonstrated that Arg⁷⁵ and Asn⁸² are important in forming the Ehp·C3d complex, but loss of these side chains did not completely disrupt Ehp/C3d binding. This suggested the presence of a second C3d-binding site in Ehp, which was mapped to the proximity of Ehp Asn⁶³. Further molecular level details of the Ehp/C3d interaction were revealed by solving the 2.7-Å crystal structure of an Ehp·C3d complex in which the low affinity site had been mutationally inactivated. Ehp potently inhibited C3b deposition onto sensitized surfaces by the alternative complement activation pathway. This inhibition was directly related to Ehp/C3d binding and was more potent than that seen for Efb-C. An altered conformation in Ehp-bound C3 was detected by monoclonal antibody C3-9, which is specific for a neoantigen exposed in activated forms of C3. Our results suggest that increased inhibitory potency of Ehp relative to Efb-C is derived from the second C3-binding site in this new protein.

Received for publication, May 23, 2007 , and in revised form, July 16, 2007.

The atomic coordinates and structure factors (code 2NOJ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the School of Biological Sciences, University of Missouri, Kansas City; Grant 2509 from the University of Missouri Research Board (to B. V. G.); and NIAID Grant AI30040 from the National Institutes of Health (to J. D. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² Present address: Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.

³ To whom correspondence should be addressed: Div. of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Rd., Kansas City, MO 64110. Tel.: 816-235-2592; Fax: 816-235-1503; E-mail: GeisbrechtB{at}umkc.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				A. Upadhyay, J. D. Burman, E. A. Clark, E. Leung, D. E. Isenman, J. M. H. van den Elsen, and S. Bagby Structure-Function Analysis of the C3 Binding Region of Staphylococcus aureus Immune Subversion Protein Sbi J. Biol. Chem., August 8, 2008; 283(32): 22113 - 22120. [Abstract] [Full Text] [PDF]

				J. D. Burman, E. Leung, K. L. Atkins, M. N. O'Seaghdha, L. Lango, P. Bernado, S. Bagby, D. I. Svergun, T. J. Foster, D. E. Isenman, et al. Interaction of Human Complement with Sbi, a Staphylococcal Immunoglobulin-binding Protein: INDICATIONS OF A NOVEL MECHANISM OF COMPLEMENT EVASION BY STAPHYLOCOCCUS AUREUS J. Biol. Chem., June 20, 2008; 283(25): 17579 - 17593. [Abstract] [Full Text] [PDF]