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J. Biol. Chem., Vol. 279, Issue 27, 28475-28482, July 2, 2004

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Structural Model of MD-2 and Functional Role of Its Basic Amino Acid Clusters Involved in Cellular Lipopolysaccharide Recognition^*

Anton Gruber, Mateja Manek¶, Hermann Wagner, Carsten J. Kirschning, and Roman Jerala¶||

From the Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany and the ^¶Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia

The receptor complex resulting from association of MD-2 and the ectodomain of Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signal transduction across the cell membrane. We prepared a tertiary structure model of MD-2, based on the known structures of homologous lipid-binding proteins. Analysis of circular dichroic spectra of purified bacterially expressed MD-2 indicates high content of -type secondary structure, in agreement with the structural model. Bacterially expressed MD-2 was able to confer LPS responsiveness to cells expressing TLR4 despite lacking glycosylation. We identified several clusters of basic residues on the surface of MD-2. Mutation of each of two clusters encompassing the residues Lys⁸⁹-Arg⁹⁰-Lys⁹¹ and Lys¹²⁵-Lys¹²⁵ significantly decreased the signal transduction of the respective MD-2 mutants either upon co-expression with TLR4 or upon addition as soluble protein into the supernatant of cells overexpressing TLR4. These basic clusters lie at the edge of the -sheet sandwich, which in cholesterol-binding protein connected to Niemann-Pick disease C2 (NPC2), dust mite allergen Der p2, and ganglioside GM2-activator protein form a hydrophobic pocket. In contrast, mutation of another basic cluster composed of Arg⁶⁹–Lys⁷², which according to the model lies further apart from the hydrophobic pocket only weakly decreased MD-2 activity. Furthermore, addition of the peptide, comprising the surface loop between Cys⁹⁵ and Cys¹⁰⁵, predicted by model, particularly in oxidized form, decreased LPS-induced production of tumor necrosis factor and interleukin-8 upon application to monocytic cells and fibroblasts, respectively, supporting its involvement in LPS signaling. Our structural model of MD-2 is corroborated by biochemical analysis and contributes to the unraveling of molecular interactions in LPS recognition.

Received for publication, January 29, 2004 , and in revised form, April 23, 2004.

The atomic coordinates and structure factors (code 1T2Z (MD-2 model)) 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 Ministry of Education, Science and Sports of Slovenia and German Research Community Grant KI 591/1-5 (to A. G.). 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.

These authors contributed equally to this work.

^|| To whom correspondence should be addressed. E-mail: roman.jerala{at}ki.si.

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