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J. Biol. Chem., Vol. 279, Issue 16, 16561-16570, April 16, 2004

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Monoclonal Antibodies Specific for the Empty Conformation of HLA-DR1 Reveal Aspects of the Conformational Change Associated with Peptide Binding^*

Gregory J. Carven, Sriram Chitta, Ivan Hilgert¶, Mia M. Rushe, Rick F. Baggio||, Michelle Palmer||, Jaime E. Arenas||, Jack L. Strominger**, Vaclav Horejsi¶, Laura Santambrogio, and Lawrence J. Stern¶¶

From the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, the Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, the ^¶Institute of Molecular Genetics, Academy of Sciences, Prague, Czech Republic CZ-16637, ^||Applied Biosystems, Cell Biology and Functional Proteomics, Bedford, Massachusetts 01730, the ^**Department of Molecular and Cellular Biology, Harvard University, Cambridge 02138, Massachusetts, the Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, and the Department of Pathology, Albert Einstein College of Medicine, New York, New York 10461

Class II major histocompatibility complex (MHC) proteins bind peptides and present them at the cell surface for interaction with CD4⁺ T cells as part of the system by which the immune system surveys the body for signs of infection. Peptide binding is known to induce conformational changes in class II MHC proteins on the basis of a variety of hydrodynamic and spectroscopic approaches, but the changes have not been clearly localized within the overall class II MHC structure. To map the peptide-induced conformational change for HLA-DR1, a common human class II MHC variant, we generated a series of monoclonal antibodies recognizing the subunit that are specific for the empty conformation. Each antibody reacted with the empty but not the peptide-loaded form, for both soluble recombinant protein and native protein expressed at the cell surface. Antibody binding epitopes were characterized using overlapping peptides and alanine scanning substitutions and were localized to two distinct regions of the protein. The pattern of key residues within the epitopes suggested that the two epitope regions undergo substantial conformational alteration during peptide binding. These results illuminate aspects of the structure of the empty forms and the nature of the peptide-induced conformational change.

Received for publication, December 31, 2003 , and in revised form, January 26, 2004.

^* This work was supported by National Institutes of Health Grants AI-48833 (to L. J. S.), AI-49524 (to J. L. S.), and AI-48832 (to L. S.). The Prague laboratory was supported by the Center of Molecular and Cellular Immunology (LN00A026), Ministry of Education, Youth and Sports of the Czech Republic. 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.

^¶¶ To whom correspondence should be addressed: Dept. of Pathology, Rm. S2-127, Worcester, MA 01655. Tel.: 508-856-1831; Fax: 508-856-0019; E-mail: lawrence.stern{at}umassmed.edu.

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