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J. Biol. Chem., Vol. 282, Issue 29, 21425-21436, July 20, 2007

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Empty Class II Major Histocompatibility Complex Created by Peptide Photolysis Establishes the Role of DM in Peptide Association^*

Gijsbert M. Grotenbreg¹², Melissa J. Nicholson¹, Kevin D. Fowler^¶3, Kathrin Wilbuer, Leah Octavio^¶, Maxine Yang^¶, Arup K. Chakraborty^¶||**3, Hidde L. Ploegh, and Kai W. Wucherpfennig⁴

From the Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02139, the Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston Massachusetts 02115, and the Departments of ^¶Chemical Engineering, ^||Chemistry, and ^**Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.

Received for publication, April 3, 2007 , and in revised form, May 17, 2007.

^* This work was supported by National Institutes of Health Grants RO1 AI057493 (to K. W. W.) and PO1 AI045757 (to K. W. W. and H. L. P.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and supplemental Fig. S1.

¹ These authors contributed equally to this work.

² Supported by a postdoctoral fellowship from the Netherlands Organization for Scientific Research.

³ Supported by National Institutes of Health Grant P01 AI071195 and Director's Pioneer Award 1DP1OD001022.

⁴ To whom correspondence should be addressed: Dr. Kai Wucherpfennig, 44 Binney St., Boston MA 02115. Tel.: 617-632-3086; Fax: 617-632-2662; E-mail: Kai_Wucherpfennig{at}dfci.harvard.edu.

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